figf5 ( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP2 - RNAi and starved for 12 h . Scale bars , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d . ( c ) Representative TEM micrographs of nonspecific - or AP2 - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bars , 5 μm . ( d ) NRK cells were starved for 4 h , stained with antibodies against AP2 and LAMP1 , and then analysed by 3D - SIM . Scale bar , 5 μm . The right panels show enlarged regions of interest from the left panel : T , top view ; L , lateral view ; B , bottom view . ( e ) Fractions from Fig . 2f were analysed by western blotting with an antibody against AP2α . The outline indicates the LAMP2 - positive fraction . ( g ) Purified autolysosomes were treated with trypsin , incubated with rat brain cytosol , washed and then analysed by western blot with antibodies against AP2α and LAMP2 . ( h ) Stripped autolysosomes were pretreated with GFP or PtdIns ( 4 , 5 ) P2 antibody and then incubated with rat brain cytosol . Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2 . ( i ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP2 - RNAi and starved for 12 h . Scale bar , 5 μm . The insets in a , c and i represent enlarged regions of interest . ( j ) Cells from i were analysed for the Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 cells from three independent experiments ) . Error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP2-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.(c) Representative TEM micrographs of nonspecific- or AP2-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.(d) NRK cells were starved for 4 h, stained with antibodies against AP2 and LAMP1, and then analysed by 3D-SIM. Scale bar, 5 μm. The right panels show enlarged regions of interest from the left panel: T, top view; L, lateral view; B, bottom view.(e) Fractions from Fig. 2f were analysed by western blotting with an antibody against AP2α. The outline indicates the LAMP2-positive fraction.(g) Purified autolysosomes were treated with trypsin, incubated with rat brain cytosol, washed and then analysed by western blot with antibodies against AP2α and LAMP2.(h) Stripped autolysosomes were pretreated with GFP or PtdIns(4,5)P2 antibody and then incubated with rat brain cytosol. Autolysosomes were washed and then analysed by western blotting with antibodies against AP2α and LAMP2.(i) NRK cell lines stably expressing LAMP1-Cherry;clathrin-GFP were transfected with nonspecific- or AP2-RNAi and starved for 12 h. Scale bar, 5 μm. The insets in a, c and i represent enlarged regions of interest. (j) Cells from i were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments). Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


figf6 ( a ) NRK cells stably expressing CFP - LC3 ; LAMP1 - YFP were transfected with nonspecific ( NS ) - or AP4 - RNAi and starved for 12 h . Scale bars , 5 μm . ( b ) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d . ( c ) Representative TEM micrographs of nonspecific - or AP4 - RNAi - transfected NRK cells after 12 h of serum starvation . Scale bars , 5 μm . ( d ) NRK cells were starved for 0 , 4 , 8 or 12 h , and isolated lysosomes / autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2 . ( e ) Purified reformation tubules were stained with antibodies against AP4 and LAMP1 . Scale bar , 2 μm . The bottom row shows enlarged regions of interest from the top panel . ( f ) NRK cells were transfected with GFP and LAMP1 - GFP . At 20 h after transfection , cells were starved for 0 or 8 h , and then collected and analysed by immunoprecipitation ( IP ) and western blotting ( WB ) of proteins as indicated , using stably expressed LAMP1 - GFP as bait . TCL , total cell lysate . The insets in a , c and g represent enlarged regions of interest . ( g ) NRK cell lines stably expressing LAMP1 - Cherry ; clathrin - GFP were transfected with nonspecific - or AP4 - RNAi and starved for 12 h . Scale bars , 5 μm . ( h ) Cells from g were analysed for the Pearson ' s co - localization coefficent ( R ( r ) ) by Olympus Fluoview . n = 30 cells from three independent experiments . Error bars indicate the s . d . Uncropped images of blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf6(a) NRK cells stably expressing CFP-LC3; LAMP1-YFP were transfected with nonspecific (NS)- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (b) Cells from a were assessed for enlarged autolysosomes in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.(c) Representative TEM micrographs of nonspecific- or AP4-RNAi-transfected NRK cells after 12 h of serum starvation. Scale bars, 5 μm.(d) NRK cells were starved for 0, 4, 8 or 12 h, and isolated lysosomes/autolysosome were analysed by western blotting using antibodies against AP4 or LAMP2.(e) Purified reformation tubules were stained with antibodies against AP4 and LAMP1. Scale bar, 2 μm. The bottom row shows enlarged regions of interest from the top panel.(f) NRK cells were transfected with GFP and LAMP1-GFP. At 20 h after transfection, cells were starved for 0 or 8 h, and then collected and analysed by immunoprecipitation (IP) and western blotting (WB) of proteins as indicated, using stably expressed LAMP1-GFP as bait. TCL, total cell lysate.The insets in a, c and g represent enlarged regions of interest. (g) NRK cell lines stably expressing LAMP1-Cherry; clathrin-GFP were transfected with nonspecific- or AP4-RNAi and starved for 12 h. Scale bars, 5 μm. (h) Cells from g were analysed for the Pearson's co-localization coefficent (R(r)) by Olympus Fluoview. n = 30 cells from three independent experiments. Error bars indicate the s.d. Uncropped images of blots are shown in Supplementary Fig. S6.


figf7 ( a ) Purified autolysosomes were treated with trypsin . The trypsin - treated ( stripped ) autolysosomes were incubated with rat brain cytosol and reaction mixture , and then analysed by western blotting . HC , heavy chain . ( b ) Scanning electron micrographs of purified autolysosomes , rat brain cytosol , trypsin - treated purified autolysosomes and trypsin - treated purified autolysosomes after the membrane budding assay . Scale bar , 250 nm . The arrows indicate buds . ( c ) Trypsin - treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP , clathrin , AP2 , AP3 , AP4 and PtdIns ( 4 , 5 ) P2 , and then analysed by western blotting . ( d ) Reactions from c were visualized by scanning electron microscopy . Scale bar , 250 nm . Arrow indicates bud . ( e ) Samples from d were quantified for the number of buds on each autolysosome . Error bars represent s . d . n = 50 autolysosomes from three independent experiments . Uncropped images of blots are shown in Supplementary Fig . S6 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf7(a) Purified autolysosomes were treated with trypsin. The trypsin-treated (stripped) autolysosomes were incubated with rat brain cytosol and reaction mixture, and then analysed by western blotting. HC, heavy chain.(b) Scanning electron micrographs of purified autolysosomes, rat brain cytosol, trypsin-treated purified autolysosomes and trypsin-treated purified autolysosomes after the membrane budding assay. Scale bar, 250 nm. The arrows indicate buds.(c) Trypsin-treated autolysosomes were incubated with rat brain cytosol and reaction mixture in the presence of antibodies against GFP, clathrin, AP2, AP3, AP4 and PtdIns(4,5)P2, and then analysed by western blotting.(d) Reactions from c were visualized by scanning electron microscopy. Scale bar, 250 nm. Arrow indicates bud. (e) Samples from d were quantified for the number of buds on each autolysosome. Error bars represent s.d. n = 50 autolysosomes from three independent experiments. Uncropped images of blots are shown in Supplementary Fig. S6.


figf8 ( a ) NRK cells were transfected with nonspecific ( NS ) - or Cltc ( clathrin heavy chain ) - RNAi and starved for 0 , 4 , 8 or 12 h . Scale bar , 5 mm . Cells were stained with antibodies against LC3 . ( b ) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified . n = 100 cells from three independent experiments . Error bars indicate the s . d .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf8(a) NRK cells were transfected with nonspecific (NS)- or Cltc (clathrin heavy chain)-RNAi and starved for 0, 4, 8 or 12 h. Scale bar, 5 mm. Cells were stained with antibodies against LC3. (b) Cells from a were assessed for LC3 puncta in a blind fashion after starvation and quantified. n = 100 cells from three independent experiments. Error bars indicate the s.d.


figf1 ( a ) Diagrams of the deletion - mutation constructs of Keap1 ( left ) and the corresponding immunoprecipitation assays ( right ) . Each Flag - tagged mouse Keap1 and mutant was expressed in HEK293T cells . At 22 h after transfection , lysates were prepared and immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - Flag , anti - p62 , anti - Nrf2 and anti - actin antibodies . Data are representative of three individual experiments . ( b ) Diagrams of the deletion - mutation constructs of p62 ( left ) and the corresponding input ( upper right ) and pull - down assay ( lower right ) . The MBP - tagged mouse p62 deletion mutants conjugated to amylose ( AM ) resins were incubated with purified GST - tagged mouse Keap1 - DC . The pulled - down complexes with the MBP - p62 mutants were subjected to SDS - PAGE and revealed by staining with Coomassie brilliant blue . The bands corresponding to MBP - p62 and its mutants are indicated by black dots . Red arrowheads indicate the band corresponding to GST - Keap1 - DC . For details of construct 14 see Supplementary Information , Fig . S3 . ( d ) Immunoprecipitation assays . Flag - tagged p62 , KIR - deleted p62 ( p62 ΔKIR ) and a p62 mutant defective in oligomerization ( p62 K7AD69A ) were expressed in primary mouse hepatocytes by the adenovirus system ( left ) or in HEK293T cells by transfection ( right ) . Cell lysates were immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - p62 and anti - Keap1 antibodies . The bands corresponding to Flag - p62 , endogenous p62 , Keap1 and actin are indicated . The data shown are representative of three separate experiments . ( e ) Interaction of endogenous p62 with Keap1 . Lysates prepared from the human hepatocellular carcinoma cell line Huh - 1 were immunoprecipitated with anti - p62 antibody or anti - Flag antibody ( negative control ) followed by immunoblotting with antibodies against p62 and Keap1 . The bands corresponding to endogenous p62 , Keap1 and IgG heavy chain ( IgG H . C . ) are indicated . The data shown are representative of three separate experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf1(a) Diagrams of the deletion-mutation constructs of Keap1 (left) and the corresponding immunoprecipitation assays (right). Each Flag-tagged mouse Keap1 and mutant was expressed in HEK293T cells. At 22 h after transfection, lysates were prepared and immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62, anti-Nrf2 and anti-actin antibodies. Data are representative of three individual experiments.(b) Diagrams of the deletion-mutation constructs of p62 (left) and the corresponding input (upper right) and pull-down assay (lower right). The MBP-tagged mouse p62 deletion mutants conjugated to amylose (AM) resins were incubated with purified GST-tagged mouse Keap1-DC. The pulled-down complexes with the MBP-p62 mutants were subjected to SDS-PAGE and revealed by staining with Coomassie brilliant blue. The bands corresponding to MBP-p62 and its mutants are indicated by black dots. Red arrowheads indicate the band corresponding to GST-Keap1-DC. For details of construct 14 see Supplementary Information, Fig. S3.(d) Immunoprecipitation assays. Flag-tagged p62, KIR-deleted p62 (p62 ΔKIR) and a p62 mutant defective in oligomerization (p62 K7AD69A) were expressed in primary mouse hepatocytes by the adenovirus system (left) or in HEK293T cells by transfection (right). Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-p62 and anti-Keap1 antibodies. The bands corresponding to Flag-p62, endogenous p62, Keap1 and actin are indicated. The data shown are representative of three separate experiments.(e) Interaction of endogenous p62 with Keap1. Lysates prepared from the human hepatocellular carcinoma cell line Huh-1 were immunoprecipitated with anti-p62 antibody or anti-Flag antibody (negative control) followed by immunoblotting with antibodies against p62 and Keap1. The bands corresponding to endogenous p62, Keap1 and IgG heavy chain (IgG H.C.) are indicated. The data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.


figf3 ( a ) A representative ITC profile of the titration of Keap1 - DC with p62M7 ( residues 168 - 391 ) . The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms . ( b ) Immunoprecipitation assays . Flag - tagged Keap1 was co - expressed with increasing concentrations of green fluorescent protein ( GFP ) - p62 ( lanes 3 - 6 ) in HEK293T cells . Cell lysates were immunoprecipitated with anti - Flag antibody . The resulting immunoprecipitates were subjected to SDS - PAGE and analysed by immunoblotting with anti - Flag , anti - p62 and anti - Nrf2 antibodies . The bands corresponding to Flag - Keap1 , endogenous p62 , Nrf2 and actin are indicated . Data are representative of three independent experiments . ( c ) The competitive p62 activity against Keap1 was measured by luciferase assay . The expression plasmids for Nrf2 , Keap1 and p62 wild - type ( w . t . ) or its mutants were transfected into Hepa1 cells along with pNqo1 - ARE reporter plasmid and pRL - TK as an internal control . At 36 h after transfection , the luciferase activity was measured in accordance with the instructions provided by the manufacturer . Assays were performed twice in triplicate . Data are means and s . d . for six determinations . ( d ) Immunoblot analysis . Flag - tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild - type and p62 − / − primary mouse hepatocytes by the adenovirus system . At 48 h after infection , total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified . The bands corresponding to Flag - p62 , endogenous p62 , Keap1 , Nrf2 , Nqo1 , actin and Lamin B are shown . Data are representative of three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 . ( e ) Quantification of mRNA levels of the detoxification enzymes Nqo1 , Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag - p62 and its mutants . Total RNAs were prepared from non - infected or infected hepatocytes and reverse transcribed into their respective cDNAs , which were used as templates in real - time PCR analysis . Values were normalized to the amount of each mRNA in the non - infected hepatocytes . The experiments were performed three times . Data are means ± s . d . for three experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf3(a) A representative ITC profile of the titration of Keap1-DC with p62M7 (residues 168-391). The upper panel shows the raw ITC thermograms and the lower panel shows the fitted binding isotherms.(b) Immunoprecipitation assays. Flag-tagged Keap1 was co-expressed with increasing concentrations of green fluorescent protein (GFP)-p62 (lanes 3-6) in HEK293T cells. Cell lysates were immunoprecipitated with anti-Flag antibody. The resulting immunoprecipitates were subjected to SDS-PAGE and analysed by immunoblotting with anti-Flag, anti-p62 and anti-Nrf2 antibodies. The bands corresponding to Flag-Keap1, endogenous p62, Nrf2 and actin are indicated. Data are representative of three independent experiments.(c) The competitive p62 activity against Keap1 was measured by luciferase assay. The expression plasmids for Nrf2, Keap1 and p62 wild-type (w.t.) or its mutants were transfected into Hepa1 cells along with pNqo1-ARE reporter plasmid and pRL-TK as an internal control. At 36 h after transfection, the luciferase activity was measured in accordance with the instructions provided by the manufacturer. Assays were performed twice in triplicate. Data are means and s.d. for six determinations.(d) Immunoblot analysis. Flag-tagged p62 and its mutants defective in interacting with Keap1 were overproduced in wild-type and p62−/− primary mouse hepatocytes by the adenovirus system. At 48 h after infection, total cell lysates and nuclear fractions were prepared and subjected to immunoblot analysis with the antibodies specified. The bands corresponding to Flag-p62, endogenous p62, Keap1, Nrf2, Nqo1, actin and Lamin B are shown. Data are representative of three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.(e) Quantification of mRNA levels of the detoxification enzymes Nqo1, Gstm1 and Cyp2a5 in hepatocytes overexpressing Flag-p62 and its mutants. Total RNAs were prepared from non-infected or infected hepatocytes and reverse transcribed into their respective cDNAs, which were used as templates in real-time PCR analysis. Values were normalized to the amount of each mRNA in the non-infected hepatocytes. The experiments were performed three times. Data are means ± s.d. for three experiments.


figf4 ( a ) Insolubilization of Keap1 in Atg7 - deficient hepatocytes . Liver homogenates from Atg7F / F : Mx1 mice on various days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions with 0 . 5 % Triton X - 100 . Each fraction was subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies . The data displayed are representative of three separate experiments . ( b ) Immunoblot analysis of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) and Atg7 p62 - deficient ( Atg7F / F : Mx1 : p62 − / − ) livers . Liver homogenates from mice of the stated genotypes at 12 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions . Each fraction was subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies . Atg7F / F mice in which Atg7 is efficiently expressed at a level similar to that in the wild - type mice were used as control . Data shown are representative of three separate experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 . ( c ) Quantitative real - time PCR analyses of Nqo1 and Gstm1 in mouse livers . Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly ( I ) • poly ( C ) . Values were normalized to the amount of mRNA in Atg7F / F liver . Data are means ± s . d . for three experiments . ( d ) Immunofluorescence analysis of the cellular localization of p62 and Keap1 . Liver sections from mice of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( top ) and anti - p62 ( middle ) antibodies . Bottom : merged images of Keap1 ( green ) and p62 ( red ) . Each inset in the Atg7 - deficient liver panels is a magnified image of the boxed region . Scale bars , 20 μm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf4(a) Insolubilization of Keap1 in Atg7-deficient hepatocytes. Liver homogenates from Atg7F/F:Mx1 mice on various days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions with 0.5% Triton X-100. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. The data displayed are representative of three separate experiments.(b) Immunoblot analysis of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 p62-deficient (Atg7F/F:Mx1:p62−/−) livers. Liver homogenates from mice of the stated genotypes at 12 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Each fraction was subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies. Atg7F/F mice in which Atg7 is efficiently expressed at a level similar to that in the wild-type mice were used as control. Data shown are representative of three separate experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.(c) Quantitative real-time PCR analyses of Nqo1 and Gstm1 in mouse livers. Total RNAs were prepared from livers of the indicated genotypes at 12 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in Atg7F/F liver. Data are means ± s.d. for three experiments.(d) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (top) and anti-p62 (middle) antibodies. Bottom: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.


figf5 ( a ) Immunoblotting of Atg7 - deficient ( Atg7F / F : Mx1 ; Atg7F / F shown here as F / F ) and Atg7 Nrf2 - deficient ( Atg7F / F : Mx1 : Nrf2 − / − ) livers . Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly ( I ) • poly ( C ) were separated into detergent - soluble and detergent - insoluble fractions . Total , soluble and insoluble fractions were subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nqo1 , Gstm1 and actin ( bottom left section ) . Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) , subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B ( as control ) ( bottom right section ) . Data were obtained from three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 . ( b ) Immunofluorescence analysis of the cellular localization of p62 and Keap1 . Liver sections from mice of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) were immunostained with anti - Keap1 ( left ) and anti - p62 ( middle ) antibodies . Right : merged images of Keap1 ( green ) and p62 ( red ) . Each inset in the Atg7 - deficient and Atg7 Nrf2 - deficient liver panels is a magnified image of the boxed region . Scale bars , 20 μm . ( c ) Quantitative real - time PCR analyses of Nqo1 , Gstm1 and Cyp2a5 in mouselivers . Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly ( I ) • poly ( C ) . Values were normalized to the amount of mRNA in the Atg7F / F liver . Data are means ± s . d . for three experiments . ( d ) Liver weight . The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly ( I ) • poly ( C ) were measured . Data are means ± s . d . for five mice from each group . Three asterisks , P 0 . 001 ( Student ' s t - test ) . ( e ) Histological analysis of the mouse liver of the indicated genotypes . At 28 days after injection of poly ( I ) • poly ( C ) , the livers were processed for haematoxylin / eosin staining . Higher - magnification views are shown in the bottom panels . CV , central vein ; P , portal vein . Scale bars , 100 μm . ( f ) Liver function tests of the mice used in d . The serum levels of aspartate aminotransferase ( AST ) , alanine aminotransferase ( ALT ) and alkaline phosphatase ( ALP ) were measured . ( IU / L , international unit per liter ) . Data are means ± s . d . for seven mice from each group . Asterisk , P 0 . 05 ; three asterisks , P 0 . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

figf5(a) Immunoblotting of Atg7-deficient (Atg7F/F:Mx1; Atg7F/F shown here as F/F) and Atg7 Nrf2-deficient (Atg7F/F:Mx1:Nrf2−/−) livers. Liver homogenates from mice of the assigned genotypes at 28 days after injection of poly(I)•poly(C) were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1, Gstm1 and actin (bottom left section). Nuclear fractions were prepared from the livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C), subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nrf2 and Lamin B (as control) (bottom right section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.(b) Immunofluorescence analysis of the cellular localization of p62 and Keap1. Liver sections from mice of the indicated genotypes at 28 days after injection of poly(I)•poly(C) were immunostained with anti-Keap1 (left) and anti-p62 (middle) antibodies. Right: merged images of Keap1 (green) and p62 (red). Each inset in the Atg7-deficient and Atg7 Nrf2-deficient liver panels is a magnified image of the boxed region. Scale bars, 20 μm.(c) Quantitative real-time PCR analyses of Nqo1, Gstm1 and Cyp2a5 in mouselivers. Total RNAs were prepared from livers of the indicated genotypes at 28 days after injection of poly(I)•poly(C). Values were normalized to the amount of mRNA in the Atg7F/F liver. Data are means ± s.d. for three experiments.(d) Liver weight. The weights of the mouse livers of the different genotypes shown at 28 days after injection of poly(I)•poly(C) were measured. Data are means ± s.d. for five mice from each group. Three asterisks, P 0.001 (Student's t-test).(e) Histological analysis of the mouse liver of the indicated genotypes. At 28 days after injection of poly(I)•poly(C), the livers were processed for haematoxylin/eosin staining. Higher-magnification views are shown in the bottom panels. CV, central vein; P, portal vein. Scale bars, 100 μm.(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP) were measured. (IU/L, international unit per liter). Data are means ± s.d. for seven mice from each group. Asterisk, P 0.05; three asterisks, P 0.001.


figf6 ( a ) Immunoblotting of Keap1 - deficient ( Keap1F / F : Alb ) and Keap1 Atg7 - deficient ( Keap1F / F : Atg7F / F : Alb ) livers . Liver homogenates from 8 - week - old mice of these genotypes were separated into detergent - soluble and detergent - insoluble fractions . Total , soluble and insoluble fractions were subjected to SDS - PAGE and analysed by immunoblotting with the indicated antibodies ( top section ) . Total lysates were subjected to SDS - PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin ( bottom section ) . Data were obtained from three independent experiments . Uncropped images of blots are shown in Supplementary Information , Fig . S11 . ( b ) Quantitative real - time PCR analyses of Nqo1 , Mrp4 , p62 and Ho - 1 in mouse livers . Total RNAs were prepared from livers of 9 - week - old indicated genotypes . Values were normalized to the amount of mRNA in the Atg7F / F liver ( control ) . Data are means ± s . d . for three experiments . ( c ) Kaplan - Meier curves of survival of Atg7F / F : Alb and Keap1F / F : Atg7F / F : Alb mice . The survival analysis of control ( n = 33 ) , Keap1F / F : Alb ( n = 15 ) , Atg7F / F : Alb ( n = 12 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 14 ) was based on a 16 - week follow - up period . ( d ) Liver weight relative to body weight was measured for the different genotypes . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 ( Student ' s t - test ) . ( e ) Histological analysis of mouse liver of the indicated genotypes . The livers from 9 - week - old mice were processed for haematoxylin / eosin staining . Scale bar , 50 μm ( left bottom panel ) ; 20 μm ( other panels ) . ( f ) Liver function tests of the mice used in d . The serum levels of aspartate aminotransferase ( AST ) and alanine aminotransferase ( ALT ) were measured . Data are means ± s . d . for Atg7F / F ( control ) ( n = 24 ) , Keap1F / F : Alb ( n = 13 ) , Atg7F / F : Alb ( n = 9 ) and Keap1F / F : Atg7F / F : Alb mice ( n = 7 ) . Two asterisks , P 0 . 01 . ( g ) Immunohistochemical analysis of cellular localization of p62 and Keap1 . Paraffin sections of the liver from 9 - week - old mice of the indicated genotypes were immunolabelled with anti - p62 ( left ) and anti - Keap1 ( right ) antibodies . Scale bars , 20 μm .

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figf6(a) Immunoblotting of Keap1-deficient (Keap1F/F:Alb) and Keap1 Atg7-deficient (Keap1F/F:Atg7F/F:Alb) livers. Liver homogenates from 8-week-old mice of these genotypes were separated into detergent-soluble and detergent-insoluble fractions. Total, soluble and insoluble fractions were subjected to SDS-PAGE and analysed by immunoblotting with the indicated antibodies (top section). Total lysates were subjected to SDS-PAGE and analysed by immunoblotting with antibodies against Nqo1 and actin (bottom section). Data were obtained from three independent experiments. Uncropped images of blots are shown in Supplementary Information, Fig. S11.(b) Quantitative real-time PCR analyses of Nqo1, Mrp4, p62 and Ho-1 in mouse livers. Total RNAs were prepared from livers of 9-week-old indicated genotypes. Values were normalized to the amount of mRNA in the Atg7F/F liver (control). Data are means ± s.d. for three experiments.(c) Kaplan-Meier curves of survival of Atg7F/F:Alb and Keap1F/F:Atg7F/F:Alb mice. The survival analysis of control (n = 33), Keap1F/F:Alb (n = 15), Atg7F/F:Alb (n = 12) and Keap1F/F:Atg7F/F:Alb mice (n = 14) was based on a 16-week follow-up period.(d) Liver weight relative to body weight was measured for the different genotypes. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01 (Student's t-test).(e) Histological analysis of mouse liver of the indicated genotypes. The livers from 9-week-old mice were processed for haematoxylin/eosin staining. Scale bar, 50 μm (left bottom panel); 20 μm (other panels).(f) Liver function tests of the mice used in d. The serum levels of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured. Data are means ± s.d. for Atg7F/F (control) (n = 24), Keap1F/F:Alb (n = 13), Atg7F/F:Alb (n = 9) and Keap1F/F:Atg7F/F:Alb mice (n = 7). Two asterisks, P 0.01.(g) Immunohistochemical analysis of cellular localization of p62 and Keap1. Paraffin sections of the liver from 9-week-old mice of the indicated genotypes were immunolabelled with anti-p62 (left) and anti-Keap1 (right) antibodies. Scale bars, 20 μm.


Figure 1E ) Representative images of prometaphase chromosomes from HeLa cells co - transfected with H2A . Z . 2 siRNA and each of the constructs in D ( green ) . Scale bar : 5μm . F ) Quantification of the percentage of cells with micronuclei from experiment ( E ) . The error bars represent the SD of three biological replicates ( Control si N = 887 ; H2A . Z . 2 si N = 1005 ; H2A . Z . 2 si + wt N = 760 ; H2A . Z . 2 si + KR N = 479 ) . Data sets were statistically analysed using Chi - square test .

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Figure 1E) Representative images of prometaphase chromosomes from HeLa cells co-transfected with H2A.Z.2 siRNA and each of the constructs in D (green). Scale bar: 5μm. F) Quantification of the percentage of cells with micronuclei from experiment (E). The error bars represent the SD of three biological replicates (Control si N=887; H2A.Z.2 si N=1005; H2A.Z.2 si + wt N=760; H2A.Z.2 si + KR N=479). Data sets were statistically analysed using Chi-square test.


Figure 2B ) Representative images of metaphases from YFP : CENP - A ( green ) HeLa cells treated with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA . The white arrowheads indicate the mis - aligned chromosomes . Scale bar : 10 μm . C ) Representative images of metaphase spreads from Control ( top ) or H2A . Z . 2 ( bottom ) siRNA - transfected HeLa cells after FISH with a Chr17 centromeric probe ( green ) . The arrowheads indicate the separated sister chromatids . Scale bar : 10 μm . D ) Representative images of HeLa cells transfected with Control ( top ) or H2A . Z . 2 ( bottom ) siRNA , fixed , and stained for Sgo1 ( green ) . Scale bar : 10 μm . E ) Quantification of Sgo1 localisation in pro - metaphase cells from the experiment in ( D ) . Error bar represents SD of two biological replicates . 35 pro - metaphase cells were analysed . J ) Representative images of HeLa YFP : CENP - A ( green ) mitotic cells after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . K ) Violin plot of centromeric CENP - A intensity of pro - metaphase / metaphase cells from the experiments in J . ( Control si N = 197 H2A . Z . 2 si N = 260 ) . The bar represents the median . Data sets were statistically analysed using the Wilcoxon rank test in R . * * = p < 0 . 01 . L ) Representative images of HeLa mitotic cells stained for CENP - C after Control ( top ) or H2A . Z . 2 ( bottom ) siRNA treatment . Scale bar : 5 μm . M ) Violin plot of centromeric CENP - C intensity of pro - metaphase / metaphase cells from the experiments in L . ( Control si N = 1083 H2A . Z . 2 si N = 686 , from 3 biological replicas ) . The bars represent the median . Data sets were statistically analysed using the Wilcoxon rank test in R . * * * = p < 0 . 0001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2B) Representative images of metaphases from YFP:CENP-A (green) HeLa cells treated with Control (top) or H2A.Z.2 (bottom) siRNA. The white arrowheads indicate the mis-aligned chromosomes. Scale bar: 10 μm. C) Representative images of metaphase spreads from Control (top) or H2A.Z.2 (bottom) siRNA-transfected HeLa cells after FISH with a Chr17 centromeric probe (green). The arrowheads indicate the separated sister chromatids. Scale bar: 10 μm. D) Representative images of HeLa cells transfected with Control (top) or H2A.Z.2 (bottom) siRNA, fixed, and stained for Sgo1 (green). Scale bar: 10 μm. E) Quantification of Sgo1 localisation in pro-metaphase cells from the experiment in (D). Error bar represents SD of two biological replicates. 35 pro-metaphase cells were analysed. J) Representative images of HeLa YFP:CENP-A (green) mitotic cells after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. K) Violin plot of centromeric CENP-A intensity of pro-metaphase/metaphase cells from the experiments in J. (Control si N=197 H2A.Z.2 si N=260). The bar represents the median. Data sets were statistically analysed using the Wilcoxon rank test in R. **=p<0.01. L) Representative images of HeLa mitotic cells stained for CENP-C after Control (top) or H2A.Z.2 (bottom) siRNA treatment. Scale bar: 5 μm. M) Violin plot of centromeric CENP-C intensity of pro-metaphase/metaphase cells from the experiments in L. (Control si N=1083 H2A.Z.2 si N=686, from 3 biological replicas). The bars represent the median. Data sets were statistically analysed using the Wilcoxon rank test in R. ***=p<0.0001.


Figure 3D ) IGV analyses of H2A . Z localisation ( from ENCODE ) on Aurora B ( green ) and c - MYC ( blue ) genes showing H2A . Z enrichment at the TSS and at the upstream region respectively ( bottom panels ) . IGV analyses of ATAC seq peaks for Control si and H2A . Z . 1 si for Aurora B and MYC genes . The number in red represents the log2Ratio H2AZ . 1 si / Control si .

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Figure 3D) IGV analyses of H2A.Z localisation (from ENCODE) on Aurora B (green) and c-MYC (blue) genes showing H2A.Z enrichment at the TSS and at the upstream region respectively (bottom panels). IGV analyses of ATAC seq peaks for Control si and H2A.Z.1 si for Aurora B and MYC genes. The number in red represents the log2Ratio H2AZ.1 si/Control si.


Figure 4E ) Representative images of Hela cell stained for Ki67 . The arrows indicate the different staining patterns quantified : strong signal ( grey arrow ) , weak signal ( light brown arrow ) and no signal ( dark brown arrow ( scale bar 10μm ) . F ) Quantification of the experiment in ( E ) . Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test . * * * = p < 0 . 001 . G ) Representative images of HeLa cells transfected with control ( top ) or H2A . Z . 1 ( bottom ) siRNA and stained for Lamin A / C ( green ) . Scale bar = 10μm .

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Figure 4E) Representative images of Hela cell stained for Ki67. The arrows indicate the different staining patterns quantified: strong signal (grey arrow), weak signal (light brown arrow) and no signal (dark brown arrow (scale bar 10μm). F) Quantification of the experiment in (E). Error bars indicate the standard deviation from 3 biological replicates Data sets were statistically analysed using a Chi square test. ***=p<0.001. G) Representative images of HeLa cells transfected with control (top) or H2A.Z.1 (bottom) siRNA and stained for Lamin A/C (green). Scale bar = 10μm.


Figure 5C ) Kaplan Meier survival curve for neuroblastoma patients with low ( red ) or high ( blue ) expression of H2AFZ ( H2A . Z . 1 ) obtained from R2 genomics .

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Figure 5C) Kaplan Meier survival curve for neuroblastoma patients with low (red) or high (blue) expression of H2AFZ (H2A.Z.1) obtained from R2 genomics.


Figure 1A - G Hematoxylin and eosin ( H & E ) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine ( PC ) initiation stage from embryonic day 50 ( E50 ) to postnatal day 10 ( PN10 ) ; ( A ' - F ' ) are magnifications of boxed regions in their corresponding figure panel . DC , deciduous canine ; PC , permanent canine ; SDL , successional dental lamina ; n = 3 . Scale bars = 100 µm . H In situ hybridization ( ISH ) showing the expression pattern of Sox2 in the initiation stage from E50 to E90 ; right figure panels are magnifications of boxed regions in left panels . Dashed lines mark the position of the successional dental lamina ( SDL ) ; green arrowhead indicates positive staining of Sox2 at the tip of the SDL . n = 3 . Scale bars = 100 µm . I Immunofluorescence ( IF ) of Ki67 at the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm . J Semi - quantification and comparison of Ki67 expression levels during E60 , E70 , and E90 stages . n = 3 . Data information : Data represent the means ± SEM . * P < 0 . 05 ( one - way ANOVA and Newman - Keuls post - hoc tests ) . K TUNEL assay showing the apoptosis status of the SDL at E60 , E70 , E90 ; right figure panels are magnifications of boxed regions in left panels . n = 3 . Scale bars = 100 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1A-G Hematoxylin and eosin (H&E) staining of the frontal sections of miniature pig mandible slices showing morphological changes during the permanent canine (PC) initiation stage from embryonic day 50 (E50) to postnatal day 10 (PN10); (A'-F') are magnifications of boxed regions in their corresponding figure panel. DC, deciduous canine; PC, permanent canine; SDL, successional dental lamina; n = 3. Scale bars = 100 µm.H In situ hybridization (ISH) showing the expression pattern of Sox2 in the initiation stage from E50 to E90; right figure panels are magnifications of boxed regions in left panels. Dashed lines mark the position of the successional dental lamina (SDL); green arrowhead indicates positive staining of Sox2 at the tip of the SDL. n = 3. Scale bars = 100 µm.I Immunofluorescence (IF) of Ki67 at the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.J Semi-quantification and comparison of Ki67 expression levels during E60, E70, and E90 stages. n = 3. Data information: Data represent the means ± SEM. *P < 0.05 (one-way ANOVA and Newman-Keuls post-hoc tests).K TUNEL assay showing the apoptosis status of the SDL at E60, E70, E90; right figure panels are magnifications of boxed regions in left panels. n = 3. Scale bars = 100 µm.


Figure 2A Three - dimensional reconstruction of serial H & E frontal sections of miniature pig mandibles at embryonic day 60 ( E60 ) and E90 ; deciduous canine ( DC ) in purple , permanent canine ( PC ) in yellow , and alveolar socket in green . The red , blue , and green arrows indicate the width of the labial alveolar socket , lingual alveolar socket , and DC , respectively . n = 3 . B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 . C The proportion of DC width relative to the total alveolar socket width during E60 and E90 . n = 3 . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 . D Micro - CT imaging of the whole mandible at E60 ; boxed region is magnified in D ' ( top panel ) . ( D ' ) Mandible slice isolated with Geomagic software . White dashed lines mark DC . E 3 - D color map ( left ) after alignment of mandible slices before and after ( sham ) surgery showing a comparison of the surface points . Coronal sections through cusp tips ( transparent squares , left ) were selected for 2 - D comparisons ( middle ) ; right panels are magnifications of yellow - boxed regions . The solid purple contour and dotted black contour indicate the pre - and post - surgery shapes , respectively . The distance between the two contours are the colored line segments showing the distance and direction of the movement . The colored ball in the 2 - D comparison marks the position of the maximum displacement . The PC position is indicated in pink . n = 3 . F Dissected mandible slice with a " U " shape .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O

Figure 2A Three-dimensional reconstruction of serial H&E frontal sections of miniature pig mandibles at embryonic day 60 (E60) and E90; deciduous canine (DC) in purple, permanent canine (PC) in yellow, and alveolar socket in green. The red, blue, and green arrows indicate the width of the labial alveolar socket, lingual alveolar socket, and DC, respectively. n = 3.B DC and labial and lingual alveolar socket widths in the horizontal plane at the bottom of the PC during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.C The proportion of DC width relative to the total alveolar socket width during E60 and E90. n = 3. Data information: Data represent the means ± SEM. Unpaired t-tests, *P < .05, **P < .01, ***P < .001.D Micro-CT imaging of the whole mandible at E60; boxed region is magnified in D' (top panel). (D') Mandible slice isolated with Geomagic software. White dashed lines mark DC. E 3-D color map (left) after alignment of mandible slices before and after (sham) surgery showing a comparison of the surface points. Coronal sections through cusp tips (transparent squares, left) were selected for 2-D comparisons (middle); right panels are magnifications of yellow-boxed regions. The solid purple contour and dotted black contour indicate the pre- and post-surgery shapes, respectively. The distance between the two contours are the colored line segments showing the distance and direction of the movement. The colored ball in the 2-D comparison marks the position of the maximum displacement. The PC position is indicated in pink. n = 3. F Dissected mandible slice with a "U" shape.


Figure 3A Diagram of the dissected miniature pig mandible slice illustrating the deciduous ( DC ) and permanent ( PC ) canine and application of compression in vitro using Flexcell FX - 5000 Compression System . B - D H & E staining of canine frontal sections from embryonic day 60 ( E60 ) ( B ) , after culturing for two days without stress ( 0 kPa ) ( C ) , or with stress ( 3 kPa ) ( D ) . ( B ' - D ' ) are magnifications of boxed regions in their corresponding figure panels . Scale bars = 50 µm . n = 6 / 6 in E60 group ; n = 12 / 12 in 0 - kPa group ; n = 4 / 8 in 3 - kPa group . F RT - qPCR of several mechanosensitive proteins ( TGFB1 , NFKB1 , EGR1 , and RUNX2 ) and of four mechanoreceptors ( ITGB1 , ITGB3 , ITGAV and ITGA2 ) in PC and surrounding soft tissue at E60 and after culturing for two days with ( 3 kPa ) or without extra mechanical stress ( 0 kPa ) . n = 3 for each group . Data represent the means ± SEM . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . =

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3A Diagram of the dissected miniature pig mandible slice illustrating the deciduous (DC) and permanent (PC) canine and application of compression in vitro using Flexcell FX-5000 Compression System.B-D H&E staining of canine frontal sections from embryonic day 60 (E60) (B), after culturing for two days without stress (0 kPa) (C), or with stress (3 kPa) (D). (B'-D') are magnifications of boxed regions in their corresponding figure panels. Scale bars = 50 µm. n = 6/6 in E60 group; n = 12/12 in 0-kPa group; n = 4/8 in 3-kPa group.F RT-qPCR of several mechanosensitive proteins (TGFB1, NFKB1, EGR1, and RUNX2) and of four mechanoreceptors (ITGB1, ITGB3, ITGAV and ITGA2) in PC and surrounding soft tissue at E60 and after culturing for two days with (3 kPa) or without extra mechanical stress (0 kPa). n = 3 for each group. Data represent the means ± SEM. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups), *P < .05, **P < .01, ***P < .001; NS, not significant. =


Figure 4A - L IF of integrin β1 , ERK1 , and RUNX2 in miniature pig canine frontal sections at embryonic day 60 ( E60 ) , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days ; ( A ' - L ' ) are magnifications of boxed regions in the corresponding figure panels . M Relative IF expression levels of integrin β1 , ERK1 , and RUNX2 during E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . Scale bars = 25 µm ( A ' , G ' ) and 50 µm ( other panels ) . Unpaired t - tests for M , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . N Morphology of the human PC at weeks 18 - 19 ( H & E staining ) ; ( N ' ) is magnification of the boxed region in ( N ) . DC , deciduous canine ; PC , permanent canine ; IEE , inner enamel epithelium ; OEE , outer enamel epithelium . Mesenchyme between DC and PC is indicated with a green arrow . O - Q ISH expression patterns of ITGB1 , ERK1 , and RUNX2 in human PCs . Green arrows indicate the mesenchyme between DC and PC . R IF of RUNX2 in human PC . Mesenchyme between DC and PC is indicated with a red arrow . Red arrow indicates the mesenchyme between DC and PC . S PC epithelium areas in E60 , E90 , and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days . Data information : Data represent the means ± SEM . n = 3 . , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . IF of RUNX2 and phospho - ERK1 / 2 ( p - ERK1 / 2 ) upon force loading with 0 or 1 . 0 g / cm2 for 2 h or after force was removed and the cells cultured for an additional 1 , 2 , and 4 hours . V Relative IF expression levels of RUNX2 and p - ERK1 / 2 between groups Data information : Data represent the means ± SEM . n = 3 . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for V , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant . W Western blots of p - ERK1 / 2 and RUNX2 levels after 1 . 0 g / cm2 was applied for 2 h , with or without anti - integrin β1 antibody and ERK1 inhibitor ( U0126 ) . X Relative expression levels of p - ERK1 / 2 , RUNX2 , and Actin in ( W ) . Data information : Data represent the means ± SEM . n = 3 . Unpaired t - tests for M ; One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for X , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS , not significant .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A-L IF of integrin β1, ERK1, and RUNX2 in miniature pig canine frontal sections at embryonic day 60 (E60), E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days; (A'-L') are magnifications of boxed regions in the corresponding figure panels. M Relative IF expression levels of integrin β1, ERK1, and RUNX2 during E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. Scale bars = 25 µm (A', G') and 50 µm (other panels). Unpaired t-tests for M , *P < .05, **P < .01, ***P < .001; NS, not significant.N Morphology of the human PC at weeks 18-19 (H&E staining); (N') is magnification of the boxed region in (N). DC, deciduous canine; PC, permanent canine; IEE, inner enamel epithelium; OEE, outer enamel epithelium. Mesenchyme between DC and PC is indicated with a green arrow.O-Q ISH expression patterns of ITGB1, ERK1, and RUNX2 in human PCs. Green arrows indicate the mesenchyme between DC and PC.R IF of RUNX2 in human PC. Mesenchyme between DC and PC is indicated with a red arrow. Red arrow indicates the mesenchyme between DC and PC.S PC epithelium areas in E60, E90, and E60 cultured under 3 kPa stress for two days and E60 cultured under 0 kPa stress for two days. Data information: Data represent the means ± SEM. n = 3. , *P < .05, **P < .01, ***P < .001; NS, not significant.IF of RUNX2 and phospho-ERK1/2 (p-ERK1/2) upon force loading with 0 or 1.0 g/cm2 for 2 h or after force was removed and the cells cultured for an additional 1, 2, and 4 hours.V Relative IF expression levels of RUNX2 and p-ERK1/2 between groups Data information: Data represent the means ± SEM. n = 3. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for V , *P < .05, **P < .01, ***P < .001; NS, not significant.W Western blots of p-ERK1/2 and RUNX2 levels after 1.0 g/cm2 was applied for 2 h, with or without anti-integrin β1 antibody and ERK1 inhibitor (U0126). X Relative expression levels of p-ERK1/2, RUNX2, and Actin in (W). Data information: Data represent the means ± SEM. n = 3. Unpaired t-tests for M; One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for X, *P < .05, **P < .01, ***P < .001; NS, not significant.


Figure 5A - C H & E staining of miniature pig canine frontal sections from embryonic day 60 ( E60 group ) ( A ) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag ( overexpression group ) ( B ) or overexpression control lentiviral vector with a Myc tag ( O - control group ) ( C ) ; ( A ' - C ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µmD - F Immunohistochemical ( IHC ) staining of Myc expression in mandible slices from the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 100 µm ( D - F ) G - I IF staining of RUNX2 expression ( yellow arrows ) in mandible slices of the E60 , overexpression , and O - control groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µmJ , K Relative expression levels of Myc and RUNX2 in the E60 , O - control , and overexpression groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for J , K , * P < . 05 , * * P < . 01 , * * * P < . 001 . L , M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector ( knockdown control , K - control ) ( L ) or RUNX2 shRNA lentiviral vector ( knockdown ) ( M ) ; ( L ' - M ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µm ( other panels ) . N Relative RUNX2 expression levels between K - control and knockdown groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for N , * P < . 05 , * * P < . 01 , * * * P < . 001 . O - Q H & E staining of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( O ' - Q ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 50 µmR - T IF staining of RUNX2 expression ( yellow arrows ) of mandible slice sections from the K - control , knockdown + force ( 3 kPa ) , and K - control + force ( 3 kPa ) groups . ( R ' - T ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Data represent the means ± SEM . n = 3 for all experiments . Scale bars = 25 µm ( R ' - T ' ) U Relative RUNX2 expression levels among the three groups Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA ( Newman - Keuls test for post - hoc comparisons between two groups ) for U , * P < . 05 , * * P < . 01 , * * * P < . 001 . V PC epithelium areas in the overexpression , O - control , knockdown , knockdown + force , K - control , and K - control + force groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for V ; , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5A-C H&E staining of miniature pig canine frontal sections from embryonic day 60 (E60 group) (A) and after the mandible slices were infected with RUNX2 overexpression lentiviral vector with a Myc tag (overexpression group) (B) or overexpression control lentiviral vector with a Myc tag (O-control group) (C); (A'-C') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmD-F Immunohistochemical (IHC) staining of Myc expression in mandible slices from the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 100 µm (D-F)G-I IF staining of RUNX2 expression (yellow arrows) in mandible slices of the E60, overexpression, and O-control groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmJ, K Relative expression levels of Myc and RUNX2 in the E60, O-control, and overexpression groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for J, K , *P < .05, **P < .01, ***P < .001.L, M IF staining of RUNX2 expression after infecting mandible slices with scrambled shRNA lentiviral vector (knockdown control, K-control) (L) or RUNX2 shRNA lentiviral vector (knockdown) (M); (L'-M') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µm (other panels).N Relative RUNX2 expression levels between K-control and knockdown groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for N , *P < .05, **P < .01, ***P < .001.O-Q H&E staining of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (O'-Q') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 50 µmR-T IF staining of RUNX2 expression (yellow arrows) of mandible slice sections from the K-control, knockdown + force (3 kPa), and K-control + force (3 kPa) groups. (R'-T') are magnifications of boxed regions in the corresponding figure panels. Data information: Data represent the means ± SEM. n = 3 for all experiments. Scale bars = 25 µm (R'-T')U Relative RUNX2 expression levels among the three groups Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA (Newman-Keuls test for post-hoc comparisons between two groups) for U, *P < .05, **P < .01, ***P < .001.V PC epithelium areas in the overexpression, O-control, knockdown, knockdown + force, K-control, and K-control + force groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for V; , *P < .05, **P < .01, ***P < .001.


Figure 6A , B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 ( E60 ) and E90 . ( A ' , B ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm . C Relative ISH expression levels of RUNX2 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests for C , * P < . 05 , * * P < . 01 , * * * P < . 001 . D , E ISH of miniature pig canine frontal sections showing expression patterns of β - catenin at E60 and E90 . ( D ' , E ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm . F Relative ISH expression levels of β - catenin in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 . G , H IF of Lef1 at E60 and E90 . ( G , H ' ) are magnifications of boxed regions in the corresponding figure panels . Data information : Scale bars = 50 µm . I Relative IF expression levels of Lef1 in epithelium ( Epi . ) and mesenchyme ( Mes . ) at E60 and E90 . Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 . J FISH of β - catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope . Boxed regions of mesenchyme are magnified in the right panels . Epithelium is indicated with a yellow arrow . Data information : Scale bars = 50 µm . K Relative FISH expression levels of β - catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60 , E90 , and E60 cultured for 12 and 24 h . Data information : Data represent the means ± SEM . n = 3 for all experiments . One - way ANOVA for K , * P < . 05 , * * P < . 01 , * * * P < . 001 . L , M IF of β - catenin at E60 and E90 ; The yellow arrows indicate the localization of β - catenin in the nucleus . Data information : Scale bars = 50 µm . N Relative IF expression levels of β - catenin in the nucleus of epithelium and mesenchyme at E60 and E90 . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 . O , P FISH of β - catenin in mandible slices infected with RUNX2 shRNA lentivirus ( knockdown ) or scrambled shRNA lentivirus ( scramsh ) via RNAscope . Data information : Scale bars = 50 µm . Q Relative FISH expression levels of β - catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups . Data information : Data represent the means ± SEM . n = 3 for all experiments . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6A, B ISH of miniature pig canine frontal sections showing expression patterns of RUNX2 at embryonic day 60 (E60) and E90. (A', B') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.C Relative ISH expression levels of RUNX2 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests for C , *P < .05, **P < .01, ***P < .001.D, E ISH of miniature pig canine frontal sections showing expression patterns of β-catenin at E60 and E90. (D', E') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.F Relative ISH expression levels of β-catenin in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.G, H IF of Lef1 at E60 and E90. (G, H') are magnifications of boxed regions in the corresponding figure panels. Data information: Scale bars = 50 µm.I Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) at E60 and E90. Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.J FISH of β-catenin in E60 and E90 mandible slices and E60 mandible slices after 12 and 24 h in culture via RNAscope. Boxed regions of mesenchyme are magnified in the right panels. Epithelium is indicated with a yellow arrow. Data information: Scale bars = 50 µm.K Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme at E60, E90, and E60 cultured for 12 and 24 h. Data information: Data represent the means ± SEM. n = 3 for all experiments. One-way ANOVA for K, *P < .05, **P < .01, ***P < .001.L, M IF of β-catenin at E60 and E90; The yellow arrows indicate the localization of β-catenin in the nucleus. Data information: Scale bars = 50 µm.N Relative IF expression levels of β-catenin in the nucleus of epithelium and mesenchyme at E60 and E90. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.O, P FISH of β-catenin in mandible slices infected with RUNX2 shRNA lentivirus (knockdown) or scrambled shRNA lentivirus (scramsh) via RNAscope. Data information: Scale bars = 50 µm.Q Relative FISH expression levels of β-catenin in the nucleus and cytoplasm of epithelium and mesenchyme in knockdown and scramsh groups. Data information: Data represent the means ± SEM. n = 3 for all experiments. Unpaired t-tests , *P < .05, **P < .01, ***P < .001.


Figure 7A , B IF of Lef1 in mandible slices at embryonic day 60 ( E60 ) infected with RUNX2 overexpression lentiviral vector ( overexpression ) or overexpression control lentiviral vector ( O - control ) . ( A ' - B ' ) are magnifications of boxed regions in the corresponding figure panels . Dashed lines mark the epithelium of PC . C Relative IF expression levels of Lef1 in epithelium ( Epi . ) and mesenchyme ( Mes . ) of the overexpression and O - control groups . Data information : Data represent the means ± SEM . Scale bars = 100 µm . n = 3 for all experiments . Unpaired t - tests for C , * * P < . 01 , * * * P < . 001 ; NS . not significant . D , E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days . ( D ' , E ' ) are magnifications of boxed regions in the corresponding figure panels . Dashed lines mark the epithelium of PC . F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure . Data information : Data represent the means ± SEM . Scale bars = 100 µm . n = 3 for all experiments . Unpaired t - tests for , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant . G Western blots of Lef1 , non - phospho - β - catenin , and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector . ( G ' ) Relative expression levels between control vector and RUNX2 overexpression groups . Data information : Data represent the means ± SEM . Unpaired t - tests , * P < . 05 , * * P < . 01 , * * * P < . 001 ; NS . not significant . H Western blots of Lef1 , non - phospho - β - catenin , and RUNX2 after DFCs were infected with scrambled shRNA ( scramsh ) or RUNX2 knockdown shRNA ( knockdown ) lentiviral vectors . ( H ' ) Relative expression levels between scramsh and knockdown groups . I Western blots of nuclear non - phospho - β - catenin and Lamin B in DFCs of RUNX2 overexpression ( O ) , control vector ( V ) , RUNX2 knockdown ( K ) , scramsh ( S ) , compressed force ( F ; 1 . 0 g / cm2 ) , and control ( C ; 0 g / cm2 ) groups . Sample were loaded in a gradient ( 7 . 5 , 15 , 30 , and 60 μg ) showing a linear relationship for each group . J Western blots of nuclear non - phospho - β - catenin and Lamin B in DFCs treated with IWR - 1 - endo . Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR - 1 - endo treatment .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A, B IF of Lef1 in mandible slices at embryonic day 60 (E60) infected with RUNX2 overexpression lentiviral vector (overexpression) or overexpression control lentiviral vector (O-control). (A'-B') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. C Relative IF expression levels of Lef1 in epithelium (Epi.) and mesenchyme (Mes.) of the overexpression and O-control groups. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for C , **P < .01, ***P < .001; NS. not significant.D, E IF of Lef1 in E60 mandible slices subjected to 3 or 0 kPa stress for two days. (D', E') are magnifications of boxed regions in the corresponding figure panels. Dashed lines mark the epithelium of PC. F Relative IF expression levels of Lef1 in the epithelium and mesenchyme after applying 3 and 0 kPa pressure. Data information: Data represent the means ± SEM. Scale bars = 100 µm. n = 3 for all experiments. Unpaired t-tests for , *P < .05, **P < .01, ***P < .001; NS. not significant.G Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with control lentiviral vector or RUNX2 overexpression lentiviral vector. (G') Relative expression levels between control vector and RUNX2 overexpression groups. Data information: Data represent the means ± SEM. Unpaired t-tests , *P < .05, **P < .01, ***P < .001; NS. not significant.H Western blots of Lef1, non-phospho-β-catenin, and RUNX2 after DFCs were infected with scrambled shRNA (scramsh) or RUNX2 knockdown shRNA (knockdown) lentiviral vectors. (H') Relative expression levels between scramsh and knockdown groups.I Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs of RUNX2 overexpression (O), control vector (V), RUNX2 knockdown (K), scramsh (S), compressed force (F; 1.0 g/cm2), and control (C; 0 g/cm2) groups. Sample were loaded in a gradient (7.5, 15, 30, and 60 μg) showing a linear relationship for each group.J Western blots of nuclear non-phospho-β-catenin and Lamin B in DFCs treated with IWR-1-endo. Relative expression levels were compared between force and no force groups and between overexpression and control groups with or without IWR-1-endo treatment.


Figure 1 ( C ) Survival curves after the injection of EL4 lymphoma cells ( n = 6 / group ) . ( H ) Ifng protein and Ifng mRNA levels ( n = 4 / group ) were determined by ELISA and quantitative PCR .

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Figure 1(C) Survival curves after the injection of EL4 lymphoma cells (n = 6/group).(H) Ifng protein and Ifng mRNA levels (n = 4/group) were determined by ELISA and quantitative PCR.


Figure 2 ( A ) m6A dot blot assay using total RNA from NK92 - siCTRL or NK92 - siFTO cells . ( D ) Western blotting for the detection of GZMB in NK92 - siCTRL and NK92 - siFTO cells after treatment with IL - 2 . ( E ) Levels of IFNγ ( n = 5 biological replicates ) and TNFα ( n = 3 biological replicates ) in the culture media 24 hours after IL - 2 treatment ( left ) or co - culture media with K562 cells for 4 hours ( right ) were determined by ELISA .

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Figure 2(A) m6A dot blot assay using total RNA from NK92-siCTRL or NK92-siFTO cells.(D) Western blotting for the detection of GZMB in NK92-siCTRL and NK92-siFTO cells after treatment with IL-2.(E) Levels of IFNγ (n = 5 biological replicates) and TNFα (n = 3 biological replicates) in the culture media 24 hours after IL-2 treatment (left) or co-culture media with K562 cells for 4 hours (right) were determined by ELISA.


Figure 3 ( B ) Western blotting analysis . NK92 - siCTRL or NK92 - siFTO cells were incubated with 20 ng / ml IL - 2 for 24 hours , washed , and cultured without IL - 2 ( IL - 2 depletion ) for the indicated times ( n = 3 ) . ( C ) A western blotting analysis of NK92 + MOCK or NK92 + FTO cells incubated with 20 ng / ml IL - 2 for the indicated times was performed ( n = 3 ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3(B) Western blotting analysis. NK92-siCTRL or NK92-siFTO cells were incubated with 20 ng/ml IL-2 for 24 hours, washed, and cultured without IL-2 (IL-2 depletion) for the indicated times (n = 3). (C) A western blotting analysis of NK92+MOCK or NK92+FTO cells incubated with 20 ng/ml IL-2 for the indicated times was performed (n = 3). D


Figure 4 ( C ) m6A methylated RNA immunoprecipitation & quantitative PCR ( MeRIP - qPCR ) analysis using a m6A antibody in NK92 - siFTO ( upper ) or NK92 + FTO ( lower ) cells compared to NK92 - siCTRL or NK92 + MOCK cells . ( D ) SOCS3 3 ' - UTR reporter assay using the AANAT reporter system . Plasmid information for the AANAT reporter system with or without the SOCS3 3 ' - UTR sequence ( upper ) . Levels of Aanat protein ( left ) and Aanat mRNA ( right ) expression in HEK - 293T cells co - transfected with plasmid - expressing AANAT reporter containing SOCS3 3 ' - UTR and siCTRL or siFTO .

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Figure 4(C) m6A methylated RNA immunoprecipitation & quantitative PCR (MeRIP-qPCR) analysis using a m6A antibody in NK92-siFTO (upper) or NK92+FTO (lower) cells compared to NK92-siCTRL or NK92+MOCK cells.(D) SOCS3 3'-UTR reporter assay using the AANAT reporter system. Plasmid information for the AANAT reporter system with or without the SOCS3 3'-UTR sequence (upper). Levels of Aanat protein (left) and Aanat mRNA (right) expression in HEK-293T cells co-transfected with plasmid-expressing AANAT reporter containing SOCS3 3'-UTR and siCTRL or siFTO.


Figure 5 ( B ) Relative mRNA expression levels of JAK / STAT target genes , IFNγ ( n = 10 biological replicates ) , GZMB ( n = 6 biological replicates ) and PRF1 ( n = 8 biological replicates ) were determined by qPCR in CD56 + - siCTRL or CD56 + - siFTO cells . ( C ) Western blotting of CD56 + - siCTRL or CD56 + - siFTO cells incubated with 20 ng / ml IL - 15 and 20 ng / ml IL - 21 for the indicated times was performed . Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated ( p - ) and total proteins ( n = 2 ~ 3 ) . Data are representative of three times independent experiments with similar results . The protein levels were expressed as the relative band density of the corresponding protein ( lower ) .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5(B) Relative mRNA expression levels of JAK/STAT target genes, IFNγ (n = 10 biological replicates), GZMB (n = 6 biological replicates) and PRF1 (n = 8 biological replicates) were determined by qPCR in CD56+-siCTRL or CD56+-siFTO cells.(C) Western blotting of CD56+-siCTRL or CD56+-siFTO cells incubated with 20 ng/ml IL-15 and 20 ng/ml IL-21 for the indicated times was performed. Cells were lysed and analyzed by immunoblotting with antibodies to the indicated phosphorylated (p-) and total proteins (n = 2~3). Data are representative of three times independent experiments with similar results. The protein levels were expressed as the relative band density of the corresponding protein (lower).


Figure 6 ( E ) The images of the ventral bioluminescence ( BLI ) following the injection of NK92 - TRC or NK92 - shFTO cells . ( G ) The survival curves after the injection of K562 - Luc cells .

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Figure 6(E) The images of the ventral bioluminescence (BLI) following the injection of NK92-TRC or NK92-shFTO cells.(G) The survival curves after the injection of K562-Luc cells.


Figure 1Negative stain TEM image of B . cereus NVH 0075 - 95 endospore , showing spore body ( SB ) , exosporium ( E ) , and endospore appendages ( Ena ) , which emerge from the endospore individually or as fiber bundles . At the distal end , Enas terminate in a single or multiple thin ruffles ( R ) . cryoEM images ( left ) and negative stain 2D class averages of single S - Ena ( E ) and L - Ena ( F ) fibers . S - Ena show ~ 100 Å diameter and a helical or staggered appearance with a ~ 37 Å rise , whereas L - Ena have a ~ 80 Å diameter and appear as stacked disks of ~ 40 Å height and ~ 60 Å translation along the fiber . L - and S - Ena show , respectively , a single or multiple ruffle ( s ) ( black arrow , labeled ' R ' ) at the distal end of fiber .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Negative stain TEM image of B. cereus NVH 0075-95 endospore, showing spore body (SB), exosporium (E), and endospore appendages (Ena), which emerge from the endospore individually or as fiber bundles. At the distal end, Enas terminate in a single or multiple thin ruffles (R).cryoEM images (left) and negative stain 2D class averages of single S-Ena (E) and L-Ena (F) fibers. S-Ena show ~100 Å diameter and a helical or staggered appearance with a ~37 Å rise, whereas L-Ena have a ~80 Å diameter and appear as stacked disks of ~40 Å height and ~60 Å translation along the fiber. L- and S-Ena show, respectively, a single or multiple ruffle(s) (black arrow, labeled 'R') at the distal end of fiber.


Figure 3SDS - PAGE of recEna1B , treated with β - mercaptoethanol or TEV protease ( to remove N - terminal 6xHis tag ) as indicated . Bands with apparent MW of ~ 13 and ~ 15 kDa correspond recEna1B monomer with ( Ena1B6His ) and without ( Ena1BC ) the 6xHis tag and TEV recognition site , respectively . In the uncleaved sample , a band running at ~ 30 kDa ( labeled * ) corresponds to a non - physiological disulfide bound S - Ena1B dimer . Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS - resistant high molecular weight complexes stuck in the stacking gel . Negative stain TEM images of rec1Ena1B oligomers formed after refolding , but prior to TEV removal of the N - terminal 6xHis tag . Close - up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S - Ena fiber ( model - right ) . Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3SDS-PAGE of recEna1B, treated with β-mercaptoethanol or TEV protease (to remove N-terminal 6xHis tag) as indicated. Bands with apparent MW of ~13 and ~15 kDa correspond recEna1B monomer with (Ena1B6His) and without (Ena1BC) the 6xHis tag and TEV recognition site, respectively. In the uncleaved sample, a band running at ~ 30 kDa (labeled *) corresponds to a non-physiological disulfide bound S-Ena1B dimer. Lower intensity in the cleaved recEna1B results from a loss of monomers to SDS-resistant high molecular weight complexes stuck in the stacking gel.Negative stain TEM images of rec1Ena1B oligomers formed after refolding, but prior to TEV removal of the N-terminal 6xHis tag. Close-up view that shows recEna1B oligomers form open crescents similar in dimensions and shape to single helical turns or arcs found in the S-Ena fiber (model - right). Steric hindrance by the 6xHis is thought to arrest recEna1B polymerization into single helical arcs.


Figure 5Transcription level of ena1A ( x ) , ena1B ( ▲ ) , ena1C ( ○ ) and dedA ( ♦ ) relative to rpoB determined by RT - qPCR during 16 hours of growth of B . cereus strain NVH 0075 - 95 . The dotted line represents the bacterial growth measured by increase in OD600 . Of note , the transcription of ena1C was surprisingly higher than ena1A and ena1B , the major components of the isolated S - Ena ( Fig EV2 ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5Transcription level of ena1A (x), ena1B (▲), ena1C (○) and dedA (♦) relative to rpoB determined by RT-qPCR during 16 hours of growth of B. cereus strain NVH 0075-95. The dotted line represents the bacterial growth measured by increase in OD600. Of note, the transcription of ena1C was surprisingly higher than ena1A and ena1B, the major components of the isolated S-Ena (Fig EV2).


Figure 6Representative negative stain images of endospores of NVH 0075 - 95 mutants lacking ena1A , ena1B , or ena1C , as well as endospores of strains complemented with the respective ena subunit expressed from plasmid ( i . e . pA , pAB and pC ) . The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B ( pAB ) due to repeated failure to transform with a plasmid holding ena1B only . Inset are 2D class averages of Enas observed on the respective mutants . Knockout of ena1A , ena1B , or ena1C results in the loss of S - Ena , a phenotype that is restored by plasmid - based complementation . Number ( top ) and length ( bottom ) of Enas found on WT , mutant ( ∆ ena1A , ∆ ena1B , or ∆ ena1C ) and plasmid complemented ( ∆ ena1A : pena1A , ∆ ena1B : pena1AB , or ∆ ena1C : pena1C ) NVH 0075 - 95 endospores . Statistics : pair - wise Mann - Whitney U tests against WT ( n : ≥ 18 spores ; n : ≥ 50 Enas ; ns : not significant , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 and * * * * p < 0 . 0001 . - - - : mean ± s . d . )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6Representative negative stain images of endospores of NVH 0075-95 mutants lacking ena1A, ena1B, or ena1C, as well as endospores of strains complemented with the respective ena subunit expressed from plasmid (i.e. pA, pAB and pC). The ena1B mutant was complemented with a plasmid carrying both ena1A and 1B (pAB) due to repeated failure to transform with a plasmid holding ena1B only. Inset are 2D class averages of Enas observed on the respective mutants. Knockout of ena1A, ena1B, or ena1C results in the loss of S-Ena, a phenotype that is restored by plasmid-based complementation.Number (top) and length (bottom) of Enas found on WT, mutant (∆ena1A, ∆ena1B, or ∆ena1C) and plasmid complemented (∆ena1A:pena1A, ∆ena1B:pena1AB, or ∆ena1C:pena1C) NVH 0075-95 endospores. Statistics: pair-wise Mann-Whitney U tests against WT (n: ≥18 spores; n: ≥50 Enas; ns: not significant, * p<0.05, ** p<0.01, *** p<0.001 and **** p<0.0001. ---: mean ± s.d.)