Figure 3Cells were pretreated with endosomal - pH interfering drugs at the indicated concentrations and subsequently infected with SARS - CoV - 2 as described in Fig . 2A and 2B in the continuous presence of NH4Cl ( A ) , chloroquine ( B ) , bafilomycin A1 ( C ) , or concanamycin B ( D ) . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to control samples that were not treated with inhibitors . n = 2 - 6 biological replicates .

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Figure 3Cells were pretreated with endosomal-pH interfering drugs at the indicated concentrations and subsequently infected with SARS-CoV-2 as described in Fig. 2A and 2B in the continuous presence of NH4Cl (A), chloroquine (B), bafilomycin A1 (C), or concanamycin B (D). The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to control samples that were not treated with inhibitors. n = 2-6 biological replicates.


Figure 4A549 * cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate ( B ) , SB412515 ( C ) , and bafilomycin A1 ( D ) . Infection with SARS - CoV - 2 ( MOI ~ 0 . 2 ) was achieved in the continuous presence of the drug . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where inhibitors had been omitted . n = 5 - 6 biological replicates .

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Figure 4A549* cells expressing or lacking TMPRSS2 were pretreated with the indicated concentrations of camostat mesylate (B), SB412515 (C), and bafilomycin A1 (D). Infection with SARS-CoV-2 (MOI ~0.2) was achieved in the continuous presence of the drug. Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where inhibitors had been omitted. n = 5-6 biological replicates.


Figure 5The timing of the MG - 132 - sensitive step during SARS - CoV - 2 infectious entry into Calu - 3 and Caco - 2 cells was assayed as detailed in D but using 60 µM MG - 132 for Caco - 2 cells and MOIs of 0 . 5 and 0 . 6 , respectively . n = 2 . A549 * cells expressing or lacking TMPRSS2 were pretreated with MG - 132 at the indicated concentrations and subsequently infected with SARS - CoV - 2 ( MOI ~ 0 . 2 ) in the continuous presence of the drug . The proportion of infected cells was quantified by flow cytometry as described in Fig . 1D , and data were normalized to that from control samples for which MG - 132 had been omitted . n = 6 biological replicates .

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Figure 5The timing of the MG-132-sensitive step during SARS-CoV-2 infectious entry into Calu-3 and Caco-2 cells was assayed as detailed in D but using 60 µM MG-132 for Caco-2 cells and MOIs of 0.5 and 0.6, respectively. n = 2.A549* cells expressing or lacking TMPRSS2 were pretreated with MG-132 at the indicated concentrations and subsequently infected with SARS-CoV-2 (MOI ~0.2) in the continuous presence of the drug. The proportion of infected cells was quantified by flow cytometry as described in Fig. 1D, and data were normalized to that from control samples for which MG-132 had been omitted. n = 6 biological replicates.


Figure 6SARS - CoV - 2 was subjected to pretreatment with trypsin and furin for 15 min at 37 ° C prior to infection of Caco - 2 and Vero cells ( MOIs of 0 . 4 and 0 . 3 , respectively ) . Infected cells were quantified by flow cytometry as described in Fig . 1D . Data were normalized to samples not pretreated with trypsin . n = 4 - 6 biological replicates . A549 * expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS - CoV - 2 at MOIs of 0 . 1 and 0 . 2 , respectively , for 24 h and then cocultured for 5 h along with target cells , not infected but prestained with CMFDA , a cytosolic green dye . Cells were subsequently treated with trypsin or furin for 5 min at 37 ° C and left to coculture for an additional hour at 37 ° C . After fixation , nuclei were stained with Hoechst ( blue ) , and infected cells were subjected to immunofluorescence staining against SARS - CoV - 2 nucleoprotein ( magenta ) . Samples were ultimately imaged by confocal fluorescence microscopy . White stars indicate syncytia with at least four nuclei . Scale bars : 100 µm . Images of microscope fields obtained in ( E ) were quantified [ A549 * : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 80 , and n ( furin ) = 60 ; A549 * TMPRSS2 : n ( no virus ) = 30 , n ( no protease ) = 80 , n ( trypsin ) = 79 , and n ( furin ) = 60 ; Vero : n ( no virus ) = 40 , n ( no protease ) = 112 , n ( trypsin ) = 114 , and n ( furin ) = 60 ] . The fusion index is given as f = 1 - [ ( number of cells in a field after fusion ) / ( number of nuclei ) ] .

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Figure 6SARS-CoV-2 was subjected to pretreatment with trypsin and furin for 15 min at 37°C prior to infection of Caco-2 and Vero cells (MOIs of 0.4 and 0.3, respectively). Infected cells were quantified by flow cytometry as described in Fig. 1D. Data were normalized to samples not pretreated with trypsin. n = 4-6 biological replicates.A549* expressing or lacking TMPRSS2 cells and Vero cells were first infected with SARS-CoV-2 at MOIs of 0.1 and 0.2, respectively, for 24 h and then cocultured for 5 h along with target cells, not infected but prestained with CMFDA, a cytosolic green dye. Cells were subsequently treated with trypsin or furin for 5 min at 37°C and left to coculture for an additional hour at 37°C. After fixation, nuclei were stained with Hoechst (blue), and infected cells were subjected to immunofluorescence staining against SARS-CoV-2 nucleoprotein (magenta). Samples were ultimately imaged by confocal fluorescence microscopy. White stars indicate syncytia with at least four nuclei. Scale bars: 100 µm. Images of microscope fields obtained in (E) were quantified [A549*: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 80, and n (furin) = 60; A549* TMPRSS2: n (no virus) = 30, n (no protease) = 80, n (trypsin) = 79, and n (furin) = 60; Vero: n (no virus) = 40, n (no protease) = 112, n (trypsin) = 114, and n (furin) = 60]. The fusion index is given as f = 1 - [(number of cells in a field after fusion)/(number of nuclei)].


Figure 7SARS - CoV - 2 particles ( MOI of 0 . 3 ) were first subjected to trypsin treatment for 15 min at 37 ° C and then exposed to buffers at the indicated pH for 10 min at 37 ° C , and vice versa . Calu - 3 and Vero cells were then infected at a MOI of 0 . 3 and analyzed by flow cytometry as described in Fig . 1D . n = 4 - 8 biological replicates .

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Figure 7SARS-CoV-2 particles (MOI of 0.3) were first subjected to trypsin treatment for 15 min at 37°C and then exposed to buffers at the indicated pH for 10 min at 37°C, and vice versa. Calu-3 and Vero cells were then infected at a MOI of 0.3 and analyzed by flow cytometry as described in Fig. 1D. n = 4-8 biological replicates.


Figure 8SARS - CoV - 2 was first treated with trypsin or thermolysin and then allowed to infect A549 * and Vero cells ( MOIs ~ 0 . 2 and 0 . 3 ) in the continuous presence of SB412515 ( cathepsin L inhibitor ) . Infected cells were quantified by flow cytometry as described in Fig . 1D , and data were normalized to samples where SB412515 had been omitted . n = 4 biological replicates .

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Figure 8SARS-CoV-2 was first treated with trypsin or thermolysin and then allowed to infect A549* and Vero cells (MOIs ~0.2 and 0.3) in the continuous presence of SB412515 (cathepsin L inhibitor). Infected cells were quantified by flow cytometry as described in Fig. 1D, and data were normalized to samples where SB412515 had been omitted. n = 4 biological replicates.


Figure 1C . Tissue distribution of the Ldb2 protein . Representative western - blot image is shown . Yellow asterisks indicate uncharacterized bands . D . Expression of Ldb2 in the cerebral cortex . DAPI - stained nuclei visualized ( right ) in the cerebral cortex . E . Expression of Ldb2 in the amygdala . LA ; lateral amygdala , BLA ; basolateral amygdala , Ce ; central nucleus of amygdala . F - G . Expression of Ldb2 in neurons . The merged images obtained with Ldb2 ( stained in green ) and NeuN ( stained in red ) antibodies are shown for cerebral cortex layers I - VI and the amygdala ( F ) . most of Ldb2 - positive cells are NeuN - positive . G . NeuN - positive cells / Ldb2 - positive cells ( left ) and Ldb2 - positive cells / NeuN - positive cells were counted in cerebral cortex layers I - VI .

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Figure 1C. Tissue distribution of the Ldb2 protein. Representative western-blot image is shown. Yellow asterisks indicate uncharacterized bands.D. Expression of Ldb2 in the cerebral cortex. DAPI-stained nuclei visualized (right) in the cerebral cortex.E. Expression of Ldb2 in the amygdala. LA; lateral amygdala, BLA; basolateral amygdala, Ce; central nucleus of amygdala.F-G. Expression of Ldb2 in neurons. The merged images obtained with Ldb2 (stained in green) and NeuN (stained in red) antibodies are shown for cerebral cortex layers I-VI and the amygdala (F). most of Ldb2-positive cells are NeuN-positive. G. NeuN-positive cells/Ldb2-positive cells (left) and Ldb2-positive cells/NeuN-positive cells were counted in cerebral cortex layers I-VI.


Figure 2Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Home cage - activity test ( A ) . Voluntary locomotor activities in the home cages were monitored for a week . Data of dark ( left ) and light ( right ) phases are indicated . Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Open - field test ( B ) Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Acoustic startle response ( C ) Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Prepulse - inhibition test ( D ) Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in , Morris water - maze test ( E ) where data in hidden test ( escape latency and moving speed , path length and no movement time ) and probe test ( path length ) are shownLdb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Y - maze test ( F ) . Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Fear - conditioning test ( G ) . Results of contextual ( left ) and cued ( right ) tests are shown . Ldb2 KO mice ( red , n = 10 - 11 ) were compared to control ( blue , n = 10 - 11 ) in Hot - plate test ( H ) .

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Figure 2Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Home cage-activity test (A). Voluntary locomotor activities in the home cages were monitored for a week. Data of dark (left) and light (right) phases are indicated.Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Open-field test (B)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Acoustic startle response (C)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Prepulse-inhibition test (D)Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in , Morris water-maze test (E) where data in hidden test (escape latency and moving speed, path length and no movement time) and probe test (path length) are shownLdb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Y-maze test (F).Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Fear-conditioning test (G). Results of contextual (left) and cued (right) tests are shown.Ldb2 KO mice (red, n = 10-11) were compared to control (blue, n = 10-11) in Hot-plate test (H).


Figure 3Locomotor activity was assessed in the same experimental set as in the open - field test . After acclimation for 120 min , mice ( n = 10 - 12 / group ) were injected ( arrows ) with methylphenidate ( MPD : 3 mg / kg , A ) and their locomotor activity was monitored . In the right panels , cumulative values for pre - and post - injection times are also shown . Locomotor activity was assessed in the same experimental set as in the open - field test . After acclimation for 120 min , mice ( n = 10 - 12 / group ) were injected ( arrows ) with MK - 801 ( 0 . 2 mg / kg , B ) and their locomotor activity was monitored . In the right panels , cumulative values for pre - and post - injection times are also shown . C . Popping behavior induced by the injection of MK - 801 ( 0 . 5 mg / kg ) was manually assessed in WT ( n = 4 ) and KO ( n = 6 ) animals . Typically , each episode is constituted by blocks of 5 - 15 successive jumping behaviors . Episode numbers in 20 min ( left ) and total duration ( right ) are shown in a box - whisker plot .

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Figure 3Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with methylphenidate (MPD: 3 mg/kg, A) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.Locomotor activity was assessed in the same experimental set as in the open-field test. After acclimation for 120 min, mice (n = 10-12/group) were injected (arrows) with MK-801 (0.2 mg/kg, B) and their locomotor activity was monitored. In the right panels, cumulative values for pre- and post-injection times are also shown.C. Popping behavior induced by the injection of MK-801 (0.5 mg/kg) was manually assessed in WT (n = 4) and KO (n = 6) animals. Typically, each episode is constituted by blocks of 5-15 successive jumping behaviors. Episode numbers in 20 min (left) and total duration (right) are shown in a box-whisker plot.


Figure 4A . Fear - conditioning test . Freezing ( % ) was monitored in the cued ( top ) and contextual ( bottom ) tests before ( pre ) and after ( post ) administration of clozapine ( left ) or haloperidol ( right ) . Data are presented as means ± S . E . M . ( n = 7 - 9 / group ) . * , p < 0 . 05 ; * * * , p < 0 . 001 ( Tukey test ) ; # , p < 0 . 05 ( t - test , KO / saline vs . KO / clozapine ) . B . Home cage - activity test . Spontaneous locomotor activity of mice chronically fed chow with or without 0 . 24 % ( w / w ) lithium carbonate for two months was monitored for 6 days in the home cage .

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Figure 4A. Fear-conditioning test. Freezing (%) was monitored in the cued (top) and contextual (bottom) tests before (pre) and after (post) administration of clozapine (left) or haloperidol (right). Data are presented as means ± S.E.M. (n = 7-9/group). *, p < 0.05; ***, p < 0.001 (Tukey test); #, p < 0.05 (t-test, KO/saline vs. KO/clozapine).B. Home cage-activity test. Spontaneous locomotor activity of mice chronically fed chow with or without 0.24% (w/w) lithium carbonate for two months was monitored for 6 days in the home cage.


Figure 5A . Scatter plots showing Venus - fluorescent intensities and the median amplitude of miniature EPSCs ( mEPSC ) in the LA of the AV - KO mice and their littermates ( AV - WT ) . B . Scatter plots showing Venus - fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV - KO and their control littermates ( AV - WT ) . Three typical pictures of Venus - positive cells with various fluorescent intensities are shown . Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude ( A ) or the mEPSC frequency ( B ) . The black dotted lines are regression lines for AV - WT mice , the red dotted lines are those for AV - KO mice , and black solid lines are those for all cells .

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Figure 5A. Scatter plots showing Venus-fluorescent intensities and the median amplitude of miniature EPSCs (mEPSC) in the LA of the AV-KO mice and their littermates (AV-WT). B. Scatter plots showing Venus-fluorescent intensities and the mean frequency of miniature EPSCs in the LA of the AV-KO and their control littermates (AV-WT). Three typical pictures of Venus-positive cells with various fluorescent intensities are shown. Lines in each panel are regression lines showing inverse correlation of the fluorescent intensity with the mEPSC amplitude (A) or the mEPSC frequency (B). The black dotted lines are regression lines for AV-WT mice, the red dotted lines are those for AV-KO mice, and black solid lines are those for all cells.


Figure 6B . Numbers of Venus - positive neurons in the lateral nucleus ( LA ) and the lateral side of the basal nucleus ( BAl ) of Naive , Unpaired , and Paired groups . Open and filled bars represent AV - WT and AV - KO mice , respectively . Positive neurons of AV - WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA ( upper panel ) . AV - KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV - KO mice were increased compared to the same groups of the AV - WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV - WT and AV - KO mice The error bars represent S . E . M . ( n = 5 / group ) . C . Numbers of Venus - positive neurons classified by fluorescence intensities in the LA of the AV - WT and AV - KO mice . Weak positive neurons were increased in the Paired group compared to the Naïve group in AV - WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV - KO The increment of the Middle Venus - positive neurons was larger in the AV - KO compared to AV -

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Figure 6B. Numbers of Venus-positive neurons in the lateral nucleus (LA) and the lateral side of the basal nucleus (BAl) of Naive, Unpaired, and Paired groups. Open and filled bars represent AV-WT and AV-KO mice, respectively. Positive neurons of AV-WT was increased only in the Paired group compared to the Naïve and Unpaired groups in the LA (upper panel). AV-KO was increased in the Unpaired as well as the Paired groups compared to the Naïve group Positive neurons both in the Unpaired and Paired AV-KO mice were increased compared to the same groups of the AV-WT mice In the BAl , positive neurons were increased both in the Unpaired and Paired groups compared to the Naïve group in both the AV-WT and AV-KO mice The error bars represent S.E.M. (n =5/group).C. Numbers of Venus-positive neurons classified by fluorescence intensities in the LA of the AV-WT and AV-KO mice. Weak positive neurons were increased in the Paired group compared to the Naïve group in AV-WT Both Weak and Middle positive neurons were increased in the Paired group compared to the Naïve group in AV-KO The increment of the Middle Venus-positive neurons was larger in the AV-KO compared to AV-WT


Figure 7Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LDB2 ( A ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively . Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LHX2 ( B ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively . Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . LMO2 / 3 / 4 ( C ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively . Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs . SSBP2 / 3 / 4 ( D ) LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively . Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs . RLIM ( E ) . LDB2 ( WT , T83N or P170L ) and candidate interactors were tagged with Flag and HA , respectively . HEK293T cells were transfected with the Flag - construct and HA - construct indicated . After Flag - LDB2 was immunoprecipitated with a Flag antibody , the immunocomplexes were analyzed with western blotting with Flag and HA antibodies . The input samples ( input ) were also analyzed . Green and black arrowhead indicate positions for Flag - Ldb2 and HA - proteins , respectively . F . Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2 . Immunocomplexes from mouse brain lysate using an antibody against Ldb2 , Lmo3 or Ssbp3 , or a control antibody were analyzed by western blotting using an anti - Ldb2 ( left ) or anti - Ssbp3 ( right ) antibody .

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Figure 7Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LDB2 (A) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LHX2 (B) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. LMO2/3/4 (C) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates , LDB2 vs. SSBP2/3/4 (D) LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.Results of coimmunoprecipitation between overexpressed LDB2 and interaction candidates LDB2 vs. RLIM (E). LDB2 (WT, T83N or P170L) and candidate interactors were tagged with Flag and HA, respectively. HEK293T cells were transfected with the Flag-construct and HA-construct indicated. After Flag-LDB2 was immunoprecipitated with a Flag antibody, the immunocomplexes were analyzed with western blotting with Flag and HA antibodies. The input samples (input) were also analyzed. Green and black arrowhead indicate positions for Flag-Ldb2 and HA-proteins, respectively.F. Coimmunoprecipitation of endogenous Ssbp3 and Lmo3 with Ldb2. Immunocomplexes from mouse brain lysate using an antibody against Ldb2, Lmo3 or Ssbp3, or a control antibody were analyzed by western blotting using an anti-Ldb2 (left) or anti-Ssbp3 (right) antibody.


Figure 8A . Human neurosphere ( bottom ) induced from iPS cells ( top ) from a healthy control subject . C . Overlap of ChIP - seq peaks of LDB2 in neurosphere cells and ChIP - seq peaks of EGR1 in human cultured cell lines ECC1 and K562 , at ARCD . Genome - wide pattern of overlap between the ChIP - seq peaks of LDB2 in neurosphere cells and the ChIP - seq peaks of EGR1 in the ECC1 cell line . Fold enrichment ( log2 ) of ChIP - seq reads against the input reads were calculated for each ChIP - seq peak region ( summit ± 1 , 000 bp ) in each category ( neurosphere specific , common , and ECC1 specific ) and displayed in a heatmap by ngs . plotE . Annotation of ChIP - seq peaks . ChIP - seq peaks of LDB2 in the neurosphere cells ( all , neurosphere specific , and common were analyzed by the PAVIS

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Figure 8A. Human neurosphere (bottom) induced from iPS cells (top) from a healthy control subject.C. Overlap of ChIP-seq peaks of LDB2 in neurosphere cells and ChIP-seq peaks of EGR1 in human cultured cell lines ECC1 and K562, at ARCD. Genome-wide pattern of overlap between the ChIP-seq peaks of LDB2 in neurosphere cells and the ChIP-seq peaks of EGR1 in the ECC1 cell line. Fold enrichment (log2) of ChIP-seq reads against the input reads were calculated for each ChIP-seq peak region (summit ± 1,000 bp) in each category (neurosphere specific, common, and ECC1 specific) and displayed in a heatmap by ngs.plotE. Annotation of ChIP-seq peaks. ChIP-seq peaks of LDB2 in the neurosphere cells (all, neurosphere specific, and common were analyzed by the PAVIS program


Figure 1 : Westernblot of producer cell lysates and concentrated reporter particles . BHK cells were transfected with either human ACE2 or empty vector , subsequently infected with VSV - reporter particles pseudotyped with chimeric spikes , luciferase was measured and normalized to no pseudotype as a readout for cell entry . Shown are the data for 3 replicates .

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Figure 1:Westernblot of producer cell lysates and concentrated reporter particles.BHK cells were transfected with either human ACE2 or empty vector, subsequently infected with VSV-reporter particles pseudotyped with chimeric spikes, luciferase was measured and normalized to no pseudotype as a readout for cell entry. Shown are the data for 3 replicates.


Figure 2 : Primate cells , were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates . human cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates . bat cells were infected with VSV - particles pseudotyped with the lineage B chimeric spike panel . Pseudotypes were either left untreated or incubated with trypsin before addition to the cells . Luciferase was measured and normalized to particles produced without pseudotype . Shown are the data for 3 replicates .

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Figure 2:Primate cells, were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.human cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.bat cells were infected with VSV-particles pseudotyped with the lineage B chimeric spike panel. Pseudotypes were either left untreated or incubated with trypsin before addition to the cells. Luciferase was measured and normalized to particles produced without pseudotype. Shown are the data for 3 replicates.


Figure 3 : Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated . Shown are data from 3 replicates . Expression and pseudotype incorporation of SARS - S - 2019 - nCoV RBD chimeras . Pseudotypes were used to infect cells expressing hACE2 , hDPP4 , hAPN , or empty vector , without protease treatment .

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Figure 3:Pseudotyped particles were either left untreated and treated with trypsin and subsequently used to infect BHK cells transfected with the coronavirus receptor indicated. Shown are data from 3 replicates.Expression and pseudotype incorporation of SARS-S-2019-nCoV RBD chimeras.Pseudotypes were used to infect cells expressing hACE2, hDPP4, hAPN, or empty vector, without protease treatment.


Figure 4 : VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector . Shown are data for 3 replicates . Westernblot of producer cell lysates and concentrated pseudotyped particles .

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Figure 4:VSV pseudotypes were generated with the indicated RBD and used to infect BHKs transfected with either human ACE2 or empty vector. Shown are data for 3 replicates.Westernblot of producer cell lysates and concentrated pseudotyped particles.


Figure 5 : Westernblot of producer cell lysates and concentrated pseudotypes particles . Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh - 7 . 5 cells . Shown are data for 3 replicates . Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2 . Shown are data for 3 replicates .

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Figure 5:Westernblot of producer cell lysates and concentrated pseudotypes particles.Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect Huh-7.5 cells. Shown are data for 3 replicates.Pseudotypes with indicated spike constructs were left untreated or treated with trypsin and subsequently used to infect BHK cells transfected with human ACE2. Shown are data for 3 replicates.


Figure 1D . BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N . benthamiana leaves . Images were taken at 60 hpi . Scale bar = 20 μm . E . Co - IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM . N . benthamiana leaves were agroinfiltrated and treated with 2 mM 3 - MA at 48 hpi , and then for IP with anti - Flag beads 12 h later .

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Figure 1D. BiFC analysis of interactions between VISP1 or VISP1mUIM and ATG8a or ATG8f in N. benthamiana leaves. Images were taken at 60 hpi. Scale bar = 20 μm.E. Co-IP experiments examining protein interactions between ATG8a with VISP1 or VISP1mUIM. N. benthamiana leaves were agroinfiltrated and treated with 2 mM 3-MA at 48 hpi, and then for IP with anti-Flag beads 12 h later.


Figure 2Confocal microscopy of YFP - ATG8e - labeled autophagic bodies in root cells of Col - 0 / YFP - ATG8e and VISP1OE / YFP - ATG8e plants . 7 - day - old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark . Scale bar = 20 μm . Puncta numbers of YFP - ATG8e - labeled autophagic bodies per 10 cells in panel A . Data points represent means of three biological repeats . Error bars indicate SD . * * * p < 0 . 001 ( Student ' s t - test ) . Western blotting analysis of accumulation of ATG8a and ATG8a - PE in leaves of 4 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE and VISP1mARM / OE plants . Representative TEM images of autophagic structures in inoculated leaves of Col - 0 and VISP1OE plants treated with mock buffer or CMV - 2blm infection at 4 dpi . The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling . Magenta arrowheads indicate autophagic bodies inside the vacuoles . Scale bar = 2 μm . Cp , chloroplast ; CW , cell wall ; V , vacuole . Autophagic structure numbers per 10 cells in panel F . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) .

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Figure 2Confocal microscopy of YFP-ATG8e-labeled autophagic bodies in root cells of Col-0/YFP-ATG8e and VISP1OE/YFP-ATG8e plants. 7-day-old seedlings were inoculated in liquid ½MS medium with 1 µM ConA for 12 h in dark. Scale bar = 20 μm.   Puncta numbers of YFP-ATG8e-labeled autophagic bodies per 10 cells in panel A. Data points represent means of three biological repeats. Error bars indicate SD. ***p < 0.001 (Student's t-test).   Western blotting analysis of accumulation of ATG8a and ATG8a-PE in leaves of 4-week-old Col-0, VISP1OE, VISP1mUIM/OE and VISP1mARM/OE plants.Representative TEM images of autophagic structures in inoculated leaves of Col-0 and VISP1OE plants treated with mock buffer or CMV-2blm infection at 4 dpi. The leaves was treated with 1 µM ConA at 4 dpi for 12 h in dark before TEM sampling. Magenta arrowheads indicate autophagic bodies inside the vacuoles. Scale bar = 2 μm. Cp, chloroplast; CW, cell wall; V, vacuole.   Autophagic structure numbers per 10 cells in panel F. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).


Figure 3Long - term symptoms of Col - 0 , VISP1OE1 , VISP1OE2 , rdr6 - 15 , and sgs3 - 1 plants inoculated with CMV - 2blm at 50 dpi . Scale bar = 2 cm . Northern blotting analyzing accumulation of genomic RNAs ( gRNAs ) in systemically leaves of Col - 0 , OE1 ( VISP1OE1 ) , OE2 ( VISP1OE2 ) , rdr6 - 15 , and sgs3 - 1 plants infected by CMV - 2blm at 14 dpi . Methylene blue - stained rRNA were used as loading controls . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D .

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Figure 3Long-term symptoms of Col-0, VISP1OE1, VISP1OE2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.Northern blotting analyzing accumulation of genomic RNAs (gRNAs) in systemically leaves of Col-0, OE1 (VISP1OE1), OE2 (VISP1OE2), rdr6-15, and sgs3-1 plants infected by CMV-2blm at 14 dpi. Methylene blue-stained rRNA were used as loading controls.   Northern blotting analyzing accumulation of RNA3-derived   vsiRNAs in the same samples as shown in panel D.


Figure 4Long - term systemic symptoms of Col - 0 , visp1 - 1 , visp1 - 2 , rdr6 - 15 , and sgs3 - 1 plants inoculated with CMV - 2blm at 50 dpi . Scale bar = 2 cm . Northern blotting detecting accumulation of genomic RNAs and RNA3 - derived vsiRNAs in systemically infected leaves at 14 dpi . Relative accumulation ( RA ) of gRNA3 was calculated from band densities . Northern blotting analyzing accumulation of RNA3 - derived vsiRNAs in the same samples as shown in panel D . Bottom values represent relative accumulation ( RA ) of RNA3 - derived vsiRNAs .

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Figure 4Long-term systemic symptoms of Col-0, visp1-1, visp1-2, rdr6-15, and sgs3-1 plants inoculated with CMV-2blm at 50 dpi. Scale bar = 2 cm.Northern blotting detecting accumulation of genomic RNAs and RNA3-derived vsiRNAs in systemically infected leaves at 14 dpi. Relative accumulation (RA) of gRNA3 was calculated from band densities.   Northern blotting analyzing accumulation of RNA3-derived vsiRNAs in the same samples as shown in panel D. Bottom values represent relative accumulation (RA) of RNA3-derived vsiRNAs.


Figure 5Co - immunoprecipitation analyses of the interactions between SGS3 , SGS3ΔCC , or SGS3CC and VISP1 or VISP1mARM . N . benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3 - MA at 48 hpi , and collected for IP with anti - Flag beads 12 h later . GST pull - down analysis of the in vitro interactions of SGS3CC with VISP1 , VISP1mARM or VISP1mUIM . GST - VISP1 , GST - VISP1mARM , GST - VISP1mUIM or GST were incubated with SGS3CC - His for IP with anti - GST beads , and detected by Western blotting with GST or His antibodies .

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Figure 5Co-immunoprecipitation analyses of the interactions between SGS3, SGS3ΔCC, or SGS3CC and VISP1 or VISP1mARM. N. benthamiana leaves were agroinfiltrated with constructs as indicated and treated with 2 mM 3-MA at 48 hpi, and collected for IP with anti-Flag beads 12 h later.GST pull-down analysis of the in vitro interactions of SGS3CC with VISP1, VISP1mARM or VISP1mUIM. GST- VISP1, GST- VISP1mARM, GST- VISP1mUIM or GST were incubated with SGS3CC-His for IP with anti-GST beads, and detected by Western blotting with GST or His antibodies.


Figure 6Confocal analysis of co - localization of CFP - NbATG8f - labelled autophagic bodies with the VISP1 - YN and SGS3 - YC bodies in N . benthamiana leaves . The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi . Scale bar = 20 μm . Confocal analysis of effect of VISP1 - Flag VISP1mARM - Flag , and VISP1mUIM - Flag on RDR6 - YN and SGS3 - YC formed bodies in N . benthamiana leaves at 60 hpi . EV : empty vector . Scale bar = 20 μm . Numbers of RDR6 - YN and SGS3 - YC formed bodies per 10 cells in E were used for quantification . Data points represent means of three biological repeats . Error bars indicate SD . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) . Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , visp1 , and sgs3 - 1 Arabidopsis plants with anti - SGS3 antibodies . Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2 - week - old Col - 0 , VISP1OE , VISP1mUIM / OE , and VISP1mARM / OE Arabidopsis plants . Western blotting detecting accumulation of the endogenous SGS3 protein in 2 - week - old VISP1 - overexpressing Col - 0 , atg5 - 1 , and atg7 - 1 mutants .

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Figure 6Confocal analysis of co-localization of CFP-NbATG8f-labelled autophagic bodies with the VISP1-YN and SGS3-YC bodies in N. benthamiana leaves. The infiltrated leaves were treated with 100 µM E64d or DMSO at 48 hpi and examined for imaging at 60 hpi. Scale bar = 20 μm.Confocal analysis of effect of VISP1-Flag VISP1mARM-Flag, and VISP1mUIM-Flag on RDR6-YN and SGS3-YC formed bodies in N. benthamiana leaves at 60 hpi. EV: empty vector. Scale bar = 20 μm.   Numbers of RDR6-YN and SGS3-YC formed bodies per 10 cells in E were used for quantification. Data points represent means of three biological repeats. Error bars indicate SD. Letters indicate significant differences (ANOVA, P < 0.05).   Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, visp1, and sgs3-1 Arabidopsis plants with anti-SGS3 antibodies.   Western blotting detecting accumulation of the endogenous SGS3 protein in leaves of 2-week-old Col-0, VISP1OE, VISP1mUIM/OE, and VISP1mARM/OE Arabidopsis plants.   Western blotting detecting accumulation of the endogenous SGS3 protein in 2-week-old VISP1-overexpressing Col-0, atg5-1, and atg7-1 mutants.


Figure 7Developmental phenotypes of rosette leaves of 5 - week - old Col - 0 , visp1 - 1 , visp1 - 2 , VISP1OE1 , VISP1OE2 , and sgs3 - 1 seedlings . Arrowheads indicate the 8th and 9th rosette leaves from the top leaves . Scale bar = 2 cm . The length / width ratios of the 8th and 9th rosette leaves of the seedlings shown in A . Every dot represents data from the length / width ratio of one leave ( n = 20 ) . Letters indicate significant differences ( ANOVA , P < 0 . 05 ) . Northern blotting detecting accumulation of TAS1 - TasiR255 , TAS2 - TasiR1511 , TAS3 - TasiR5 ′ D8 , miR173 , and miR390 in leaves of Arabidopsis plants shown in panel A . Values represent relative accumulation ( RA ) of siRNAs and values in Col - 0 plants were set as 1 . rRNA served as loading controls .

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 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

Figure 7Developmental phenotypes of rosette leaves of 5-week-old Col-0, visp1-1, visp1-2, VISP1OE1, VISP1OE2, and sgs3-1 seedlings. Arrowheads indicate the 8th and 9th rosette leaves from the top leaves. Scale bar = 2 cm.   The length/width ratios of the 8th and 9th rosette leaves of the seedlings shown in A. Every dot represents data from the length/width ratio of one leave (n = 20). Letters indicate significant   differences (ANOVA, P < 0.05).Northern blotting detecting accumulation of TAS1-TasiR255, TAS2-TasiR1511, TAS3-TasiR5′D8, miR173, and miR390 in leaves of Arabidopsis plants shown in panel A. Values represent relative accumulation (RA) of siRNAs and values in Col-0 plants were set as 1. rRNA served as loading controls.


Figure 1 B - C Competitor donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood . In the control group , competitor donor cells are WT HSCs . In the deficient group , competitor donor cells are uMT - / - or NSG HSCs Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test ) D - E WT donor derived Gr , B , CD4 T , and CD8 T cells in the peripheral blood Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test ) F - G Total production of Gr , B , CD4 T , and CD8 T cells in the peripheral blood ( derived from WT HSCs , competitor HSCs , helper whole bone marrow cells , and residual host cells ) shown as percentages of the total number of white blood cells ( WBCs ) . Data information : Data are shown as percentages of the total number of white blood cells ( WBCs ) . Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed and two - sample equal variance Student " s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1 B-C Competitor donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood. In the control group, competitor donor cells are WT HSCs. In the deficient group, competitor donor cells are uMT-/- or NSG HSCs Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)  D-E WT donor derived Gr, B, CD4 T, and CD8 T cells in the peripheral blood Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)  F-G Total production of Gr, B, CD4 T, and CD8 T cells in the peripheral blood (derived from WT HSCs, competitor HSCs, helper whole bone marrow cells, and residual host cells) shown as percentages of the total number of white blood cells (WBCs). Data information: Data are shown as percentages of the total number of white blood cells (WBCs). Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05, **p<0.01, ***p<0.001 (two-tailed and two-sample equal variance Student"s t-test)


Figure 2 A - B Total numbers of barcoded WT clones that give rise to granulocytes ( Gr ) , B cells , CD4 T cells , and CD8 T cells Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test ) C - J Number of HSC clones that produce different amounts of Gr , B , CD4 T , and CD8 T cells . We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows : Clonal abundance = 100 % * ( Each cell population ( Gr , B , CD4T or CD8T cells ) % WBCs ) * ( Donor % Each cell population ) * ( GFP % Donor cells ) * ( number of reads for each barcode ) / ( total reads of all barcodes Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test ) K - L Production of Gr , B , CD4 T , and CD8 T cells by expanding WT clones that produce more than 0 . 1 % of WBCs in each lineage . Data information : Data were collected at month 7 post - transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 ( two - tailed and two - sample equal variance Student " s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2 A-B Total numbers of barcoded WT clones that give rise to granulocytes (Gr), B cells, CD4 T cells, and CD8 T cells Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)  C-J Number of HSC clones that produce different amounts of Gr, B, CD4 T, and CD8 T cells. We combined the sequencing data with the flow cytometry data to calculate clonal abundance for each clone as follows: Clonal abundance =100%*(Each cell population (Gr, B, CD4T or CD8T cells) % WBCs) * (Donor % Each cell population) * (GFP % Donor cells) * (number of reads for each barcode) / (total reads of all barcodes Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)  K-L Production of Gr, B, CD4 T, and CD8 T cells by expanding WT clones that produce more than 0.1% of WBCs in each lineage. Data information: Data were collected at month 7 post-transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 (two-tailed and two-sample equal variance Student"s t-test)


Figure 4 A - B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow ( BM ) cells . Shown are hematopoietic stem cells ( HSCs ) , Flk2 - and Flk2 + multipotent progenitors ( MPP ) , common lymphoid progenitors ( CLP ) , common myeloid progenitors ( CMP ) , megakaryocyte - erythroid progenitors ( MEP ) , and granulocyte - monocyte progenitors ( GMP ) Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s t - test ) C - D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells . Data information : Data were collected at month 7 post transplantation for the uMT - / - group and month 8 post - transplantation for the NSG group , and presented as mean ± SEM . n = 7 mice for the uMT - / - group and n = 6 for the NSG group . * p < 0 . 05 and * * p < 0 . 01 ( two - tailed and two - sample equal variance Student " s t - test )

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4 A-B Total amount of progenitors as a percentage of ckit and Il7rα enriched bone marrow (BM) cells. Shown are hematopoietic stem cells (HSCs), Flk2- and Flk2+ multipotent progenitors (MPP), common lymphoid progenitors (CLP), common myeloid progenitors (CMP), megakaryocyte-erythroid progenitors (MEP), and granulocyte-monocyte progenitors (GMP) Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)  C-D WT donor derived progenitors as a percentage of ckit and Il7rα enriched BM cells. Data information: Data were collected at month 7 post transplantation for the uMT-/- group and month 8 post-transplantation for the NSG group, and presented as mean ± SEM. n=7 mice for the uMT-/- group and n=6 for the NSG group. *p<0.05 and **p< 0.01 (two-tailed and two-sample equal variance Student"s t-test)


f1a , Immunoprecipitation of Flag - beclin 1 constructs with Nef - HA in HeLa cells 24 h post - transfection . b , GFP - LC3 - positive dots ( autophagosomes ) in MCF7 cells expressing GFP - LC3 and Flag - beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h . d , Biochemical assessment of autophagy ( p62 and LC3 immunoblots ) in peptide - treated HeLa cells ( 3 h ) . e , f , Representative images ( e ) and quantification of GFP - LC3 - positive dots ( f ) in peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) . Scale bars , 20 µm . g , GFP - LC3 - positive dots in siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 30 µM , 3 h ) . h , Model of Tat - beclin 1 peptide ( left ) based on corresponding elements of the beclin 1 evolutionarily conserved domain ( ECD ) structure ( centre ) . Essential phenylalanine side chains , magenta ; positions of solubility mutations , pink ; lipid interaction site , yellow . ECD surface representation ( right ) illustrates exposure of corresponding peptide ( cyan ) . i , p62 and LC3 immunoblots in peptide - treated HeLa cells ( 3 h ) . In b , f , g , bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . * P 0 . 05 , * * P 0 . 01 ; t - test .

B-PANEL_START 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 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 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 O O O O O

f1a, Immunoprecipitation of Flag-beclin 1 constructs with Nef-HA in HeLa cells 24 h post-transfection.b, GFP-LC3-positive dots (autophagosomes) in MCF7 cells expressing GFP-LC3 and Flag-beclin 1 constructs grown in either normal medium or starved in EBSS for 2 h.d, Biochemical assessment of autophagy (p62 and LC3 immunoblots) in peptide-treated HeLa cells (3 h).e, f, Representative images (e) and quantification of GFP-LC3-positive dots (f) in peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h). Scale bars, 20 µm.g, GFP-LC3-positive dots in siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (30 µM, 3 h).h, Model of Tat-beclin 1 peptide (left) based on corresponding elements of the beclin 1 evolutionarily conserved domain (ECD) structure (centre). Essential phenylalanine side chains, magenta; positions of solubility mutations, pink; lipid interaction site, yellow. ECD surface representation (right) illustrates exposure of corresponding peptide (cyan).i, p62 and LC3 immunoblots in peptide-treated HeLa cells (3 h). In b, f, g, bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. *P 0.05, **P 0.01; t-test.


f2a , HeLa cells were treated with biotin - conjugated peptides ( 30 μM , 3 h ) and proteins bound to peptides were analysed by immunoblot with anti - GAPR - 1 . b - T - B , biotin - Tat - beclin 1 ; b - T - S , biotin - Tat - scrambled . b , Immunoprecipitation of Flag - beclin 1 with GAPR - 1 - Myc in HeLa cells 24 h post - transfection . c , GFP - LC3 - positive dots in GAPR - 1 siRNA - transfected peptide - treated HeLa / GFP - LC3 cells ( 20 & amp ; amp ; mgr ; M , 3 h ) with or without 100 nM bafilomycin A1 . Bars represent mean ± s . e . m . of triplicate samples ( 50 - 100 cells per sample ) . Similar results were observed in three independent experiments . NC , non - silencing control . d , Localization of beclin 1 and GM130 ( a Golgi marker ) in HeLa cells stably transduced with empty vector ( left panel ) or GAPR - 1 - Myc ( middle panel ) or transfected with GAPR - 1 siRNA ( right panel ) and treated with peptide ( 20 μM , 1 h ) . Scale bar , 20 μm . e , Immunoblot of beclin 1 in post - nuclear supernatant ( PNS ) and Golgi - enriched fractions in HeLa cells stably transduced with empty vector or GAPR - 1 - Myc after peptide treatment ( 20 μM , 2 h ) . f , WIPI2 dots in cells in the experimental conditions shown in d . Bars represent mean ± s . e . m . for 100 - 150 cells . * P 0 . 05 , * * * P 0 . 001 ; t - test . † † † P 0 . 001 ; two - way ANOVA for comparison of magnitude of changes between groups .

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 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 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 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 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

f2a, HeLa cells were treated with biotin-conjugated peptides (30 μM, 3 h) and proteins bound to peptides were analysed by immunoblot with anti-GAPR-1. b-T-B, biotin-Tat-beclin 1; b-T-S, biotin-Tat-scrambled.b, Immunoprecipitation of Flag-beclin 1 with GAPR-1-Myc in HeLa cells 24 h post-transfection.c, GFP-LC3-positive dots in GAPR-1 siRNA-transfected peptide-treated HeLa/GFP-LC3 cells (20 &amp;mgr;M, 3 h) with or without 100 nM bafilomycin A1. Bars represent mean ± s.e.m. of triplicate samples (50-100 cells per sample). Similar results were observed in three independent experiments. NC, non-silencing control.d, Localization of beclin 1 and GM130 (a Golgi marker) in HeLa cells stably transduced with empty vector (left panel) or GAPR-1-Myc (middle panel) or transfected with GAPR-1 siRNA (right panel) and treated with peptide (20 μM, 1 h). Scale bar, 20 μm.e, Immunoblot of beclin 1 in post-nuclear supernatant (PNS) and Golgi-enriched fractions in HeLa cells stably transduced with empty vector or GAPR-1-Myc after peptide treatment (20 μM, 2 h).f, WIPI2 dots in cells in the experimental conditions shown in d. Bars represent mean ± s.e.m. for 100-150 cells. *P 0.05, ***P 0.001; t-test. †††P 0.001; two-way ANOVA for comparison of magnitude of changes between groups.


f3a , Percentage of cells with small htt103Q aggregates ( left ) and number of aggregates per cell ( right ) in HeLa cells expressing doxycycline ( Dox ) - repressible CFP - htt103Q after daily treatment with doxycycline or peptide ( 20 μM , 4 h per day ) for 2 days . Bars represent mean ± s . e . m . of triplicate samples ( 60 - 120 cells per sample ) . Similar results were observed in three independent experiments . b , Filter trap assays for htt103Q large and small aggregates in HeLa / htt103Q cells . c , Viral titres in HeLa cells infected with 0 . 1 plaque - forming units ( p . f . u . ) per cell of SINV , CHIKV or WNV ( strain TX02 ) and treated with peptide ( 10 μM , 4 - 8 h post - infection ) . Values represent geometric mean ± s . e . m . for triplicate samples of supernatants collected 18 h post - infection ( SINV ) or 24 h post - infection ( CHIKV and WNV ) . Similar results were observed in three independent experiments . d , Bacteriacolony - forming units ( c . f . u . ) in primary BMDMs infected with L . monocytogenes ΔactA mutant strain DPL - 4029 for 30 min and treated with peptide ( 10 µM from 0 to 2 h post - infection ) . Bars represent mean ± s . e . m . of triplicate samples . Similar results were observed in three independent experiments . e , HIV - 1 p24 antigen release in primary human MDMs infected with HIV - 1 24 h after initiation of daily peptide treatment . Values represent mean ± s . e . m . of triplicate samples . Similar results were observed in MDMs from three independent donors . f , HIV - 1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA ( shNS ) or ATG5 shRNA ( shATG5 ) and treated daily with peptide ( 5 µM ) . Values represent mean ± s . e . m . of triplicate wells . Similar results were observed in MDMs from two independent donors . g , LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV - 1 infection . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; t - test .

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 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 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 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

f3a, Percentage of cells with small htt103Q aggregates (left) and number of aggregates per cell (right) in HeLa cells expressing doxycycline (Dox)-repressible CFP-htt103Q after daily treatment with doxycycline or peptide (20 μM, 4 h per day) for 2 days. Bars represent mean ± s.e.m. of triplicate samples (60-120 cells per sample). Similar results were observed in three independent experiments.b, Filter trap assays for htt103Q large and small aggregates in HeLa/htt103Q cells.c, Viral titres in HeLa cells infected with 0.1 plaque-forming units (p.f.u.) per cell of SINV, CHIKV or WNV (strain TX02) and treated with peptide (10 μM, 4-8 h post-infection). Values represent geometric mean ± s.e.m. for triplicate samples of supernatants collected 18 h post-infection (SINV) or 24 h post-infection (CHIKV and WNV). Similar results were observed in three independent experiments.d, Bacteriacolony-forming units (c.f.u.) in primary BMDMs infected with L. monocytogenes ΔactA mutant strain DPL-4029 for 30 min and treated with peptide (10 µM from 0 to 2 h post-infection). Bars represent mean ± s.e.m. of triplicate samples. Similar results were observed in three independent experiments.e, HIV-1 p24 antigen release in primary human MDMs infected with HIV-1 24 h after initiation of daily peptide treatment. Values represent mean ± s.e.m. of triplicate samples. Similar results were observed in MDMs from three independent donors.f, HIV-1p24 antigen release in MDMs transduced with nonspecific scrambled shRNA (shNS) or ATG5 shRNA (shATG5) and treated daily with peptide (5 µM). Values represent mean ± s.e.m. of triplicate wells. Similar results were observed in MDMs from two independent donors.g, LC3 immunoblot of MDMs transduced with the indicated shRNA at day 0 and day 10 after HIV-1 infection. *P  0.05; **P  0.01; ***P  0.001; t-test.


f4a , GFP - LC3 - positive dots in tissues of 6 - week - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . A minimum of ten fields was counted per tissue section . Bars represent mean ± s . e . m . for three mice . Similar results were observed in three independent experiments . b , p62 immunoblot of brains of 5 - day - old GFP - LC3 mice treated with the indicated peptide ( 20 mg kg − 1 i . p . , 6 h ) . c , Survival curves of 5 - day - old C57BL / 6J mice infected with CHIKV ( 106 p . f . u . s . c . ) and treated daily with peptide ( 15 mg kg − 1 i . p . beginning 1 day post - infection ) . d - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice . gd - f , Representative images of WNV envelope antigen and TdT - mediated dUTP nick end labelling ( TUNEL ) staining ( d ) ( T , Tat alone ) , quantification of cell death in brain ( e ) , and survival curves ( f ) for 5 - day - oldC57BL / 6Jmice infected with WNV ( Egypt strain 101 , 1 p . f . u . intracerebral ( i . c . ) ) and treated daily with peptide ( D - amino acid forms , 20mgkg − 1 i . p . beginning 1day post - infection ) . Images in d are from cerebral cortex day 6 post - infection . Similar results were observed in all regions of the brain for three mice per group . Scale bar , 20μm . Bars in e represent mean ± s . e . m . TUNEL - positive cells per unit area of brain for three mice . gg , Geometric mean + s . e . m . viral titres of WNV - infected mousebrains day 6 post - infection . Values represent combined data for 12 - 20 mice per treatment group from 10 to 12 litters . Data in c and f represent combined survival probabilities for three and four independent litters , respectively , in each group . Similar results were observed in each independent experiment . * P 0 . 05 ; * * P 0 . 01 ; * * * P 0 . 001 ; NS , not significant ; t - test .

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 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 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 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 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

f4a, GFP-LC3-positive dots in tissues of 6-week-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h). A minimum of ten fields was counted per tissue section. Bars represent mean ± s.e.m. for three mice. Similar results were observed in three independent experiments.b, p62 immunoblot of brains of 5-day-old GFP-LC3 mice treated with the indicated peptide (20 mg kg−1 i.p., 6 h).c, Survival curves of 5-day-old C57BL/6J mice infected with CHIKV (106 p.f.u. s.c.) and treated daily with peptide (15 mg kg−1 i.p. beginning 1 day post-infection).d-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e) and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. gd-f, Representative images of WNV envelope antigen and TdT-mediated dUTP nick end labelling (TUNEL) staining (d) (T, Tat alone), quantification of cell death in brain (e), and survival curves (f) for 5-day-oldC57BL/6Jmice infected with WNV (Egypt strain 101, 1 p.f.u. intracerebral (i.c.)) and treated daily with peptide (D-amino acid forms, 20mgkg−1 i.p. beginning 1day post-infection). Images in d are from cerebral cortex day 6 post-infection. Similar results were observed in all regions of the brain for three mice per group. Scale bar, 20μm. Bars in e represent mean±s.e.m. TUNEL-positive cells per unit area of brain for three mice. gg, Geometric mean + s.e.m. viral titres of WNV-infected mousebrains day 6 post-infection. Values represent combined data for 12-20 mice per treatment group from 10 to 12 litters. Data in c and f represent combined survival probabilities for three and four independent litters, respectively, in each group. Similar results were observed in each independent experiment. *P 0.05; **P 0.01; ***P 0.001; NS, not significant; t-test.


Figure 2A - B Clearance of a target with a consensus PAM by direct interference . Overlay of fluorescent and phase contrast time - lapse images . Presence of the target plasmid is tracked by its YFP production ( A ) . Reconstructed lineage traces of the imaged population ( A ) from induction of the CRISPR - Cas system over time ( grey ) lineages show some variation in plasmid clearance times ( coloured ) ( B ) . E - F Clearance of a matching target with a mutated PAM via priming . Overlay of fluorescent and phase contrast time - lapse images as in ( A ) . Reconstructed lineage traces of the imaged population ( E ) ( grey ) . Lineages show large variations in the time taken to clear the plasmid ( coloured ) ( F ) .

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 O O O O O O O O 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 2A-B Clearance of a target with a consensus PAM by direct interference. Overlay of fluorescent and phase contrast time-lapse images. Presence of the target plasmid is tracked by its YFP production (A). Reconstructed lineage traces of the imaged population (A) from induction of the CRISPR-Cas system over time (grey) lineages show some variation in plasmid clearance times (coloured) (B).E-F Clearance of a matching target with a mutated PAM via priming. Overlay of fluorescent and phase contrast time-lapse images as in (A). Reconstructed lineage traces of the imaged population (E) (grey). Lineages show large variations in the time taken to clear the plasmid (coloured) (F).


Figure 1 ( C ) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A ( ant . A ) . ( D , F ) Mean FRET efficiency measured over time in the ( D ) AP boundary ( APB ) or ( F ) DV boundary ( DVB ) and non - organizer regions ( NO ) . Shaded regions indicate standard deviation ( SD ) ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . Black arrows indicate the addition of the drug .

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Figure 1(C) Timelapse of ATP sensor FRET efficiency across the wing pouch after addition of 10 μM antimycin A (ant.A).(D, F) Mean FRET efficiency measured over time in the (D) AP boundary (APB) or (F) DV boundary (DVB) and non-organizer regions (NO). Shaded regions indicate standard deviation (SD); dots indicate mean per timepoint, and solid lines indicate the fit to the mean. Black arrows indicate the addition of the drug.


Figure 2 ( A ) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A ( ant . A ) , 100 μM 3 - Bromopyruvate ( 3BP ) , and 50 mM 2 - deoxy - D - glucose ( 2DG ) . ( B ) Mean FRET efficiency of organizer ( ORG ) regions and non - organizer ( NO ) regions analyzed over time . Shaded regions indicate SD ; dots indicate mean per timepoint , and solid lines indicate the fit to the mean . ( C ) Fit parameters of individual time traces for organizer and non - organizer regions . Each dot represents data from one disc , and lines connect the corresponding regions of the same disc . Box plots summarize the data : boxes encompass the 2nd - 3rd quartiles , with whiskers indicating the 1st and 4th quartiles and the red line indicating the median . * = p - value < 0 . 05 , ns = not significant p - value using a Kruskal - Wallis test ( n = 26 discs ) . ( D ) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition . ( E ) Mean FRET efficiency time trace of the entire wing pouch ; shaded region indicates SD ; black arrows indicate the addition of the drugs . Brackets indicate the mean FRET values before and 2h after 2DG addition ( 1 - 10 min and 121 - 130 min , respectively ) . The distribution of mean values within these brackets are shown as violin plots in ( F ) ( solid line indicates median , dotted indicate quartiles ) . These were compared using an unpaired t - test ( ns = not significant p - value , n = 17 discs ) including ( G ) Welch ' s correction and estimation plot ( n = 17 discs for each group ) . In ( G ) , horizontal dotted lines run through the mean values for the two samples ( before 2DG and 2hr after 2DG ) ; on the right , the difference between the two means is plotted on a separate axis ( right ) , where the bars indicate the upper and lower 95 % confidence intervals .

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 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

Figure 2(A) Timelapse of ATP sensor FRET efficiency after combined addition of 10 μM antimycin A (ant.A), 100 μM 3-Bromopyruvate (3BP), and 50 mM 2-deoxy-D-glucose (2DG). (B) Mean FRET efficiency of organizer (ORG) regions and non-organizer (NO) regions analyzed over time. Shaded regions indicate SD; dots indicate mean per timepoint, and solid lines indicate the fit to the mean. (C) Fit parameters of individual time traces for organizer and non-organizer regions. Each dot represents data from one disc, and lines connect the corresponding regions of the same disc. Box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median. * = p-value < 0.05, ns = not significant p-value using a Kruskal-Wallis test (n=26 discs).(D) Time lapse montage of ATP sensor FRET efficiency in the wing pouch after 2DG addition. (E) Mean FRET efficiency time trace of the entire wing pouch; shaded region indicates SD; black arrows indicate the addition of the drugs. Brackets indicate the mean FRET values before and 2h after 2DG addition (1-10 min and 121-130 min, respectively). The distribution of mean values within these brackets are shown as violin plots in (F) (solid line indicates median, dotted indicate quartiles). These were compared using an unpaired t-test (ns = not significant p-value, n = 17 discs) including (G) Welch's correction and estimation plot (n=17 discs for each group). In (G), horizontal dotted lines run through the mean values for the two samples (before 2DG and 2hr after 2DG); on the right, the difference between the two means is plotted on a separate axis (right), where the bars indicate the upper and lower 95% confidence intervals.


Figure 3 ( C ) Timelapse of ATP sensor FRET efficiency in the apGalts > PtcRNAi wing pouch after 10 μM antimycin A ( ant . A ) addition . Red dashed line indicates the position of the AP boundary . ( D , F ) Mean FRET efficiency analyzed over time in the ( D ) dorsal anterior ( DA ) and ventral anterior ( VA ) sub - compartments or the ( F ) dorsal anterior ( DA ) and dorsal posterior ( DP ) sub - compartments of apGalts > PtcRNAi wing discs . Shaded regions indicate SD ; dots represent the mean per timepoint ; solid lines illustrate a fit of the mean data . ( I ) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition . ( J ) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings . Shaded regions indicate SD ; dots represent the average per timepoint ; solid lines illustrate a fit of the mean data .

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 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 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

Figure 3(C) Timelapse of ATP sensor FRET efficiency in the apGalts>PtcRNAi wing pouch after 10 μM antimycin A (ant.A) addition. Red dashed line indicates the position of the AP boundary.(D, F) Mean FRET efficiency analyzed over time in the (D) dorsal anterior (DA) and ventral anterior (VA) sub-compartments or the (F) dorsal anterior (DA) and dorsal posterior (DP) sub-compartments of apGalts>PtcRNAi wing discs. Shaded regions indicate SD; dots represent the mean per timepoint; solid lines illustrate a fit of the mean data.(I) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition.(J) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings. Shaded regions indicate SD; dots represent the average per timepoint; solid lines illustrate a fit of the mean data.


Figure 4 ( A ) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants . To highlight the variability in this experiment , we show example discs from two separate experiments . ( B , D ) Mean FRET efficiency measured over time in non - organizers compared to ( B ) Anterior - Posterior boundary ( APB ) or ( D ) all organizer regions . Shaded regions indicate SD ; dots are the means per each timepoint , and the solid lines indicate the fit to the mean .

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

Figure 4(A) Timelapse montage of ATP sensor FRET efficiency upon induction of hypoxia in wing disc explants. To highlight the variability in this experiment, we show example discs from two separate experiments.(B, D) Mean FRET efficiency measured over time in non-organizers compared to (B) Anterior-Posterior boundary (APB) or (D) all organizer regions. Shaded regions indicate SD; dots are the means per each timepoint, and the solid lines indicate the fit to the mean.


Figure 1C DNA breaks were measured using alkaline comet assay in BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with 2 µM pyridostatin ( PDS ) for 16 hours and released into fresh medium without pyridostatin . Representative images are shown . Scale bar represents 100 µm . D Quantification of DNA breaks shown in ( C ) . Graph and error bars represent the mean and SEM of n = 3 independent experiments . A minimum of 50 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; NS , P > 0 . 5 . E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in ( C ) . A minimum of 200 cells were analysed per condition per experiment . Graph and error bars represent the mean and SEM of n = 3 independent experiments . P values were calculated using an unpaired two - tailed t - test . * * * , P ≤ 0 . 001 ; NS , P > 0 . 5 . F BRCA2 - deficient ( - BRCA2 ) human DLD1 cells were treated with 2 µM pyridostatin ( PDS ) for 24 hours . Next , pyridostatin was removed and cells were released in a medium containing 250 nM NU - 7441 ( NU ) . Whole - cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown . SMC1 was used as a loading control . KAP1 phosphorylation site is indicated in red . G Dose - dependent viability assays of BRCA2 - proficient ( + BRCA2 ) or - deficient ( - BRCA2 ) human DLD1 cells treated with pyridostatin ( PDS ) and NU - 7441 at the indicated concentrations for six days . Graphs represent average values obtained from of n = 3 independent experiments , each performed in technical triplicates .

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 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 O O O O O 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 1C DNA breaks were measured using alkaline comet assay in BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with 2 µM pyridostatin (PDS) for 16 hours and released into fresh medium without pyridostatin. Representative images are shown. Scale bar represents 100 µm. D Quantification of DNA breaks shown in (C). Graph and error bars represent the mean and SEM of n = 3 independent experiments. A minimum of 50 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; NS, P > 0.5. E Quantification of γH2AX foci visualised using immunofluorescence staining in cells treated as in (C). A minimum of 200 cells were analysed per condition per experiment. Graph and error bars represent the mean and SEM of n = 3 independent experiments. P values were calculated using an unpaired two-tailed t-test. ***, P ≤ 0.001; NS, P > 0.5.F BRCA2-deficient (-BRCA2) human DLD1 cells were treated with 2 µM pyridostatin (PDS) for 24 hours. Next, pyridostatin was removed and cells were released in a medium containing 250 nM NU-7441 (NU). Whole-cell extracts were prepared at the indicated timepoints after release and immunoblotted as shown. SMC1 was used as a loading control. KAP1 phosphorylation site is indicated in red.G Dose-dependent viability assays of BRCA2-proficient (+BRCA2) or -deficient (-BRCA2) human DLD1 cells treated with pyridostatin (PDS) and NU-7441 at the indicated concentrations for six days. Graphs represent average values obtained from of n = 3 independent experiments, each performed in technical triplicates.


Figure 2Quantitative RT - PCR of cells grown as in ( A ) and treated with 10 µM pyridostatin ( PDS ) for 3 days was performed using primers specific for the indicated genes . mRNA levels are expressed relative to GAPDH and to - DOX ( + BRCA2 ) cells . Error bars represent the SEM of n = 3 independent experiments , each performed in technical triplicate . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * * , P ≤ 0 . 001 . BRCA2 + / + and BRCA2 - / - RPE - 1 cells were treated with 10 μM pyridostatin ( PDS ) for 2 days . Whole - cell extracts were immunoblotted as indicated . KAP1 , IRF3 and STAT1 phosphorylation sites are shown in red . Tubulin and GAPDH were used as loading controls . H1299 + shBRCA2DOX cells treated as in ( B ) were fixed and prepared for immunofluorescence with antibody against cGAS . DNA was counterstained with DAPI . Scale bar represents 20 µm . Quantification of cGAS - positive micronuclei per cells shown in ( E ) . Graph and error bars represent the mean and SEM of of n = 3 independent experiments . A minimum of 250 cells were analysed per condition per experiment . P values were calculated using an unpaired two - tailed t - test . * , P ≤ 0 . 05 ; * * , P ≤ 0 . 01 ; NS , P > 0 . 5 .

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Figure 2Quantitative RT-PCR of cells grown as in (A) and treated with 10 µM pyridostatin (PDS) for 3 days was performed using primers specific for the indicated genes. mRNA levels are expressed relative to GAPDH and to -DOX (+BRCA2) cells. Error bars represent the SEM of n = 3 independent experiments, each performed in technical triplicate. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; ***, P ≤ 0.001.BRCA2+/+ and BRCA2-/- RPE-1 cells were treated with 10 μM pyridostatin (PDS) for 2 days. Whole-cell extracts were immunoblotted as indicated. KAP1, IRF3 and STAT1 phosphorylation sites are shown in red. Tubulin and GAPDH were used as loading controls.H1299+shBRCA2DOX cells treated as in (B) were fixed and prepared for immunofluorescence with antibody against cGAS. DNA was counterstained with DAPI. Scale bar represents 20 µm. Quantification of cGAS-positive micronuclei per cells shown in (E). Graph and error bars represent the mean and SEM of of n = 3 independent experiments. A minimum of 250 cells were analysed per condition per experiment. P values were calculated using an unpaired two-tailed t-test. *, P ≤ 0.05; **, P ≤ 0.01; NS, P > 0.5.