Figure 5A . Comparison of LPS and full length S1 spike protein - mediated changes in cytokines in THP1 macrophages . Data information , , data are shown as the mean of three technical replicates . Error bars denote SEM . D . Representative H & E images showing Ponatinib alleviates LPS - induced inflammatory cell infiltration , septal thickening , alveolar edema in mouse lungs . Scale bars 100 µm ( upper ) and 50 µm ( lower ) . E . Plots showing pre - treatment ( 60 min ) with Ponatinib ( 35 mg / Kg ) inhibits LPS - induced cytokine release in BALF . Cytokine levels were measured using Luminex . Data information , data are mean of five biological replicates from two independent studies , error bars denote SEM . ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 , Welch ' s t - test .

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Figure 5A. Comparison of LPS and full length S1 spike protein-mediated changes in cytokines in THP1 macrophages. Data information, , data are shown as the mean of three technical replicates. Error bars denote SEM.D. Representative H&E images showing Ponatinib alleviates LPS-induced inflammatory cell infiltration, septal thickening, alveolar edema in mouse lungs. Scale bars 100 µm (upper) and 50 µm (lower).E. Plots showing pre-treatment (60 min) with Ponatinib (35 mg/Kg) inhibits LPS-induced cytokine release in BALF. Cytokine levels were measured using Luminex. Data information, data are mean of five biological replicates from two independent studies, error bars denote SEM. ∗p < 0.05, ∗∗p < 0.01, Welch's t-test.


Figure 1 ( A ) CP20 and OVCAR4 cells were transfected with either non - target miRNA control ( miR - CTL ) , miR - 15a , or miR - 195 . 72h following transfection , cells were lysed and immunoblotted for detection of MICU1 , BMI1 , BCL2 , and MFN2 . GAPDH and VDAC were used as loading control . ( B ) miR - 195 expression in FTE188 and ovarian cell lines as determined by RT - qPCR normalized with U6 . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test . ( C ) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting . Actin is used as the loading control . ( D ) Anti - miR - 195 was transfected in the FTE188 cell line , level of miR - 195 was measured using RT - qPCR ( left ) and MICU1 levels were measured using immunoblotting ( right ) . Data are mean ± S . D . , n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test .

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 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 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

Figure 1(A) CP20 and OVCAR4 cells were transfected with either non-target miRNA control (miR-CTL), miR-15a, or miR-195. 72h following transfection, cells were lysed and immunoblotted for detection of MICU1, BMI1, BCL2, and MFN2. GAPDH and VDAC were used as loading control.(B) miR-195 expression in FTE188 and ovarian cell lines as determined by RT-qPCR normalized with U6. Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.(C) Expression of MICU1 in FTE188 and various ovarian cell lines as determined by immunoblotting. Actin is used as the loading control.(D) Anti-miR-195 was transfected in the FTE188 cell line, level of miR-195 was measured using RT-qPCR (left) and MICU1 levels were measured using immunoblotting (right). Data are mean ± S.D., n=3 (Biological repeats). *P<0.05, Student's t-test.


Figure 2 ( A , B ) CP20 , OVCAR4 , and OVSAHO cells were transfected with non - target micro RNA control ( miR - CTL ) or miR - 195 , 24 h post - transfection , cells were recounted and plated as single cells for anchorage - dependent ( A ) or anchorage - independent ( B ) clonal growth . After 10 ( CP20 ) or 14 ( OVCAR4 and OVSAHO ) days , colonies were quantified using an Optronix GelCount colony counter . The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control ( miR - CTL ) . ( C ) Migration of miR - CTL , miR - 195 , and miR - 195 + MICU1 ( pLYS1 - MICU1 - Flag ) transfected cells , towards serum gradient was examined and the number of cells per field was counted . The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control ( miR - CTL ) . ( D ) Invasion of miR - CTL , miR - 195 , and miR - 195 + MICU1 cells through fibronectin - coated filters was examined using Boyden chamber and number of cells per field was counted . Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control ( miR - CTL ) .

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 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

Figure 2(A, B) CP20, OVCAR4, and OVSAHO cells were transfected with non-target micro RNA control (miR-CTL) or miR-195, 24 h post-transfection, cells were recounted and plated as single cells for anchorage-dependent (A) or anchorage-independent (B) clonal growth. After 10 (CP20) or 14 (OVCAR4 and OVSAHO) days, colonies were quantified using an Optronix GelCount colony counter. The left panel shows representative images of the colonies and the right panel depicts a graphical representation of data presented as percent clonal growth relative to the control (miR-CTL).(C) Migration of miR-CTL, miR-195, and miR-195+ MICU1 (pLYS1-MICU1-Flag) transfected cells, towards serum gradient was examined and the number of cells per field was counted. The left panel shows representative images and the right panel depicts a graphical representation of relative migration compared to the control (miR-CTL).(D) Invasion of miR-CTL, miR-195, and miR-195 + MICU1 cells through fibronectin-coated filters was examined using Boyden chamber and number of cells per field was counted. Left panel shows representative images and the right panel depicts a graphical representation of relative invasion compared to the control (miR-CTL).


Figure 3 ( B ) MICU1 3 ' UTR in LightSwitch ™ 3 ′ UTR Reporter Vector ( wild type or miR - 195 binding site deleted ) was co - transfected with either non - target miR control ( miR - CTL ) or different concentrations of miR - 195 . Post 24h transfection luciferase ( Renilla ) activity was measured . Relative light units ( RLU ) compared to miR - CTL were plotted . Data are mean ± S . D . n = 3 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test . CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in miR - CTL and miR - 195 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated . CP20 cells were transfected as indicated , 48 h post transfection 4x106 cells were permeabilized , and extra - mitochondrial calcium ( [ Ca2 + ] out ) clearance was measured , representative traces of [ Ca2 + ] out ‑ clearance in control siRNA ( siCTL ) and siMICU1 transfected cells . [ Ca2 + ] out pulses and FCCP were delivered as indicated . ( E ) Bar graph illustrating the number of Ca2 + pulses handled by control miR , miR - 195 , siCTL and siMICU1 transfected cells . Mean ± SEM ; n = 3 - 4 ( Biological repeats ) . * P < 0 . 05 , Student ' s t - test . ( F ) Representative [ Ca2 + ] ( m ) traces after addition of FCCP ( 10 µM ) in permeabilized CP20 cells transfected with non - target miR - CTL or miR - 195 . ( G ) Quantification of resting matrix [ Ca2 + ] ( m ) after addition of FCCP . n = 5 ( Biological repeats ) * P < 0 . 05 , Student ' s t - test , ( H ) Representative traces of mitochondrial membrane potential ( ∆ Ψμ ) in CP20 cells transfected with miR - CTL or miR - 195 . ( I ) Quantification of relative ∆ Ψμ . ( n = 5 , Biological repeats ) . ( J ) Intracellular lactate was measured in miR - CTL , miR - 195 , siCTL , and siMICU1 expressing OVCAR4 , CP20 , and OVSAHO cells . n = 3 , biological repeats * P < 0 . 05 , Student ' s t - test . ( K & L ) miR - CTL , miR - 195 , or miR - 195 + pLYS1 - MICU1 - Flag transfected cells were stained with MitoSOX Red , and mitochondrial ROS levels were determined by flow cytometry . The histogram shows representative staining and bar graph ( right ) shows results of three independent experiments . Mean ± SEM ; . n = 3 , biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

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 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 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 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 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 O O O O O O O O O O O

Figure 3(B) MICU1 3'UTR in LightSwitch™ 3′UTR Reporter Vector (wild type or miR-195 binding site deleted) was co-transfected with either non- target miR control (miR-CTL) or different concentrations of miR-195. Post 24h transfection luciferase (Renilla) activity was measured. Relative light units (RLU) compared to miR-CTL were plotted. Data are mean ± S.D. n=3 (Biological repeats). *P<0.05, Student's t-test.CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in miR-CTL and miR-195 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.CP20 cells were transfected as indicated, 48 h post transfection 4x106 cells were permeabilized, and extra-mitochondrial calcium ([Ca2+]out) clearance was measured, representative traces of [Ca2+]out‑ clearance in control siRNA (siCTL) and siMICU1 transfected cells. [Ca2+]out pulses and FCCP were delivered as indicated.(E) Bar graph illustrating the number of Ca2+ pulses handled by control miR, miR-195, siCTL and siMICU1 transfected cells. Mean ±SEM; n=3-4 (Biological repeats). *P<0.05, Student's t-test.(F) Representative [Ca2+](m) traces after addition of FCCP (10 µM) in permeabilized CP20 cells transfected with non-target miR-CTL or miR-195. (G) Quantification of resting matrix [Ca2+](m) after addition of FCCP. n=5 (Biological repeats) *P<0.05, Student's t-test, (H) Representative traces of mitochondrial membrane potential (∆Ψμ) in CP20 cells transfected with miR-CTL or miR-195. (I) Quantification of relative ∆Ψμ. (n=5, Biological repeats). (J) Intracellular lactate was measured in miR-CTL, miR-195, siCTL, and siMICU1 expressing OVCAR4, CP20, and OVSAHO cells. n=3, biological repeats *P<0.05, Student's t-test.(K&L) miR-CTL, miR-195, or miR-195 + pLYS1-MICU1-Flag transfected cells were stained with MitoSOX Red, and mitochondrial ROS levels were determined by flow cytometry. The histogram shows representative staining and bar graph (right) shows results of three independent experiments. Mean ± SEM; . n=3, biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


Figure 4 ( A & B ) Stable cell lines for CP20 and OVCAR4 expressing GFP - miR - 195 or non - target miR - GFP were generated using lentiviral mediated transduction and were selected using puromycin ; representative images are shown . Scale bars , 50 µm . ( C ) Relative miR - 195 expression was measured using RT - qPCR in stable cell lines , normalized with U6 . Mean ± SEM ; n = 3 , Biological repeats . * P < 0 . 05 , Student ' s t - test . ( D ) CP20 and OVCAR4 stable cell lines were transfected as either empty vector ( EV ) or pLYS1 - MICU1 - Flag ( MICU1 ) and relative MICU1 levels were evaluated using immunoblotting . ( E , F ) CP20 and OVCAR4 stable cell lines transfected as in ( D ) were counted and re - plated for anchorage - dependent ( E ) and independent clonal growth ( F ) . Quantification compared to the miR - CTL stable cells transfected with EV is shown . Mean ± SD ; n = 3 , Biological repeats . * P < 0 . 05 when comparing with miR - CTL , # P < 0 . 05 when comparing with miR - 195 by Student ' s t - test .

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 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 O O O O O O O O O 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) Stable cell lines for CP20 and OVCAR4 expressing GFP-miR-195 or non-target miR-GFP were generated using lentiviral mediated transduction and were selected using puromycin; representative images are shown. Scale bars, 50 µm.(C) Relative miR-195 expression was measured using RT-qPCR in stable cell lines, normalized with U6. Mean ± SEM; n=3, Biological repeats. *P<0.05, Student's t-test.(D) CP20 and OVCAR4 stable cell lines were transfected as either empty vector (EV) or pLYS1-MICU1-Flag (MICU1) and relative MICU1 levels were evaluated using immunoblotting.(E, F) CP20 and OVCAR4 stable cell lines transfected as in (D) were counted and re-plated for anchorage-dependent (E) and independent clonal growth (F). Quantification compared to the miR-CTL stable cells transfected with EV is shown. Mean ± SD; n=3, Biological repeats. *P<0.05 when comparing with miR-CTL, # P<0.05 when comparing with miR-195 by Student's t-test.


Figure 5 ( A ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 cells . Mice injected with the miR - 195 expressing cells ( miR - 195 ) had significantly smaller tumor volumes than control mice ( miR - CTL ) . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test . ( B ) Tumor doubling time was significantly longer in the mice injected with miR - 195 expressing cells than in control mice . Data are mean ± S . D , n = 10 . * P < 0 . 05 , Student ' s t - test . ( C ) Percent survival based on humane endpoint criteria , was calculated by the Kaplan - Meier method and P values determined by Log Rank Test . ( D ) Four tumor samples from each group were analyzed for the expression of MICU1 , pPDH , PDH , PARP , and BCL2 using immunoblotting . α tubulin was used as the loading control . Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( E ) , CD31 positive vessels ( Scale Bar = 100 μm ) and TUNEL staining ( Scale Bar = 50 μm ) Representative histology images of tumors from mice xenografts of CP20 - GPF - miR - CTL ( miR - CTL ) or CP20 - GPF - miR - 195 ( miR - 195 ) cells with Ki67 expression ( Scale Bar = 50 μm ) ( F ) graph showing quantification of each marker in tumors from the experimental mice ; values are mean ± SD , n = 10 , * P < 0 . 05 , Student ' s t - test . ( G ) Female athymic mice were injected subcutaneously with either CP20 GFP - miR - CTL or CP20 - GFP - miR - 195 or CP20 - GFP - miR - 195 + MICU1 cells and tumor volume was measured , values are mean ± SE , n = 10 . * P < 0 . 05 , Student ' s t - test .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O 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

Figure 5(A) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 cells. Mice injected with the miR-195 expressing cells (miR-195) had significantly smaller tumor volumes than control mice (miR-CTL). Data are mean ± S.D, n=10. *P<0.05, Student's t-test.(B) Tumor doubling time was significantly longer in the mice injected with miR-195 expressing cells than in control mice. Data are mean ± S.D, n=10. *P<0.05, Student's t-test.(C) Percent survival based on humane endpoint criteria, was calculated by the Kaplan-Meier method and P values determined by Log Rank Test.(D) Four tumor samples from each group were analyzed for the expression of MICU1, pPDH, PDH, PARP, and BCL2 using immunoblotting. α tubulin was used as the loading control.Representative histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (E), CD31 positive vessels (Scale Bar = 100 μm) and TUNEL staining (Scale Bar = 50 μm)Representative histology images of tumors from mice xenografts of CP20-GPF-miR-CTL (miR-CTL) or CP20-GPF-miR-195 (miR-195) cells with Ki67 expression (Scale Bar = 50 μm) (F) graph showing quantification of each marker in tumors from the experimental mice; values are mean ±SD, n=10, *P<0.05, Student's t-test.(G) Female athymic mice were injected subcutaneously with either CP20 GFP-miR-CTL or CP20- GFP-miR-195 or CP20- GFP-miR-195 + MICU1 cells and tumor volume was measured, values are mean ±SE, n=10. *P<0.05, Student's t-test.


Figure 1Percentage of circulating highly differentiated CD4 T cells within CD4 cells ( upper graph ) in age - matched healthy donors or NSCLC patients before undergoing immunotherapies . G1 and G2 , groups of patients classified according to high THD cells ( G1 , > 40 % CD4 THD cells ) and low THD cells ( G2 , < 40 % CD4 THD cells ) . Relevant statistical comparisons are shown by the test of Mann - Whitney . In green , objective responders ( OR ) . In red , no OR . Below the graph , correlation of objective responses to G1 and G2 groups by the Fisher ´ s exact test . Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 ( blue ) or G2 ( red ) profile . Dotted lines represents the limit to define significant progression ( upper line ) or significant regression ( lower line ) . Kaplan - Meier plot for PFS in patients treated with immunotherapies stratified only by G1 ( green ) and G2 ( red ) CD4 T cell profiles . Patients starting therapy with a G2 profile had an overall response rate ( ORR ) of 0 % and 82 % of them experienced progression or death by week 9 . ORR was 44 . 8 % for G1 patients , and the 12 - week PFS was 50 . 2 % .

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 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

Figure 1Percentage of circulating highly differentiated CD4 T cells within CD4 cells (upper graph) in age-matched healthy donors or NSCLC patients before undergoing immunotherapies. G1 and G2, groups of patients classified according to high THD cells (G1, >40% CD4 THD cells) and low THD cells (G2, <40% CD4 THD cells). Relevant statistical comparisons are shown by the test of Mann-Whitney. In green, objective responders (OR). In red, no OR. Below the graph, correlation of objective responses to G1 and G2 groups by the Fisher´s exact test.Waterfall plot of change in lesion size in patients with measurable disease classified as having a G1 (blue) or G2 (red) profile. Dotted lines represents the limit to define significant progression (upper line) or significant regression (lower line).Kaplan-Meier plot for PFS in patients treated with immunotherapies stratified only by G1 (green) and G2 (red) CD4 T cell profiles. Patients starting therapy with a G2 profile had an overall response rate (ORR) of 0% and 82% of them experienced progression or death by week 9. ORR was 44.8% for G1 patients, and the 12-week PFS was 50.2%.


Figure 2The scatter plot shows PD - 1 expression after co - culture of CD4 T cells from healthy donors or NSCLC patients , as indicated , with A459 - SC3 lung cancer cells . Relevant statistical comparisons with the test of Mann - Whitney are indicated . Upper graphs , flow cytometry density plots of PD - 1 and LAG - 3 co - expression in CD4 T cells from healthy donors , a G1 responder ( G1 R ) , a G1 non - responder ( G1 NR ) and a G2 non - responder as indicated , following stimulation with A549 - SC3 cells . Percentage of expressing cells are indicated within each quadrant . Below , same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co - express PD - 1 and LAG - 3 . Relevant statistical comparisons are shown with the test of Mann - Whitney . Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right , after stimulation with A549 - SC3 cells . Vertical dotted line indicates the cut - off value of positive vs negative Ki67 expression . The percentage of Ki67 - expressing CD4 T cells is shown within the histograms . Below , same data represented as a scatter plot from a sample of G1 and G2 donors as indicated , with relevant statistical comparisons with the test Mann - Whitney . Proliferation of CD4 T cells stimulated by A549 - SC3 cells from the indicated patient groups . CD28 expression is shown together with the proliferation marker ki67 . Percentages of cells within each quadrant are shown . Same as in ( D ) but in the presence of an isotype control antibody or an anti - PD - 1 antibody with the equivalent sequence to pembrolizumab . The effects on CD4 T cells from a G1 and a G2 patient are shown , divided in CD28 high or low / negative subsets as indicated . Relevant statistical comparisons are shown with paired Student ´ s t test . Top , flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy , activated by incubation with A549 - SC3 cells . Below , same as above but as a dot - plot graph . A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples .

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Figure 2The scatter plot shows PD-1 expression after co-culture of CD4 T cells from healthy donors or NSCLC patients, as indicated, with A459-SC3 lung cancer cells. Relevant statistical comparisons with the test of Mann-Whitney are indicated.Upper graphs, flow cytometry density plots of PD-1 and LAG-3 co-expression in CD4 T cells from healthy donors, a G1 responder (G1 R), a G1 non-responder (G1 NR) and a G2 non-responder as indicated, following stimulation with A549-SC3 cells. Percentage of expressing cells are indicated within each quadrant. Below, same as in the upper graphs but as a scatter plot of the percentage of CD4 T cells that simultaneously co-express PD-1 and LAG-3. Relevant statistical comparisons are shown with the test of Mann-Whitney.Upper flow cytometry histograms of Ki67 expression in CD4 T cells from the representative subjects as indicated on the right, after stimulation with A549-SC3 cells. Vertical dotted line indicates the cut-off value of positive vs negative Ki67 expression. The percentage of Ki67-expressing CD4 T cells is shown within the histograms. Below, same data represented as a scatter plot from a sample of G1 and G2 donors as indicated, with relevant statistical comparisons with the test Mann-Whitney.Proliferation of CD4 T cells stimulated by A549-SC3 cells from the indicated patient groups. CD28 expression is shown together with the proliferation marker ki67. Percentages of cells within each quadrant are shown.Same as in (D) but in the presence of an isotype control antibody or an anti-PD-1 antibody with the equivalent sequence to pembrolizumab. The effects on CD4 T cells from a G1 and a G2 patient are shown, divided in CD28 high or low/negative subsets as indicated. Relevant statistical comparisons are shown with paired Student´s t test.Top, flow cytometry density plots of Ki67 expression in CD4 T cells from representative G1 or G2 patients after three cycles of therapy, activated by incubation with A549-SC3 cells. Below, same as above but as a dot-plot graph. A comparison between proliferating CD4 T cells before and after therapy is shown in unpaired patient samples.


Figure 3Scatter plot graph with the percentage of lung cancer - specific systemic CD4 T cells quantified by an autologous DC - based antigen presentation assay ( See Materials and Methods ) , in a sample of G1 and G2 patients as indicated . Objective responses ( OR ) are shown in green . In red , patients with no OR . The scatter plot graph on the left represents the percentage of CD4 THD cells within lung - cancer specific CD4 T cells in a sample of patients from the indicated G1 / G2 groups . On the right , same as left but representing the percentage of CD28 + CD4 T cells within lung - cancer specific CD4 T cells . Objective responders ( OR ) are shown in green .

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Figure 3Scatter plot graph with the percentage of lung cancer-specific systemic CD4 T cells quantified by an autologous DC-based antigen presentation assay (See Materials and Methods), in a sample of G1 and G2 patients as indicated. Objective responses (OR) are shown in green. In red, patients with no OR.The scatter plot graph on the left represents the percentage of CD4 THD cells within lung-cancer specific CD4 T cells in a sample of patients from the indicated G1/G2 groups. On the right, same as left but representing the percentage of CD28+ CD4 T cells within lung-cancer specific CD4 T cells. Objective responders (OR) are shown in green.


Figure 4Column graphs representing the percentage of CD4 T cells from NSCLC patients or age - matched healthy donors as represented in the graph , expressing the indicated cytokines after T cell stimulation with anti - CD3 / anti - CD28 antibodies . Data on total CD4 ( left graph ) , CD28 + subsets ( center graph ) and CD28negative subsets ( right graph ) are shown . Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates ( patients ) . representing CD4 T cells expressing only one cytokinerepresenting CD4 T cells expressing only cytokine , twoCD4 T cells expressing only three cytokines

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Figure 4Column graphs representing the percentage of CD4 T cells from NSCLC patients or age-matched healthy donors as represented in the graph, expressing the indicated cytokines after T cell stimulation with anti-CD3/anti-CD28 antibodies. Data on total CD4 (left graph), CD28+ subsets (center graph) and CD28negative subsets (right graph) are shown. Error bars correspond to standard deviations and bars represent means from 5 independent biological replicates (patients).representing CD4 T cells expressing only one cytokinerepresenting CD4 T cells expressing only cytokine, twoCD4 T cells expressing only three cytokines simultaneously


Figure 5Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . Dot plots of the percentage of Ki67 + proliferating CD8 T cells after ex vivo activation by A549 - SC3 cells . CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti - PD - 1 therapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . Green , objective responders ( OR ) and red , no ORs . Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549 - SC3 cells from the indicated patient groups , before the start of immunotherapy . Relevant statistical comparisons are shown with the test of Mann - Whitney . but in the presence of an isotype control antibody or an anti - PD - 1 antibody molecularly equivalent to pembrolizumab . Relevant statistical comparisons are shown with comparisons carried out with paired Student ´ s t test . Change in CD8 CD28 + T cells from baseline to post - therapy in G1 patients ( left ) or in G2 patients ( right ) . Statistical comparisons were carried out with paired Student ´ s t test .

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Figure 5Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney.Dot plots of the percentage of Ki67+ proliferating CD8 T cells after ex vivo activation by A549-SC3 cells. CD8 T cells were obtained from samples of G1 or G2 patients before immunotherapy and after three cycles of anti-PD-1 therapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. Green, objective responders (OR) and red, no ORs.Expression of the proliferation marker Ki67 in CD8 T cells stimulated ex vivo by A549-SC3 cells from the indicated patient groups, before the start of immunotherapy. Relevant statistical comparisons are shown with the test of Mann-Whitney. but in the presence of an isotype control antibody or an anti-PD-1 antibody molecularly equivalent to pembrolizumab. Relevant statistical comparisons are shown with comparisons carried out with paired Student´s t test.Change in CD8 CD28+ T cells from baseline to post-therapy in G1 patients (left) or in G2 patients (right). Statistical comparisons were carried out with paired Student´s t test.


Figure 6Scatter plots of PD - 1 / LAG - 3 - expressing CD8 T cells after activation by A459 - SC3 cells in a sample of G1 and G2 patients within CD28 + and CD28negative populations as indicated in the figure . Dot - plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated . Dot - plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy , activated ex vivo by A549 - SC3 cells in the presence of the indicated antibodies . " Dual " represents the addition of both anti - PD - 1 and anti - LAG - 3 antibodies . Appropriate statistical comparisons are shown within the graph with two - way paired ANOVA . Data from CD28 + and CD28negative subsets are represented as indicated . for CD8 T cells .

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Figure 6Scatter plots of PD-1/LAG-3-expressing CD8 T cells after activation by A459-SC3 cells in a sample of G1 and G2 patients within CD28+ and CD28negative populations as indicated in the figure.Dot-plot representing the percentage of proliferating CD4 T cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated.Dot-plot representing the percentage of proliferating cells from a sample of G2 patients before starting immunotherapy, activated ex vivo by A549-SC3 cells in the presence of the indicated antibodies. "Dual" represents the addition of both anti-PD-1 and anti-LAG-3 antibodies. Appropriate statistical comparisons are shown within the graph with two-way paired ANOVA. Data from CD28+ and CD28negative subsets are represented as indicated. for CD8 T cells.


Figure 7Kaplan - Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD - L1 + tumors ( blue ) and remaining patients for whom their PD - L1 tumor status is known ( red ) . Same as ( A ) but including all patients in the study cohort . Remaining patients ( red ) also included G1 patients with PD - L1 low / negative tumors , G1 patients with unknown PD - L1 tumor status and G2 patients with either PD - L1 + or PD - L1 negative tumors .

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Figure 7Kaplan-Meier plot for PFS in patients undergoing immune checkpoint inhibitor therapies stratified by G1 / PD-L1+ tumors (blue) and remaining patients for whom their PD-L1 tumor status is known (red).Same as (A) but including all patients in the study cohort. Remaining patients (red) also included G1 patients with PD-L1 low/negative tumors, G1 patients with unknown PD-L1 tumor status and G2 patients with either PD-L1+ or PD-L1 negative tumors.


Figure 1B Equilibrium titration of Hsf1 binding to Alexa Fluor ® 488 - labeled HSE - DNA . Fluorescence polarization plotted versus the Hsf1 trimer concentration . KD = 4 . 7 ± 1 . 9 nM ( n = 4 ) . C Hsf1 rapidly switches between different HSE - containing double stranded DNA oligonucleotides . Trimeric Hsf1 was pre - incubated with Alexa Fluor ® 488 - labeled HSE - DNA in MgCl2 - free buffer . At timepoint 0 ( arrow ) buffer , 4 mM MgCl2 , 2 mM ATP + 4 mM MgCl2 , control DNA ( DNACtrl at 10 - fold molar excess over HSE - DNA ) , and HSE - containing DNA was added as indicated and fluorescence polarization followed over time . D Trimeric Hsf1 were bound to HSE - DNA and the indicated components added at timepoint 0 . E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70 . Rates were determinedThe rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on temperature ( for 25 ° C n = 6 , for 37 ° C n = 5 , T - test , p = 0 . 011 ) ( F ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of Hsc70 and DnaJB1 ( n = 3 ) ( G ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of ATP ( n = 3 ) ( H ) The rate of Hsc70 / DnaJB1 - mediated dissociation of Hsf1 from HSE - DNA depends on concentration of the nucleotide exchange factor Apg2 ( HSPA4 ) ( n = 3 , ANOVA , * * , p < 0 . 01 ; * * * * , p < 0 . 0001 ) ( I ) . Shown are mean ± SD .

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Figure 1B Equilibrium titration of Hsf1 binding to Alexa Fluor® 488-labeled HSE-DNA. Fluorescence polarization plotted versus the Hsf1 trimer concentration. KD = 4.7 ± 1.9 nM (n = 4).C Hsf1 rapidly switches between different HSE-containing double stranded DNA oligonucleotides. Trimeric Hsf1 was pre-incubated with Alexa Fluor® 488-labeled HSE-DNA in MgCl2-free buffer. At timepoint 0 (arrow) buffer, 4 mM MgCl2, 2 mM ATP+4 mM MgCl2, control DNA (DNACtrl at 10-fold molar excess over HSE-DNA), and HSE-containing DNA was added as indicated and fluorescence polarization followed over time.D Trimeric Hsf1 were bound to HSE-DNA and the indicated components added at timepoint 0.E Hsp70 dissociates Hsf1 from DNA with similar rates as Hsc70. Rates were determinedThe rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on temperature (for 25°C n = 6, for 37°C n = 5, T-test, p = 0.011) (F)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of Hsc70 and DnaJB1 (n = 3) (G)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of ATP (n = 3) (H)The rate of Hsc70/DnaJB1-mediated dissociation of Hsf1 from HSE-DNA depends on concentration of the nucleotide exchange factor Apg2 (HSPA4) (n = 3, ANOVA, **, p < 0.01; ****, p < 0.0001) (I). Shown are mean ± SD.


Figure 2A , B Standard Hsc70 / DnaJB1 - mediated dissociation reaction of Hsf1 from Alexa Fluor ® 488 - labeled HSE - DNA monitored by fluorescence polarization ( A ) . The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x - axis , the reaction stopped by addition of blue - native loading buffer and stored on ice until separation by blue - native PAGE , blotted onto a PVDF membrane and detected with an Hsf1 - specific antiserum ( B ) . Lanes 1 , purified Hsf1 trimer ( T ) ; 2 , purified Hsf1 monomer ( M ) ; 3 - 11 , samples from the Hsc70 / DnaJB1 - mediated Hsf1 dissociation reaction ( 0 to 80 min ) ; 12 - 15 , Dissociation reaction incubated for 80 min missing individual components as indicated . HO , higher order oligomers ; T , trimer ; D , dimer ; M , mono mer . C Quantification of the amounts of Hsf1 species of the blot shown in ( B ) and five similar blots . For each lane the intensities of the two areas indicated by the brackets to the right were integrated ; upper bracket , DNA bound timers and higher order oligomers ( Hsf1b ) ; lower bracket , monomers and Hsf1 - species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA ( Hsf1d ) . Shown are means ± SD ( n = 3 ) . Inset , deoligomerization rate as determined by fitting an exponential decay function to the data . Shown are means ± SD ( n = 3 )

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Figure 2A,B Standard Hsc70/DnaJB1-mediated dissociation reaction of Hsf1 from Alexa Fluor® 488-labeled HSE-DNA monitored by fluorescence polarization (A). The same reaction mixture was incubated for different time intervals as indicated by the arrows below the x-axis, the reaction stopped by addition of blue-native loading buffer and stored on ice until separation by blue-native PAGE, blotted onto a PVDF membrane and detected with an Hsf1-specific antiserum (B). Lanes 1, purified Hsf1 trimer (T); 2, purified Hsf1 monomer (M); 3-11, samples from the Hsc70/DnaJB1-mediated Hsf1 dissociation reaction (0 to 80 min); 12-15, Dissociation reaction incubated for 80 min missing individual components as indicated. HO, higher order oligomers; T, trimer; D, dimer; M, monomer. C Quantification of the amounts of Hsf1 species of the blot shown in (B) and five similar blots. For each lane the intensities of the two areas indicated by the brackets to the right were integrated; upper bracket, DNA bound timers and higher order oligomers (Hsf1b); lower bracket, monomers and Hsf1-species possibly bound by Hsc70 or DnaJB1 or both dissociated from DNA (Hsf1d). Shown are means ± SD (n = 3). Inset, deoligomerization rate as determined by fitting an exponential decay function to the data. Shown are means ± SD (n = 3)


Figure 3C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159 - 168 ( left ) , 169 - 175 ( middle ) , and 176 - 189 ( right ) . Representative plots of 3 independent experiments . E Relative deuteron incorporation of the low ( solid bars ) and high ( open bars ) exchanging subpopulations .

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Figure 3C Fit of two Gaussian distributions to the peak intensities for peptic peptides 159-168 (left), 169-175 (middle), and 176-189 (right). Representative plots of 3 independent experiments.E Relative deuteron incorporation of the low (solid bars) and high (open bars) exchanging subpopulations.


Figure 4B KD values for the indicated peptides as determined by fluorescence polarization . Peptides N74 - Q86 - C ( NMYGFRKVVHIEQC ) , P223 - H235 - C ( PKYSRQFSLEHVHC ) and A343 - R352 - C ( ASVTALTDARC ) represent the most hydrophobic part of HX protected segments 78 - 98 , 229 - 252 , and 347 - 375 , respectively ; N200 - K208 - C ( NRILGVKRKC ) , N200 - K208 - C _ SSS ( NRSSGSKRKC ) , G204 - N214 - C ( GVKRKIPLMLNC ) , and G204 - N214 - C _ SSS ( GVKRKSPSMSNC ) represent the two potential Hsc70 binding sites in segment 202 - 213 in original sequence or with hydrophobic residues replaced by serine ; S461 - Q471 - C ( SGKQLVHYTAQC ) and Y468 - S480 - C ( YTAQPLFLLDPGSC ) represent the two potential Hsc70 binding sites in segment 442 - 474 . C Hsf1 ( 1 - 408 ) with a deleted TAD is dissociated from HSE - DNA by Hsc70 , DnaJB1 and ATP with similar rates as Hsf1wt . Fluorescence polarization assays containing Alexa Fluor ® 488 - HSE - DNA bound Hsf1 ( 1 - 408 ) trimers and the indicated components . D The HR - B proximal Hsc70 binding site is essential for Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 .

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Figure 4B KD values for the indicated peptides as determined by fluorescence polarization. Peptides N74-Q86-C (NMYGFRKVVHIEQC), P223-H235-C (PKYSRQFSLEHVHC) and A343-R352-C (ASVTALTDARC) represent the most hydrophobic part of HX protected segments 78-98, 229-252, and 347-375, respectively; N200-K208-C (NRILGVKRKC), N200-K208-C_SSS (NRSSGSKRKC), G204-N214-C (GVKRKIPLMLNC), and G204-N214-C_SSS (GVKRKSPSMSNC) represent the two potential Hsc70 binding sites in segment 202-213 in original sequence or with hydrophobic residues replaced by serine; S461-Q471-C (SGKQLVHYTAQC) and Y468-S480-C (YTAQPLFLLDPGSC) represent the two potential Hsc70 binding sites in segment 442-474.C Hsf1(1-408) with a deleted TAD is dissociated from HSE-DNA by Hsc70, DnaJB1 and ATP with similar rates as Hsf1wt. Fluorescence polarization assays containing Alexa Fluor® 488-HSE-DNA bound Hsf1(1-408) trimers and the indicated components.D The HR-B proximal Hsc70 binding site is essential for Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1.


Figure 5A The rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1 depends on the number of HR - B proximal Hsc70 binding sites in the Hsf1 trimer . Hsf1wt ( wt ) and Hsf1 ∆ ( 202 - 213 ) ( ∆ ) monomers were mixed in the indicated ratios and heat shocked at 42 ° C for 10 min to form mixtures of different homo - and heterotrimers as indicated with the cartoons ( the red line and the arrow head indicate the Hsc70 binding site ) . Numbers indicate the relative fraction of the different species . B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA - bound Hsf1 . Hsf1 _ t10 / 20 / 30 , region 202 - 213 moved by 10 , 20 , or 30 residues towards the C - terminus . C Anti - FLAG antibodies are not able to dissociate HSE - DNA - bound Hsf1 . Red lines in the cartoon indicate the Hsc70 binding site ; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK . Hsf1 _ i201 / 211 / 221 , FLAG - epitope inserted after residue 201 , 211 or 221 . Anti - FLAG antibodies were split in halfmers by incubation with 2 mM DTTAnti - FLAG antibody halfmers bound to FLAG - epitope containing Hsf1 variants . Hsf1wt and FLAG - insertion variants were analyzed by blue - native gel electrophoresis in the absence or presence of DTT - treated anti - FLAG antibodies as indicated and Hsf1 detected by immunoblotting ; t - bound , FLAG antibody - bound Hsf1 trimers ; m - bound , FLAG antibody bound Hsf1 monomer . F Trimer - to - monomer ratio of freshly purified Hsf1wt and Hsf1 - I190S , I194S in the absence of heat shock , determined by gel filtration and BN - GelG Rate of Hsc70 / DnaJB1 - mediated dissociation of HSE - DNA bound Hsf1wt and Hsf1 - I190S ,

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 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 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 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

Figure 5A The rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1 depends on the number of HR-B proximal Hsc70 binding sites in the Hsf1 trimer. Hsf1wt (wt) and Hsf1∆(202-213) (∆) monomers were mixed in the indicated ratios and heat shocked at 42°C for 10 min to form mixtures of different homo- and heterotrimers as indicated with the cartoons (the red line and the arrow head indicate the Hsc70 binding site). Numbers indicate the relative fraction of the different species.B Moving the Hsc70 binding site away from the trimerization domain reduces the rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA-bound Hsf1. Hsf1_t10/20/30, region 202-213 moved by 10, 20, or 30 residues towards the C-terminus.C Anti-FLAG antibodies are not able to dissociate HSE-DNA-bound Hsf1. Red lines in the cartoon indicate the Hsc70 binding site; blue lines and arrow head indicate the inserted FLAG epitope DYKDDDDK. Hsf1_i201/211/221, FLAG-epitope inserted after residue 201, 211 or 221. Anti-FLAG antibodies were split in halfmers by incubation with 2 mM DTTAnti-FLAG antibody halfmers bound to FLAG-epitope containing Hsf1 variants. Hsf1wt and FLAG-insertion variants were analyzed by blue-native gel electrophoresis in the absence or presence of DTT-treated anti-FLAG antibodies as indicated and Hsf1 detected by immunoblotting; t-bound, FLAG antibody-bound Hsf1 trimers; m-bound, FLAG antibody bound Hsf1 monomer.F Trimer-to-monomer ratio of freshly purified Hsf1wt and Hsf1-I190S,I194S in the absence of heat shock, determined by gel filtration and BN-GelG Rate of Hsc70/DnaJB1-mediated dissociation of HSE-DNA bound Hsf1wt and Hsf1-I190S,I194S


Figure 6A Schematics of the used system . MEFs , in which HSF1 on chromosome 8 was deleted , were stably transfected with mTag - BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence , indicating the gates used for analysis . Numbers indicate the fraction ( % ) of cells ( forward scatter singlets ) in each gate . Color indicates the cell density from dark blue ( low ) to red ( high ) . C - H Geometric mean of BFP ( C , E ) and GFP fluorescence ( D , F , H ) of cells grown at 37 ° C ( non - stress , NS ) or incubated for 1 h at 43 ° C with a recovery period of 3 h at 37 ° C ( heat shocked , HS ) in the GFPhigh ( C , D ) , GFPlow ( E , F ) , and GFPlow - BFPhigh ( H ) gates . ( G ) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow - BFPhigh gate ( fluorescence * fraction forward scatter singlets ) . Note that the GFPlow gate also comprises the GFPlow - BFPhigh gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

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Figure 6A Schematics of the used system. MEFs, in which HSF1 on chromosome 8 was deleted, were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. B Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high).C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) of cells grown at 37°C (non-stress, NS) or incubated for 1 h at 43°C with a recovery period of 3 h at 37°C (heat shocked, HS) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh (H) gates. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


Figure 7A Schematics of the used system . HeLa cells were stably transfected with mTag - BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP , which is translated from an IRES element . Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence , indicating the gates used for analysis . Numbers indicate the fraction ( % ) of cells ( forward scatter singlets ) in each gate . Color indicates the cell density from dark blue ( low ) to red ( high ) . NS , cells grown at 37 ° C ; HS , cells heat shocked at 43 ° C for 1 h with a recovery period of 3 h at 37 ° C . C - H Geometric mean of BFP ( C , E ) and GFP fluorescence ( D , F , H ) in the GFPhigh ( C , D ) , GFPlow ( E , F ) , and GFPlow - BFPhigh43 ( H ) gates . Of note , the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments . ( G ) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow - BFPhigh43 gate ( fluorescence * fraction forward scatter singlets ) . Note that the GFPlow gate also comprises the GFPlow - BFPhigh37 and that the GFPlow - BFPhigh37 gate also comprises the GFPlow - BFPhigh43 gate . Hsf1 - 202SSS , Hsf1 - I202S , L203S , V205S ; Hsf1 - 209SSS , Hsf1 - I209S , L211S , L213S ; Hsf1 - 427SSSS , Hsf1 - M427S , L429S , L432S , L436S ; Hsf1 - 465SSS , Hsf1 - L465S , V466S , Y468S ; Hsf1 - 473SSSS , Hsf1 - L473S , F474S , L475S ,

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 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

Figure 7A Schematics of the used system. HeLa cells were stably transfected with mTag-BFP expressed under the control of the HSPA6 promoter and subsequently transiently transfected with plasmids that expressed HSF1wt or mutant variants in an artificial operon with hrGFP, which is translated from an IRES element. Exemplary flow cytometry data plotting BFP fluorescence versus GFP fluorescence, indicating the gates used for analysis. Numbers indicate the fraction (%) of cells (forward scatter singlets) in each gate. Color indicates the cell density from dark blue (low) to red (high). NS, cells grown at 37°C; HS, cells heat shocked at 43°C for 1 h with a recovery period of 3 h at 37°C.C-H Geometric mean of BFP (C, E) and GFP fluorescence (D, F, H) in the GFPhigh (C, D), GFPlow (E, F), and GFPlow-BFPhigh43 (H) gates. Of note, the number of data points show the number of experiments in which a significant number of cells was found in this gate and missing bars indicate that cells did not fall within this gate in more than two out of four experiments. (G) Geometric mean of BFP fluorescence weighted by the relative number of cells in the GFPlow-BFPhigh43 gate (fluorescence * fraction forward scatter singlets). Note that the GFPlow gate also comprises the GFPlow-BFPhigh37 and that the GFPlow-BFPhigh37 gate also comprises the GFPlow-BFPhigh43 gate. Hsf1-202SSS, Hsf1-I202S,L203S,V205S; Hsf1-209SSS, Hsf1-I209S,L211S,L213S; Hsf1-427SSSS, Hsf1-M427S,L429S,L432S,L436S; Hsf1-465SSS, Hsf1-L465S,V466S,Y468S; Hsf1-473SSSS, Hsf1-L473S,F474S,L475S,L476S


Figure 1At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA ( 10 nM each ) , the intracellular localization of TfnR in HeLa cells was examined ( shown as green ) . Nuclear DNA was stained with Hoechst 33342 ( shown as blue ) . Control knockdown ( left panel ) , Rab8a knockdown ( center panel ) , and BAG6 knockdown ( right panel ) . Intracellular localization of Ptc1 ( green ) in HeLa cells . Nuclei were stained with Hoechst 33342 ( shown as blue ) . Knockdown of Rab8a ( with Rab8a siRNA # 1 , # 2 , and # 3 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells . Knockdown of BAG6 ( with BAG6 siRNA # 1 ) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells .

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Figure 1At 72 h after transfection with siRNA duplexes for BAG6 or control siRNA (10 nM each), the intracellular localization of TfnR in HeLa cells was examined (shown as green). Nuclear DNA was stained with Hoechst 33342 (shown as blue). Control knockdown (left panel), Rab8a knockdown (center panel), and BAG6 knockdown (right panel).Intracellular localization of Ptc1 (green) in HeLa cells. Nuclei were stained with Hoechst 33342 (shown as blue).Knockdown of Rab8a (with Rab8a siRNA#1, #2, and #3) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.Knockdown of BAG6 (with BAG6 siRNA#1) stimulated the accumulation and stabilization of Ptc1 protein in HEK293 cells.


Figure 2BAG6 protein co - precipitated Rab8a , while neither Rab7 nor Rab11a were co - precipitated with BAG6 . Flag - tagged Rab8a , Rab7 , Rab11a , and luciferase - CL1 ( Lc - CL1 ; a positive control ) were expressed in HeLa cells and the cells were treated with 10 μM MG - 132 for 4 h . Flag - immunoprecipitates were blotted with anti - BAG6 and anti - Flag antibodies , respectively . Note that all cells used were treated with 10 μM MG - 132 for 4 h . S - tagged BAG6 pull - down efficiently co - precipitated Rab8a ( T22N ) , a GDP - bound mutant , while BAG6 scarcely co - precipitated Rab8a ( Q67L ) , a constitutively active mutant . Co - precipitation of Rab8a WT with BAG6 was used as a standard . MG - 132 ( 10 μM ) was included in the cell culture for 4 h , as indicated . Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L , and that this was partly due to increased degradation , as will be shown later . S - BAG6 stands for N - terminally S - tagged BAG6 proteinAnti - Flag signals in ( C ) were quantified , and relative signal intensities are presented . The value of the WT Rab8a signal with MG - 132 was defined as 1 . 0 . Note that all signal intensities of Flag - tag were normalized by that of the Rab8a input signals . A series of Flag - Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag - Rab8a . Note that all cells used were treated with 10 μM MG - 132 for 4 h . Deficiency of Rabin8 , a GEF for Rab8a , enhanced the physical interaction between BAG6 and WT Rab8a proteins . Flag - tagged WT Rab8a was expressed in Rabin8 siRNA - treated cells , and Flag immunoprecipitates were probed with an anti - BAG6 antibody . Note that all cells used were treated with 10 μM MG - 132 for 4 h .

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 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 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

Figure 2BAG6 protein co-precipitated Rab8a, while neither Rab7 nor Rab11a were co-precipitated with BAG6. Flag-tagged Rab8a, Rab7, Rab11a, and luciferase-CL1 (Lc-CL1; a positive control) were expressed in HeLa cells and the cells were treated with 10 μM MG-132 for 4 h. Flag-immunoprecipitates were blotted with anti-BAG6 and anti-Flag antibodies, respectively. Note that all cells used were treated with 10 μM MG-132 for 4 h.S-tagged BAG6 pull-down efficiently co-precipitated Rab8a (T22N), a GDP-bound mutant, while BAG6 scarcely co-precipitated Rab8a (Q67L), a constitutively active mutant. Co-precipitation of Rab8a WT with BAG6 was used as a standard. MG-132 (10 μM) was included in the cell culture for 4 h, as indicated. Note that the T22N mutant protein was expressed at lower levels than either WT or Q67L, and that this was partly due to increased degradation, as will be shown later. S-BAG6 stands for N-terminally S-tagged BAG6 proteinAnti-Flag signals in (C) were quantified, and relative signal intensities are presented. The value of the WT Rab8a signal with MG-132 was defined as 1.0. Note that all signal intensities of Flag-tag were normalized by that of the Rab8a input signals.A series of Flag-Rab8a mutants were immunoprecipitated and quantified the amount of endogenous BAG6 that were coprecipitated with Flag-Rab8a. Note that all cells used were treated with 10 μM MG-132 for 4 h.Deficiency of Rabin8, a GEF for Rab8a, enhanced the physical interaction between BAG6 and WT Rab8a proteins. Flag-tagged WT Rab8a was expressed in Rabin8 siRNA-treated cells, and Flag immunoprecipitates were probed with an anti-BAG6 antibody. Note that all cells used were treated with 10 μM MG-132 for 4 h.


Figure 3C - terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition . The N - terminal GTPase domain of Rab8a was essential for BAG6 recognition . A series of Flag - tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S - tagged BAG6 and treated with ( + ) or without ( - ) protease inhibitors for 4 h . Flag - Rab8a substrates were immunoprecipitated and probed with an anti - BAG6 antibody . Hydrophobicity recognition domain of BAG6 ( N465 ) was critical for Rab8a recognition . A schematic representation of the BAG6 - truncated proteins used in this experiment is shown in the upper panel . Numbers denote the corresponding amino acids of mammalian BAG6 . Positions of UBL , BUILD , and DUF3538 domains , which are all linked to hydrophobicity recognition by BAG6 [ 37 ] , are indicated . WT , wild - type ; ∆ N465 , N - terminal 465 residues - deleted mutant ; and Ν465 , C - terminal 689 residues - deleted mutant . CHX chase experiments show that GDP - bound Rab8a ( T22N ) was a highly labile protein , while the GTP - bound active mutant ( Q67L ) was a stable protein ( E ) . Instability of the T22N mutant was not perturbed by the C204S mutation ( F ) . Actin was used as a loading control . N - terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor , while the ∆ N100 fragment was not . Rab8a ( T22N ) was polyubiquitinated in the presence of the proteasome inhibitor . Asterisks indicate non - specific bands .

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

Figure 3C-terminal geranylgeranylation of Rab8a was dispensable for BAG6 recognition.The N-terminal GTPase domain of Rab8a was essential for BAG6 recognition. A series of Flag-tagged truncated fragments of WT Rab8a were expressed in HeLa cells with S-tagged BAG6 and treated with (+) or without (-) protease inhibitors for 4 h. Flag-Rab8a substrates were immunoprecipitated and probed with an anti-BAG6 antibody.Hydrophobicity recognition domain of BAG6 (N465) was critical for Rab8a recognition. A schematic representation of the BAG6-truncated proteins used in this experiment is shown in the upper panel. Numbers denote the corresponding amino acids of mammalian BAG6. Positions of UBL, BUILD, and DUF3538 domains, which are all linked to hydrophobicity recognition by BAG6 [37], are indicated. WT, wild-type; ∆N465, N-terminal 465 residues-deleted mutant; and Ν465, C-terminal 689 residues-deleted mutant.CHX chase experiments show that GDP-bound Rab8a (T22N) was a highly labile protein, while the GTP-bound active mutant (Q67L) was a stable protein (E). Instability of the T22N mutant was not perturbed by the C204S mutation (F). Actin was used as a loading control.N-terminal 100 residue fragment of the Rab8a GTPase domain was sensitive to the proteasome inhibitor, while the ∆N100 fragment was not.Rab8a (T22N) was polyubiquitinated in the presence of the proteasome inhibitor. Asterisks indicate non-specific bands.


Figure 4Kyte - Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N - 3IS mutant . The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant ( indicated within the red box ) . The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins . Substitution of the hydrophobic residues of Switch I with hydrophilic residues ( T22N 3IS ) abolished its binding to BAG6 protein . Rab8a ( T22N - 3IS ) mutant protein was highly stable , while Rab8a ( T22N ) protein was quite unstable in HeLa cells . Actin was used as a loading control . Rab8a ( T22N - 3IS ) mutant was not subject to polyubiquitin co - precipitation , while Rab8a ( T22N ) protein was .

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 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 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

Figure 4Kyte-Doolittle hydrophobicity plots of the complete amino acid sequence of human WT Rab8a and T22N-3IS mutant. The hydrophobicity peak within WT Switch I was abolished in the 3IS mutant (indicated within the red box). The numbers on the horizontal axis denote the corresponding amino acid positions in these proteins.Substitution of the hydrophobic residues of Switch I with hydrophilic residues (T22N 3IS) abolished its binding to BAG6 protein.Rab8a (T22N-3IS) mutant protein was highly stable, while Rab8a (T22N) protein was quite unstable in HeLa cells. Actin was used as a loading control.Rab8a (T22N-3IS) mutant was not subject to polyubiquitin co-precipitation, while Rab8a (T22N) protein was.


Figure 5Rab8a ( T22N ) protein accumulated in BAG6 - knockdown cells . HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA . At 48 h after siRNA transfection , Flag tagged - Rab8a ( T22N ) was expressed in the cells . At 24 h after Rab8a ( T22N ) transfection , the cells were chased with 50 μg / mL CHX and harvested at the indicated time after CHX addition . Actin was used as a loading control . Anti - Flag blot signals in the control or BAG6 siRNA - treated cells were quantified , and relative signal intensities after CHX addition were calculated . The value of the Flag signal at 0 h was defined as 1 . 0 . Note that all signal intensities of the Flag - tag were normalized by that of actin , a loading control , in each sample . Polyubiquitin modification of Rab8a was abolished in BAG6 - knockdown cells . Flag - Rab8a ( T22N ) immunoprecipitates were blotted with an anti - polyubiquitin antibody ( FK2 , left panel ) . As a negative control , siRNA # 1scr was used . Anti - polyubiquitin signals co - precipitated with Rab8a ( T22N ) ( a representative example is shown in the left panel ) were quantified ( right panel ) . Note that the intensities of the co - precipitated polyubiquitin signal were normalized both by the input ubiquitin - signal and bait Flag - signal . Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a . Right panel shows a merged image of TfnR staining ( shown as green ) , Rab8a ( T22N ) staining ( magenta ) , and Hoechst 33342 nuclear staining ( blue ) .

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 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

Figure 5Rab8a (T22N) protein accumulated in BAG6-knockdown cells. HeLa cells were transfected with siRNA duplexes for BAG6 or control siRNA. At 48 h after siRNA transfection, Flag tagged-Rab8a (T22N) was expressed in the cells. At 24 h after Rab8a (T22N) transfection, the cells were chased with 50 μg/mL CHX and harvested at the indicated time after CHX addition. Actin was used as a loading control.Anti-Flag blot signals in the control or BAG6 siRNA-treated cells were quantified, and relative signal intensities after CHX addition were calculated. The value of the Flag signal at 0 h was defined as 1.0. Note that all signal intensities of the Flag-tag were normalized by that of actin, a loading control, in each sample.Polyubiquitin modification of Rab8a was abolished in BAG6-knockdown cells. Flag-Rab8a (T22N) immunoprecipitates were blotted with an anti-polyubiquitin antibody (FK2, left panel). As a negative control, siRNA#1scr was used. Anti-polyubiquitin signals co-precipitated with Rab8a (T22N) (a representative example is shown in the left panel) were quantified (right panel). Note that the intensities of the co-precipitated polyubiquitin signal were normalized both by the input ubiquitin-signal and bait Flag-signal.Defective distribution of the endosomal protein TfnR in HeLa cells with the excess accumulation of the inactive form of Rab8a. Right panel shows a merged image of TfnR staining (shown as green), Rab8a (T22N) staining (magenta), and Hoechst 33342 nuclear staining (blue).


Figure 6A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti - BAG6 antibody . Co - precipitation of Rab8a ( T22N ) with BAG6 was used as a positive control for this experiment . Quantification of BAG6 co - precipitation with Rab family proteins . The graph shows the quantities of BAG6 protein in the respective anti - Flag precipitates that were normalized by the amount of Flag - Rab bait proteins . The value of BAG6 co - precipitation with Rab8a ( T22N ) was defined as 1 . 0 . The quantities of the respective immunosignals were also normalized to the actin signal of each sample , and represent the median calculated from at least 3 independent biological replicates . The number of independent biological replicates was as follows : Rab1a , n = 4 ; Rab1b , n = 5 ; Rab2b , n = 3 ; Rab3a , n = 4 ; Rab4 , n = 4 ; Rab5a , n = 3 ; Rab5b , n = 4 ; Rab6a , n = 5 ; Rab6b , n = 4 ; Rab7a , n = 3 ; Rab8a , n = 14 ; Rab9b , n = 4 ; Rab10 , n = 6 ; Rab11a , n = 3 ; Rab11b , n = 3 ; Rab12 , n = 2 ; Rab13 , n = 6 ; Rab14 , n = 3 ; Rab15 , n = 5 ; Rab35 , n = 3 .

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Figure 6A series of major Rab family proteins were immunoprecipitated from HeLa cell extracts and then probed with an anti-BAG6 antibody. Co-precipitation of Rab8a (T22N) with BAG6 was used as a positive control for this experiment.Quantification of BAG6 co-precipitation with Rab family proteins. The graph shows the quantities of BAG6 protein in the respective anti-Flag precipitates that were normalized by the amount of Flag-Rab bait proteins. The value of BAG6 co-precipitation with Rab8a (T22N) was defined as 1.0. The quantities of the respective immunosignals were also normalized to the actin signal of each sample, and represent the median calculated from at least 3 independent biological replicates. The number of independent biological replicates was as follows: Rab1a, n = 4; Rab1b, n = 5; Rab2b, n = 3; Rab3a, n = 4; Rab4, n = 4; Rab5a, n = 3; Rab5b, n = 4; Rab6a, n = 5; Rab6b, n = 4; Rab7a, n = 3; Rab8a, n = 14; Rab9b, n = 4; Rab10, n = 6; Rab11a, n = 3; Rab11b, n = 3; Rab12, n = 2; Rab13, n = 6; Rab14, n = 3; Rab15, n = 5; Rab35, n = 3.


Figure 7BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Representative images of the trans - Golgi membrane protein Stx6 ( green ) in BAG6 - suppressed CHO cells with a Chinese hamster - specific siRNA ( cBAG6 siRNA # 2 ) . Scale bar : 10 μm ( A ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) . BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers . Images of the cis - Golgi membrane protein GS28 and the cis - Golgi matrix protein GM130 in BAG6 - suppressed CHO cells with another Chinese hamster - specific siRNA ( cBAG6 siRNA # 5 ) . GS28 ( green ) and GM130 ( red ) are indicated by arrowheads . Scale bar : 10 μm . ( B ) . Fluorescent signals were detected using a laser scanning confocal microscopy system . Nuclei were stained by Hoechst ( blue ) . Glycosylation of the IL - 2Rα transmembrane protein was not reduced by BAG6 knockdown . Flag - tagged WT IL - 2Rα protein was expressed in HeLa cells with ( + ) or without ( - ) BAG6 siRNA , and was immunoprecipitated with an anti - Flag antibody . The precipitates were incubated with ( + ) or without ( - ) 10 unit of the deglycosylation enzyme PNGase F , and subjected to Western blot analysis with an anti - Flag antibody . Low - mobility ( indicated as glycosylated ) and high mobility ( indicated as non - glycosylated ) signals of WT IL - 2Rα are indicated . Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S . D . calculated from 10 independent biological replicates ( E ) . Defects in the distribution of cell surface glycoproteins in BAG6 - suppressed cells . Lectin GS - II - derived cell surface signals were counted using ImageJ software . SRP54 knockdown was used as a positive control for this experiment t - test ) . Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization . BAG6 siRNA down - regulated the cell surface expression of glycoproteins ( 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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

Figure 7BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Representative images of the trans-Golgi membrane protein Stx6 (green) in BAG6-suppressed CHO cells with a Chinese hamster-specific siRNA (cBAG6 siRNA#2). Scale bar: 10 μm (A). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).BAG6 knockdown induced the abnormal distribution of Golgi apparatus markers. Images of the cis-Golgi membrane protein GS28 and the cis-Golgi matrix protein GM130 in BAG6-suppressed CHO cells with another Chinese hamster-specific siRNA (cBAG6 siRNA#5). GS28 (green) and GM130 (red) are indicated by arrowheads. Scale bar: 10 μm. (B). Fluorescent signals were detected using a laser scanning confocal microscopy system. Nuclei were stained by Hoechst (blue).Glycosylation of the IL-2Rα transmembrane protein was not reduced by BAG6 knockdown. Flag-tagged WT IL-2Rα protein was expressed in HeLa cells with (+) or without (-) BAG6 siRNA, and was immunoprecipitated with an anti-Flag antibody. The precipitates were incubated with (+) or without (-) 10 unit of the deglycosylation enzyme PNGase F, and subjected to Western blot analysis with an anti-Flag antibody. Low-mobility (indicated as glycosylated) and high mobility (indicated as non-glycosylated) signals of WT IL-2Rα are indicated.Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. The graph quantitatively displays the number of fluorescence counts per cell as the mean ± S.D. calculated from 10 independent biological replicates (E).Defects in the distribution of cell surface glycoproteins in BAG6-suppressed cells. Lectin GS-II-derived cell surface signals were counted using ImageJ software. SRP54 knockdown was used as a positive control for this experiment t-test). Cell surface fluorescent signals were detected by confocal microscopy without plasma membrane permeabilization. BAG6 siRNA down-regulated the cell surface expression of glycoproteins (F).


figf1 ( a ) Densitometric analysis relative to actin of A53T α - synuclein clearance in stable inducible PC12 cell line expressing A53T α - synuclein . Transgene expression was induced with doxycycline for 48 h , and then switched off ( by removing doxycycline ) , with drug ( all 1 μM ) or DMSO ( control ) treatment for 24 h . Control condition is set to 100 % . Error bars show s . e . m . ( b ) Densitometric analysis relative to actin of soluble EGFP - HDQ74 clearance in stable inducible PC12 cell line expressing EGFP - HDQ74 . Transgene expression was induced with doxycycline for 8 h , and then switched off ( by removing doxycycline ) with drug ( all 1 μM ) or DMSO ( control ) treatment for 96 h . Control condition is set to 100 % . Error bars show s . e . m . ( c ) SK - N - SH cells transfected with EGFP - HDQ74 construct for 4 h were treated with drugs ( all 1 μM ) or DMSO ( control ) for 48 h post - transfection . The proportions of EGFP - positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1 . Error bars show 95 % confidence interval . ( d ) PC12 cells were treated with clonidine , minoxidil and verapamil ( all 1 μM ) or DMSO ( control ) for 24 h . Endogenous LC3 - II levels were detected with anti - LC3 antibody and quantified relative to actin . Rapamycin ( 0 . 2 μM ) was positive control . Error bars show s . e . m . ( e ) The proportions of EGFP - positive cells with EGFP - HDQ74 aggregates in wild - type ( Atg5 + / + ) and knockout ( Atg5 − / − ) Atg5 MEFs , transfected with EGFP - HDQ74 for 4 h and then treated for 48 h with drugs ( all 1 μM ) or DMSO ( control ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant . ( a ) Densitometric analysis relative to actin of A53T α - synuclein clearance in stable inducible PC12 cell line expressing A53T α - synuclein . Transgene expression was induced with doxycycline for 48 h , and then switched off ( by removing doxycycline ) , with drug ( all 1 μM ) or DMSO ( control ) treatment for 24 h . Control condition is set to 100 % . Error bars show s . e . m .

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 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 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 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

figf1(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.(b) Densitometric analysis relative to actin of soluble EGFP-HDQ74 clearance in stable inducible PC12 cell line expressing EGFP-HDQ74. Transgene expression was induced with doxycycline for 8 h, and then switched off (by removing doxycycline) with drug (all 1 μM) or DMSO (control) treatment for 96 h. Control condition is set to 100%. Error bars show s.e.m.(c) SK-N-SH cells transfected with EGFP-HDQ74 construct for 4 h were treated with drugs (all 1 μM) or DMSO (control) for 48 h post-transfection. The proportions of EGFP-positive cells with aggregates or cell death were expressed as odds ratios and the control was taken as 1. Error bars show 95% confidence interval.(d) PC12 cells were treated with clonidine, minoxidil and verapamil (all 1 μM) or DMSO (control) for 24 h. Endogenous LC3-II levels were detected with anti-LC3 antibody and quantified relative to actin. Rapamycin (0.2 μM) was positive control. Error bars show s.e.m.(e) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in wild-type (Atg5+/+) and knockout (Atg5−/−) Atg5 MEFs, transfected with EGFP-HDQ74 for 4 h and then treated for 48 h with drugs (all 1 μM) or DMSO (control). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.(a) Densitometric analysis relative to actin of A53T α-synuclein clearance in stable inducible PC12 cell line expressing A53T α-synuclein. Transgene expression was induced with doxycycline for 48 h, and then switched off (by removing doxycycline), with drug (all 1 μM) or DMSO (control) treatment for 24 h. Control condition is set to 100%. Error bars show s.e.m.


figf2 ( a ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 1 μM rilmenidine , 1 mM dibutyl - cAMP ( db - cAMP ) , 24 μM forskolin or 500 μM 2 ′ 5 ′ - dideoxyadenosine ( 2 ′ 5 ′ ddA ) for 24 h . Note that control clearance levels for a given substrate may vary from figure to figure , due to different exposures of blots , which aid illustration of agents that either increase or retard clearance . ( b ) The proportions of EGFP - positive cells with EGFP - HDQ74 aggregates in SK - N - SH ( for rilmenidine ) and COS - 7 ( for db - cAMP , forskolin and 2 ′ 5 ′ ddA ) cells as in Figure 1c , treated with concentrations used in a for 48 h post - transfection . Error bars show 95 % confidence interval . ( c ) Endogenous LC3 - II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2 ′ 5 ′ ddA for 24 h . ( d ) A53T α - synuclein clearance in stable PC12 cells as in Figure 1a , treated with or without 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP ( 1 μM or 10 μM ) . ( e ) The proportions of EGFP - positive COS - 7 cells with aggregates as in Figure 1c , treated with or without 10 μM 8 - CPT - 2Me - cAMP or 6 - Bnz - cAMP for 48 h post - transfection . Error bars show 95 % confidence interval . ( f ) COS - 7 cells , transfected for 4 h with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) , dominant - negative S17N Rap2B ( DN Rap2B ) or wild - type PLC - ε ( 1 : 3 ratio ) , were assessed for the proportions of EGFP - positive cells with aggregates after 48 h . Error bars show 95 % confidence interval . ( g ) Endogenous LC3 - II levels in COS - 7 cells transfected with a dominant - negative Rap2B ( DN Rap2B ) for 4 h were assessed after 48 h expression . ( h ) The proportions of EGFP - positive COS - 7 cells with aggregates , transfected with EGFP - HDQ74 along with empty vector ( pcDNA3 . 1 ) or dominant - negative Rap2B ( 1 : 3 ratio ) for 4 h , were treated with or without 10 μM 8 - CPT - 2Me - cAMP for 48 h . Error bars show 95 % confidence interval . ( i ) COS - 7 cells , transfected with EGFP - HDQ74 and dsRed2 or cytosolic dsRed2 - IP3 kinase A ( 1 : 3 ratio ) for 4 h , were assessed at 48 h post - transfection for aggregation and cell death ( as in Fig . 1c ) . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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 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 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 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 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

figf2(a) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 1 μM rilmenidine, 1 mM dibutyl-cAMP (db-cAMP), 24 μM forskolin or 500 μM 2′5′-dideoxyadenosine (2′5′ddA) for 24 h. Note that control clearance levels for a given substrate may vary from figure to figure, due to different exposures of blots, which aid illustration of agents that either increase or retard clearance.(b) The proportions of EGFP-positive cells with EGFP-HDQ74 aggregates in SK-N-SH (for rilmenidine) and COS-7 (for db-cAMP, forskolin and 2′5′ddA) cells as in Figure 1c, treated with concentrations used in a for 48 h post-transfection. Error bars show 95% confidence interval.(c) Endogenous LC3-II levels in PC12 cells treated with 1 μM rilmenidine or 500 μM 2′5′ddA for 24 h.(d) A53T α-synuclein clearance in stable PC12 cells as in Figure 1a, treated with or without 8-CPT-2Me-cAMP or 6-Bnz-cAMP (1 μM or 10 μM).(e) The proportions of EGFP-positive COS-7 cells with aggregates as in Figure 1c, treated with or without 10 μM 8-CPT-2Me-cAMP or 6-Bnz-cAMP for 48 h post-transfection. Error bars show 95% confidence interval.(f) COS-7 cells, transfected for 4 h with EGFP-HDQ74 along with empty vector (pcDNA3.1), dominant-negative S17N Rap2B (DN Rap2B) or wild-type PLC-ε (1:3 ratio), were assessed for the proportions of EGFP-positive cells with aggregates after 48 h. Error bars show 95% confidence interval.(g) Endogenous LC3-II levels in COS-7 cells transfected with a dominant-negative Rap2B (DN Rap2B) for 4 h were assessed after 48 h expression.(h) The proportions of EGFP-positive COS-7 cells with aggregates, transfected with EGFP-HDQ74 along with empty vector (pcDNA3.1) or dominant-negative Rap2B (1:3 ratio) for 4 h, were treated with or without 10 μM 8-CPT-2Me-cAMP for 48 h. Error bars show 95% confidence interval.(i) COS-7 cells, transfected with EGFP-HDQ74 and dsRed2 or cytosolic dsRed2-IP3 kinase A (1:3 ratio) for 4 h, were assessed at 48 h post-transfection for aggregation and cell death (as in Fig. 1c). Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.


figf3 ( a ) SK - N - SH cells were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Calpain activity was detected by immunoblotting with anti - calpain small subunit antibody . Densitometry analysis is relative to actin . Error bars show s . e . m . ( b ) SK - N - SH cells transfected with EGFP - HDQ74 for 4 h , then pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 48 h post - transfection , were assessed for the proportion of EGFP - positive cells with EGFP - HDQ74 aggregates . Error bars show 95 % confidence interval . ( c ) SK - N - SH cells were pretreated with or without 10 , 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM ( ± ) - Bay K8644 for 4 h . Endogenous LC3 - II levels were detected by immunoblotting with anti - LC3 antibody . ( d ) Comparison between calpain activity , EGFP - HDQ74 aggregation and autophagosomes as seen in a - c . The control condition for all the assessments was set at 100 % . Calpastatin lowered the increased calpain activity and EGFP - HDQ74 aggregation caused by ( ± ) - Bay K8644 in a dose - dependent manner with a simultaneous increase in LC3 - II levels ( autophagosomes ) . ( e ) COS - 7 cells transfected with empty vector ( pcDNA3 . 1 ) or wild - type PLC - ε for 4 h and treated with or without 50 μM calpeptin for 48 h post - transfection were assessed for the proportion of EGFP - positive cells with EGFP - HDQ74 aggregates . Error bars show 95 % confidence interval . * * * P 0 . 001 ; * * P 0 . 01 ; * P 0 . 05 ; NS , nonsignificant .

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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O 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) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Calpain activity was detected by immunoblotting with anti-calpain small subunit antibody. Densitometry analysis is relative to actin. Error bars show s.e.m.(b) SK-N-SH cells transfected with EGFP-HDQ74 for 4 h, then pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 48 h post-transfection, were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval.(c) SK-N-SH cells were pretreated with or without 10, 15 or 20 μM calpastatin for 15 min followed by addition of 1 μM (±)-Bay K8644 for 4 h. Endogenous LC3-II levels were detected by immunoblotting with anti-LC3 antibody.(d) Comparison between calpain activity, EGFP-HDQ74 aggregation and autophagosomes as seen in a-c. The control condition for all the assessments was set at 100%. Calpastatin lowered the increased calpain activity and EGFP-HDQ74 aggregation caused by (±)-Bay K8644 in a dose-dependent manner with a simultaneous increase in LC3-II levels (autophagosomes).(e) COS-7 cells transfected with empty vector (pcDNA3.1) or wild-type PLC-ε for 4 h and treated with or without 50 μM calpeptin for 48 h post-transfection were assessed for the proportion of EGFP-positive cells with EGFP-HDQ74 aggregates. Error bars show 95% confidence interval. ***P 0.001; **P 0.01; *P 0.05; NS, nonsignificant.