Figure 2 ( B ) Representative immunohistochemical analysis with anti - pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan - keratin positive areas ( mm2 ) of individual lesions ( dots ) formed by 6 HKCs strains of the two ancestries . n ( tumors per ancestry ) = 8 . * p < 0 . 05 . Horizontal line : median . Two - tailed unpaired t - test . Scale bar : 500μm . Double immunofluorescence analysis of tumors formed by six HKC strains , as in the previous panels , with antibodies against Ki67 ( C ) together with anti - pan - keratin antibodies for tumor cells identification . Shown are representative images and quantification of results of individual lesions ( dots ) . Digitally acquired images were used to determine the % of Ki67 positive cells ( C ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 and K1 staining : n ( tumors per ancestry ) = 24 , 22 and 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal line : median , two - tailed unpaired t test . Scale bars : 100μm . Double immunofluorescence analysis of tumors formed by six HKC strains , as in the previous panels , with antibodies against keratin 10 and 1 ( K10 , K1 ) ( D ) together with anti - pan - keratin antibodies for tumor cells identification . Shown are representative images and quantification of results of individual lesions ( dots ) . Digitally acquired images were used to determine the % of K1 / K10 positivity ( D ) in pan - keratin positive areas , examining 3 - 5 fields in each case . For Ki67 , K10 and K1 staining : n ( tumors per ancestry ) = 24 , 22 and 6 , respectively . * * * * p < 0 . 0001 ; * * p < 0 . 005 , horizontal line : median , two - tailed unpaired t test . Scale bars : 100μm .

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 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 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

Figure 2(B) Representative immunohistochemical analysis with anti-pan keratin antibodies of a pair of tumor lesions formed by parallel injections of White versus Black HKC strains together with quantification of the pan-keratin positive areas (mm2) of individual lesions (dots) formed by 6 HKCs strains of the two ancestries. n(tumors per ancestry) = 8. *p<0.05. Horizontal line: median. Two-tailed unpaired t-test. Scale bar: 500μm.Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against Ki67 (C) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of Ki67 positive cells (C) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.Double immunofluorescence analysis of tumors formed by six HKC strains, as in the previous panels, with antibodies against keratin 10 and 1 (K10, K1) (D) together with anti-pan-keratin antibodies for tumor cells identification. Shown are representative images and quantification of results of individual lesions (dots). Digitally acquired images were used to determine the % of K1/K10 positivity (D) in pan-keratin positive areas, examining 3-5 fields in each case. For Ki67, K10 and K1 staining: n(tumors per ancestry) = 24, 22 and 6, respectively. ****p<0.0001; **p<0.005, horizontal line: median, two-tailed unpaired t test. Scale bars: 100μm.


Figure 4 ( B ) Kaplan Meier plots of long - term overall survival of H / NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males ( darkgreen and lightgreen lines ) and females ( red and orange lines ) , showing statistically significant association of elevated HSD17B7 expression with poorer patient survival . Statistical significance was calculated by the log - rank test between the groups . High _ female : n = 57patients . Low _ female : n = 79 patients . High _ male : n = 203 patients . Low _ male : n = 181 patients . ( G ) Immunofluorescence ( IF ) analysis of HSD17B7 protein expression in White and Black HKCs . Shown are representative images as well as IF signal quantification at the level of individual cells ( dots ) for the indicated strains . 19 - 46 cells were counted in each case . n ( HKC strains per ancestry ) = 5 , * * * p < 0 . 0005 , horizontal line : median , 2 - tailed unpaired t - test . Unit of Y - axis : pixels . Scale bar : 20 μm .

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 O O O O O O O O O O O O O O O O O O O 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(B) Kaplan Meier plots of long-term overall survival of H/NSCC patients from the TCGA dataset divided on the basis of high versus low HSD17B7 expression levels relative to its median expression value among males (darkgreen and lightgreen lines) and females (red and orange lines), showing statistically significant association of elevated HSD17B7 expression with poorer patient survival. Statistical significance was calculated by the log-rank test between the groups. High_female: n=57patients. Low_female: n= 79 patients. High_male: n= 203 patients. Low_male: n= 181 patients.(G) Immunofluorescence (IF) analysis of HSD17B7 protein expression in White and Black HKCs. Shown are representative images as well as IF signal quantification at the level of individual cells (dots) for the indicated strains. 19-46 cells were counted in each case. n(HKC strains per ancestry) = 5, ***p<0.0005, horizontal line: median, 2-tailed unpaired t-test. Unit of Y-axis: pixels. Scale bar: 20 μm.


Figure 5 ( D - F ) Intradermal tumorigenicity assays with HKCs ( 269W ) stably infected with an HSD17B7 overexpressing lentivirus ( HSD17B7 - oe ) versus control ( Ctrl ) . Experimental conditions were the same as in Fig 2 . Mice were sacrificed 15 days after injection . Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti - pan keratin antibodies ( D , ) as well as % of Ki67 positive cells ( E ) and K1 positivity ( F ) of pan - keratin positive areas . For ( D ) , n ( tumors per condition ) = 9 ; for ( E and F ) n ( 3 fields per tumor , 5 tumors per condition ) = 15 . * p < 0 . 05 , * * * * p < 0 . 0001 . Horizontal line : median . 2 - tail unpaired t - test . Scale bar : 500μm ( D ) , 100μm ( F ) . ( G , H ) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 ( HSD17B7 - oe ) versus control ( Ctrl ) . Mice were sacrificed 15 days after injection . Shown are representative images and quantification of tumor cells density , assessed by immunohistochemical staining with anti - pan keratin antibodies ( G ) as well as % of Ki67 positive cells in pan - keratin positive areas ( H ) . For ( G ) , n ( tumors per condition ) = 4 ; for ( H ) , n ( 3 - 5 fields per tumor , 4 tumors per condition ) 15 , 19 . * p < 0 . 05 ; Horizontal line : median . One - tailed paired t - test . Scale bar in ( G , H ) : 100μm .

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 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

Figure 5(D-F) Intradermal tumorigenicity assays with HKCs (269W) stably infected with an HSD17B7 overexpressing lentivirus (HSD17B7-oe) versus control (Ctrl). Experimental conditions were the same as in Fig 2. Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density assessed by immunohistochemical staining with anti-pan keratin antibodies (D,) as well as % of Ki67 positive cells (E) and K1 positivity (F) of pan-keratin positive areas. For (D), n(tumors per condition) = 9; for (E and F) n(3 fields per tumor, 5 tumors per condition) = 15. *p<0.05, ****p<0.0001. Horizontal line: median. 2-tail unpaired t-test. Scale bar: 500μm (D), 100μm (F).(G, H) Intradermal tumorigenicity assays with SCC13 cells overexpressing HSD17B7 (HSD17B7-oe) versus control (Ctrl). Mice were sacrificed 15 days after injection. Shown are representative images and quantification of tumor cells density, assessed by immunohistochemical staining with anti-pan keratin antibodies (G) as well as % of Ki67 positive cells in pan-keratin positive areas (H). For (G), n(tumors per condition) = 4; for (H), n(3-5 fields per tumor, 4 tumors per condition) 15, 19. *p<0.05; Horizontal line: median. One-tailed paired t-test. Scale bar in (G, H): 100μm.


Figure 6 ( D ) Colony forming assays of the indicated SCC cell lines plus / minus HSD17B7 silencing as in the previous panels . Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in ( A ) . Data are represented as number of colonies + / - SD . n ( dishes per condition ) = 3 * p < 0 . 05 , * * p < 0 . 01 ; p * * * * < 0 . 0001 , 2 - tailed unpaired 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

Figure 6(D) Colony forming assays of the indicated SCC cell lines plus/minus HSD17B7 silencing as in the previous panels. Cells plated in triplicate dishes 5 days after lentiviral vector infection and selection were tested for clonogenic capability as in (A). Data are represented as number of colonies +/- SD. n(dishes per condition)= 3 *p<0.05, **p<0.01; p****<0.0001, 2-tailed unpaired t-test.


Figure 8 ( C , D ) SCC13 cells stably infected with an HSD17B7 - expressing lentivirus ( HSD - oe ) versus empty vector control ( Ctrl ) ( C ) or two HSD17B7 - silencing lentivirus ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) ( D ) were analyzed as in the previous panels . Shown are individual values for 3 parallel cultures per condition together with mean + / - SD . * * p < 0 . 005 ; * * * p < 0 . 0005 ; * * * * p < 0 . 0001 . 2 - tailed unpaired t - test . Additional metabolic measurement experiments of SCC cells plus / minus HSD17B7 overexpression and silencing , in the context of additional manipulations are shown in Fig 9 .

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

Figure 8(C, D) SCC13 cells stably infected with an HSD17B7-expressing lentivirus (HSD-oe) versus empty vector control (Ctrl) (C) or two HSD17B7-silencing lentivirus (shHSD2, shHSD4) versus vector control (shCtrl) (D) were analyzed as in the previous panels. Shown are individual values for 3 parallel cultures per condition together with mean +/- SD. **p<0.005; ***p<0.0005; ****p<0.0001. 2-tailed unpaired t-test. Additional metabolic measurement experiments of SCC cells plus/minus HSD17B7 overexpression and silencing, in the context of additional manipulations are shown in Fig 9.


Figure 9 ( C ) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type ( HSD - wt ) and Y193G and K197G mutated ( HSD - mut ) forms versus empty vector control ( Ctrl ) as in ( B ) were assayed for levels of Electron Transport Chain activity , ATP and mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * p < 0 . 005 ; * * * p < 0 . 0005 . 2 - tailed unpaired t - test . Individual experimental values are provided in Dataset EV3 . ( D ) SCC13 cells were infected with two HSD17B7 silencing lentiviruses ( shHSD2 , shHSD4 ) versus vector control ( shCtrl ) followed , 3 days after infection and antibiotic selection , by treatment with Zymosterol ( 2 . 5uM ) or ethanol vehicle alone for 48hours . Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity , ATP and mtROS production as in Fig 8A . Data are displayed as average values of triplicate measurements together with mean + / - SD . n ( dishes per condition ) = 3 . * * * * p < 0 . 0001 , 2 - tailed unpaired t - test . Individual experimental values are provided in Dataset EV3 .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 9(C) Purified mitochondrial preparation from SCC13 cells infected with lentiviruses expressing wild type (HSD-wt) and Y193G and K197G mutated (HSD-mut) forms versus empty vector control (Ctrl) as in (B) were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. **p<0.005; ***p<0.0005. 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.(D) SCC13 cells were infected with two HSD17B7 silencing lentiviruses (shHSD2, shHSD4) versus vector control (shCtrl) followed, 3 days after infection and antibiotic selection, by treatment with Zymosterol (2.5uM) or ethanol vehicle alone for 48hours. Purified mitochondrial preparation were assayed for levels of Electron Transport Chain activity, ATP and mtROS production as in Fig 8A. Data are displayed as average values of triplicate measurements together with mean +/- SD. n(dishes per condition)= 3. ****p<0.0001, 2-tailed unpaired t-test. Individual experimental values are provided in Dataset EV3.


Figure 1A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( A ) left untreated or treated with increasing concentrations of LLO for 15 min . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( B ) left uninfected ( U ) or infected with wt or Δhly Lm for 1 h . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( A - C ) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates ( WCL ) and Gp96 IP fractions ( IP Gp96 ) of HeLa cells : ( C ) infected with wt Lm for the indicated time points . ( A , C ) Quantifications of NMHCIIA in IP Gp96 are the mean ± SEM ( n ≥ 3 ) ( a . u . - arbitrary units ) . ( D ) Confocal microscopy images of HeLa cells : ( D ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) , immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) and stained with DAPI ( blue ) . Arrows point to various NMHCIIA - Gp96 positive cortical bundles in different cell . Insets show high magnification image ( E ) Quantification of Pearson ' s correlation coefficient ( PCC ) within : uninfected cells ( U ) , LLO - treated cells , cortical NMHCIIA bundles or equivalent size control cellular ROI . ( Ctrl ROI ) Data are mean ± SEM ( n = 6 ) ; p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( F ) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA ( green ) , Gp96 ( red ) Lm ( blue ) and stained with DAPI ( white ) . Arrows indicate NMHCIIA - Gp96 cortical bundles at cortical sites close to wt Lm . ( D and F ) Scale bar - 10 μm . See also Figs EV1 and EV2 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 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 1A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (A) left untreated or treated with increasing concentrations of LLO for 15 min. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (B) left uninfected (U) or infected with wt or Δhly Lm for 1 h. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(A-C) Immunoblots of Gp96 and NMHCIIA levels from whole cell lysates (WCL) and Gp96 IP fractions (IP Gp96) of HeLa cells: (C) infected with wt Lm for the indicated time points. (A, C) Quantifications of NMHCIIA in IP Gp96 are the mean±SEM (n≥3) (a.u. - arbitrary units).(D) Confocal microscopy images of HeLa cells: (D) left untreated or treated with LLO (0.5 nM, 15 min), immunolabelled for NMHCIIA (green), Gp96 (red) and stained with DAPI (blue). Arrows point to various NMHCIIA-Gp96 positive cortical bundles in different cell. Insets show high magnification image (E) Quantification of Pearson's correlation coefficient (PCC) within: uninfected cells (U), LLO-treated cells, cortical NMHCIIA bundles or equivalent size control cellular ROI. (Ctrl ROI) Data are mean±SEM (n=6); p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.05, **p<0.01, ***p<0.001(F) Confocal microscopy images of HeLa cells infected with wt or Δhly Lm for 6 h immunolabelled for NMHCIIA (green), Gp96 (red) Lm (blue) and stained with DAPI (white). Arrows indicate NMHCIIA-Gp96 cortical bundles at cortical sites close to wt Lm. (D and F) Scale bar - 10 μm. See also Figs EV1 and EV2.


Figure 2 ( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( A ) GFP - NMHCIIA . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( A ) Arrows indicate NMHCIIA bundles at PM blebbing sites . ( A - B ) Sequential frames of time - lapse confocal microscopy sequence of LLO - treated HeLa cells expressing ( B ) simultaneously GFP - NMHCIIA and mcherry - KDEL . LLO was added to culture medium 10 seconds before t0 . DIC - differential interference contrast . ( B ) Highlights depicting ER structures within NMHCIIA bundles and PM blebs . Arrows indicate cortical ER surrounding NMHCIIA accumulations , arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs ( C ) Confocal microscopy images of HeLa cells left untreated ( Control ) or treated with LLO ( 0 . 5 nM 15 min ) . Cells were stained with FITC - WGA ( Plasma membrane , PM - red ) and immunolabelled for NMHCIIA ( green ) . Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated ( 1 ) or detached ( 2 ) from the cell body . ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( D ) LLO pre - incubated with cholesterol ( LLOCHT ) ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( E ) LLO in medium supplemented with 140 mM K + ( HighK + ) and LLO in Ca2 + - free medium Ca2 + ( Ca2 + free ) ( D - F ) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with : LLO ( 0 . 5 nM , 15 min ) ( LLO ) and ( F ) LLO in the presence of 25 μM blebbistatin ( BB ) . ( G ) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER - KDEL ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . ( H ) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA ( green ) and Gp96 ( red ) . Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA . ( I ) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) , SLO ( 1 . 5 μg / ml , 30 min ) ( SLO ) or aerolysin ( 0 . 2 nM , 40 min ) ( AL ) .

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 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 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 2(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (A) GFP-NMHCIIA . LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (A) Arrows indicate NMHCIIA bundles at PM blebbing sites.(A-B) Sequential frames of time-lapse confocal microscopy sequence of LLO-treated HeLa cells expressing (B) simultaneously GFP-NMHCIIA and mcherry-KDEL. LLO was added to culture medium 10 seconds before t0. DIC - differential interference contrast. (B) Highlights depicting ER structures within NMHCIIA bundles and PM blebs. Arrows indicate cortical ER surrounding NMHCIIA accumulations, arrowheads indicate contact points between ER vesicles and NMHCIIA cables and asterisks point to ER vacuoles within PM blebs(C) Confocal microscopy images of HeLa cells left untreated (Control) or treated with LLO (0.5 nM 15 min). Cells were stained with FITC-WGA (Plasma membrane, PM-red) and immunolabelled for NMHCIIA (green). Insets show PM blebs and arrows indicate recruitment of NMHCIIA bundles to PM blebs associated (1) or detached (2) from the cell body.(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (D) LLO pre-incubated with cholesterol (LLOCHT)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (E) LLO in medium supplemented with 140 mM K+(HighK+) and LLO in Ca2+-free medium Ca2+ (Ca2+free)(D-F) Immunoblots of Gp96 and NMHCIIA levels from Gp96 IP of HeLa cells left untreated (U) or treated with: LLO (0.5 nM, 15 min) (LLO) and (F) LLO in the presence of 25 μM blebbistatin (BB).(G) Confocal microscopy images of HeLa cells treated as indicated and immunolabelled for ER-KDEL (blue), NMHCIIA (green) and Gp96 (red).(H) Confocal microscopy images of HeLa cells treated with aerolysin and immunolabelled for NMHCIIA (green) and Gp96 (red). Insets show NMHCIIA bundles and arrows indicate association between Gp96 and NMHCIIA.(I) Immunoblots of NMHCIIA and Gp96 levels from Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min), SLO (1.5 μg/ml, 30 min) (SLO) or aerolysin (0.2 nM, 40 min) (AL).


Figure 3 ( A ) Confocal microscopy images of shCtrl , shGp96 or shNMHCIIA HeLa cells treated with LLO ( 0 . 5 nM , 15 min ) and immunolabelled for KDEL - proteins ( blue ) , NMHCIIA ( green ) and Gp96 ( red ) . Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells . Scale bar - 10 μm . ( B - D ) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with ( B ) LLO ( 0 . 5 nM , 15 min ) , ( C ) aerolysin ( 0 . 2 nM , 40 min ) or ( D ) with wt Lm . Values are the mean ± SEM ( n ≥ 3 ) , p - values were calculated using ( B - C ) one - way - ANOVA with Dunnett post hoc analyses and ( D ) two - tailed un - paired Student ' s t - test , * p < 0 . 5 , * * * p < 0 . 001 . For shNMHCIIA , bundles were detected following actin staining ( Fig EV3C ) . ( E - F ) Number of ( E ) blebs per cell or ( F ) retracting blebs per cell evaluated by time - lapse microscopy analysis of LLO - treated shCtrl or shGp96 cells . shCtrl n = 32 cells and shGp96 n = 40 cells , p - values were calculated using two - tailed un - paired Student ' s t - test * p < 0 . 5 . ( G ) Sequential frames of time - lapse microscopy analysis of LLO - treated HeLa cells expressing GFP - NMHCIIA ( shCtrl , shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl - BB ) . LLO was added to culture medium 10 seconds before t0 . ( H ) Immunoblots of Ser19 - phosphorylated MRLC ( pMRLC ) , MRLC and actin levels from shCtrl or shGp96 cells left untreated ( U ) or treated with 0 . 1 nM LLO for the indicated time points . Quantification of pMRLC levels corresponds to the mean ± SEM ( n ≥ 3 ) , p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 , * * * p < 0 . 001 . See also Appendix Fig S2 and Fig EV3 .

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

Figure 3(A) Confocal microscopy images of shCtrl, shGp96 or shNMHCIIA HeLa cells treated with LLO (0.5 nM, 15 min) and immunolabelled for KDEL-proteins (blue), NMHCIIA (green) and Gp96 (red). Insets show compact NMHCIIA bundles in shCtrl cells and dispersed NMHCIIA bundles in shGp96 cells. Scale bar - 10 μm. (B-D) Quantification of the % of cells harbouring NMHCIIA bundles after incubation with (B) LLO (0.5 nM, 15 min), (C) aerolysin (0.2 nM, 40 min) or (D) with wt Lm. Values are the mean ±SEM (n≥3), p-values were calculated using (B-C) one-way-ANOVA with Dunnett post hoc analyses and (D) two-tailed un-paired Student's t-test, *p<0.5, ***p<0.001. For shNMHCIIA, bundles were detected following actin staining (Fig EV3C).(E-F) Number of (E) blebs per cell or (F) retracting blebs per cell evaluated by time-lapse microscopy analysis of LLO-treated shCtrl or shGp96 cells. shCtrl n=32 cells and shGp96 n=40 cells, p-values were calculated using two-tailed un-paired Student's t-test *p<0.5. (G) Sequential frames of time-lapse microscopy analysis of LLO-treated HeLa cells expressing GFP-NMHCIIA (shCtrl, shGp96 and shCtrl with 25 μM Blebbistatin - shCtrl-BB). LLO was added to culture medium 10 seconds before t0.(H) Immunoblots of Ser19-phosphorylated MRLC (pMRLC), MRLC and actin levels from shCtrl or shGp96 cells left untreated (U) or treated with 0.1 nM LLO for the indicated time points. Quantification of pMRLC levels corresponds to the mean ± SEM (n≥3), p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01, ***p<0.001. See also Appendix Fig S2 and Fig EV3.


Figure 4 ( Ai ) Confocal microscopy images of LLO - treated HeLa cells immunolabelled for the indicated proteins . Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles . ( Aii ) Quantification of Pearson ' s correlation coefficient ( PCC ) between NMHCIIA and the indicated proteins within entire LLO - treated cells , cortical bundles or equivalent size control cellular ROI ( Ctrl ROI ) . For Calpain 2 cortical bundles were defined by actin staining . Data are representative of one experiment repeated 3 independent times with similar results . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 ; * * * p < 0 . 001 , ns - non significant ( B ) Immunoblots of NMHCIIA , Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with LLO ( 0 . 5 nM , 15 min ) ( LLO ) . ( C ) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated ( U ) or treated with 25 μg / ml of Wnt5a for the indicated time . ( D ) Confocal microscopy images of HeLa cells untreated ( Mock ) or treated with 25 μg / ml of Wnt5a for 30 min . Cells were immunolabelled for ER - KDEL ( blue ) , Gp96 ( red ) and NMHCIIA ( green ) . Arrows indicate polarised localization of NMHCIIA , ER - KDEL and Gp96 in Wnt5a - treated cells . ( E - F ) Confocal microscopy images of shCtlr , shGp96 or shNMHCIIA HeLa cells stained for actin ( red ) and DAPI ( blue ) and immunolabelled for ( E ) NMHCIIA ( green ) or ( F ) focal adhesion kinase ( FAK ) ( green ) . ( A , D - F ) Scale bar - 10 μm . Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points . Quantification of the % of cells with ( G ) stress fibers or ( H ) focal adhesion points labelled by FAK . ( I - J ) Wound closure assay . ( I ) Sequential frames of time - lapse microscopy of HeLa cells grown to confluence separated by a stopper . Stopper was removed at t0 and cellular migration was imaged for the indicated times . ( J ) Quantification of the % of wound area occupied by migrating cells over time stopper removal ( upper panel ) and respective rate of closure ( lower panel ) .

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

Figure 4(Ai) Confocal microscopy images of LLO-treated HeLa cells immunolabelled for the indicated proteins. Insets show colocalization between NMHCIIA and WRAMP components at cortical bundles. (Aii) Quantification of Pearson's correlation coefficient (PCC) between NMHCIIA and the indicated proteins within entire LLO-treated cells, cortical bundles or equivalent size control cellular ROI (Ctrl ROI). For Calpain 2 cortical bundles were defined by actin staining. Data are representative of one experiment repeated 3 independent times with similar results. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses ** p<0.01; *** p<0.001, ns - non significant(B) Immunoblots of NMHCIIA, Gp96 and Filamin A levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with LLO (0.5 nM, 15 min) (LLO).(C) Immunoblots of NMHCIIA and Gp96 levels from WCL and Gp96 IP of HeLa cells left untreated (U) or treated with 25 μg/ml of Wnt5a for the indicated time.(D) Confocal microscopy images of HeLa cells untreated (Mock) or treated with 25 μg/ml of Wnt5a for 30 min. Cells were immunolabelled for ER-KDEL (blue), Gp96 (red) and NMHCIIA (green). Arrows indicate polarised localization of NMHCIIA, ER-KDEL and Gp96 in Wnt5a-treated cells.(E-F) Confocal microscopy images of shCtlr, shGp96 or shNMHCIIA HeLa cells stained for actin (red) and DAPI (blue) and immunolabelled for (E) NMHCIIA (green) or (F) focal adhesion kinase (FAK) (green). (A, D-F) Scale bar - 10 μm. Insets show sites with focal adhesion points and arrows indicate stress fibers or distinct focal adhesion points. Quantification of the % of cells with (G) stress fibers or (H) focal adhesion points labelled by FAK.(I-J) Wound closure assay. (I) Sequential frames of time-lapse microscopy of HeLa cells grown to confluence separated by a stopper. Stopper was removed at t0 and cellular migration was imaged for the indicated times. (J) Quantification of the % of wound area occupied by migrating cells over time stopper removal (upper panel) and respective rate of closure (lower panel).


Figure 5 ( A ) Epifluorescence microscopy images of supernatants from LLO - treated ( 0 . 5 nM 15 min ) shCtlr , shGp96 or shNMHCIIA HeLa cells collected into poly - lysine - coated cover slips , fixed and stained with FITC - WGA ( green ) and DAPI ( blue ) . Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei . Scale bar - 10 μm . ( B ) Quantification of released PM blebs ( total FITCWGA / field ) . Values are the means ± SEM ( n ≥ 3 ) . * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( C ) 0 . 5 nM LLO for the indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( D ) 1 . 5 or 3 nM LLO for 15 min ; or ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . ( C - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa cells . Cells were treated with ( E ) 0 . 1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times . Levels of PI incorporation by untreated cells were subtracted from all LLO - treated samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 .

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

Figure 5(A) Epifluorescence microscopy images of supernatants from LLO-treated (0.5 nM 15 min) shCtlr, shGp96 or shNMHCIIA HeLa cells collected into poly-lysine-coated cover slips, fixed and stained with FITC-WGA (green) and DAPI (blue). Insets show PM blebs of variable sizes and DAPI staining confirms the absence of cell nuclei. Scale bar - 10 μm. (B) Quantification of released PM blebs (total FITCWGA/field). Values are the means ± SEM (n≥3). ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (C) 0.5 nM LLO for the indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (D) 1.5 or 3 nM LLO for 15 min; or (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.(C-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa cells. Cells were treated with (E) 0.1 nM LLO for 10 min followed by LLO washed out and recovery for indicated times. Levels of PI incorporation by untreated cells were subtracted from all LLO-treated samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001.


Figure 6 ( A - B ) Epifluorecence microscopy images of HeLa cells ( A ) left untreated or treated with LLO ( 0 . 5 nM , 15 min ) . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria . ( A - B ) Epifluorecence microscopy images of HeLa cells ( B ) infected with GFP - expressing wt Lm for 6 h . Cells were incubated with exofacial - PS probe ( Alexa - 568 annexin A5 ) ( red ) 30 min prior fixation , immunolabelled for NMHCIIA ( green ) and stained with DAPI ( blue ) . Insets show PM damage marked by exofacial - PS sites . Arrows indicate exofacial - PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles ; arrowheads indicate NMHCIIA bundles associated with exofacial - PS sites without detectable bacteria . ( C ) Quantification of the % of infected cells with exofacial - PS sites in HeLa cells infected with wt Lm for 6 h . Data are the mean ± SEM ( n = 3 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 . ( D - E ) Flow cytometry analysis of the % of PI - positive shCtlr , shGp96 or shNMHCIIA HeLa and RAW264 . 7 cells , infected with GFP - expressing wt or Δhly Lm for 6 h . The levels of PI incorporation by uninfected cells were subtracted from infected samples . Values are the mean ± SEM ( n ≥ 4 ) and p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * p < 0 . 5 , * * p < 0 . 01 , * * * p < 0 . 001 . PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria ( Fig Appendix Fig S3 ) .

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 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 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

Figure 6(A-B) Epifluorecence microscopy images of HeLa cells (A) left untreated or treated with LLO (0.5 nM, 15 min). Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(A-B) Epifluorecence microscopy images of HeLa cells (B) infected with GFP-expressing wt Lm for 6 h. Cells were incubated with exofacial-PS probe (Alexa-568 annexin A5) (red) 30 min prior fixation, immunolabelled for NMHCIIA (green) and stained with DAPI (blue). Insets show PM damage marked by exofacial-PS sites. Arrows indicate exofacial-PS associated with intracellular bacteria and in shCtrl cells with NMHCIIA bundles; arrowheads indicate NMHCIIA bundles associated with exofacial-PS sites without detectable bacteria.(C) Quantification of the % of infected cells with exofacial-PS sites in HeLa cells infected with wt Lm for 6 h. Data are the mean ±SEM (n=3) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5.(D-E) Flow cytometry analysis of the % of PI-positive shCtlr, shGp96 or shNMHCIIA HeLa and RAW264.7 cells, infected with GFP-expressing wt or Δhly Lm for 6 h. The levels of PI incorporation by uninfected cells were subtracted from infected samples. Values are the mean ± SEM (n≥4) and p-values were calculated using one-way-ANOVA with Tukey post hoc analyses *p<0.5, **p<0.01, ***p<0.001. PM permeability was assessed in cells harbouring equivalent numbers of intracellular bacteria (Fig Appendix Fig S3).


Figure 7 ( A ) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates ( WCL ) and IP fractions ( IP Gp96 ) of extracts of zebrafish larvae 3 dpf uninfected ( U ) or infected with wt or ∆ hly Lm ( low dose 200 - 1500 CFU ) for 24h . ( B ) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control ( Ctrl - mo ) or Gp96 morpholino oligonucleotides ( Gp96 mo ) . ( C ) Confocal microscopy images of zebrafish larvae ( Ctrl mo or Gp96 mo ) infected ( low dose ) in the tail muscle , with the GFP - expressing wt or Δhly Lm for 24 h , immunolabelled for KDEL - proteins ( green ) , and stained with phalloidin ( actin , red ) and DAPI ( white ) . Images show actin rich structures at the cell cortex ( arrows ) and ER - KDEL puncta ( arrowheads ) in larvae infected with wt strain . ( D ) CFU counts per zebrafish larvae ( Ctrl or Gp96 mo ) infected with wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm and analysed at 0 , 24 , 48 hpi . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) and each circle represents 1 larvae . p - values were calculated using one - way - ANOVA with Tukey post hoc analyses * * p < 0 . 01 . ( E ) Survival curves of zebrafish larvae ( Ctrl - mo or Gp96 - mo ) infected with a wt ( low dose ) or Δhly ( high dose > 10 , 000 CFU ) Lm . Results are the mean ± SEM ( n ≥ 3 ) . wt infection of Ctrl and Gp96 mo , n = 28 larvae ; Δhly infection of Gp96 mo n = 13 . p - values were calculated using Log Rank test , * * p < 0 . 01 . ( F ) CFU counts per zebrafish larvae infected with a high dose of ∆ hly Lm at different times post - infection . Results are mean ± SEM ( n ≥ 3 ) ( horizontal bars ) ; each point represents 1 larvae . See also Fig EV5 .

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 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 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 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 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

Figure 7(A) Immunoblots of NMHCIIA and Gp96 levels from whole cell lysates (WCL) and IP fractions (IP Gp96) of extracts of zebrafish larvae 3 dpf uninfected (U) or infected with wt or ∆hly Lm (low dose 200-1500 CFU) for 24h.(B) Immunoblots of Gp96 and tubulin levels from zebrafish 3 dpf larvae injected with control (Ctrl-mo) or Gp96 morpholino oligonucleotides (Gp96 mo).(C) Confocal microscopy images of zebrafish larvae (Ctrl mo or Gp96 mo) infected (low dose) in the tail muscle, with the GFP-expressing wt or Δhly Lm for 24 h, immunolabelled for KDEL-proteins (green), and stained with phalloidin (actin, red) and DAPI (white). Images show actin rich structures at the cell cortex (arrows) and ER-KDEL puncta (arrowheads) in larvae infected with wt strain.(D) CFU counts per zebrafish larvae (Ctrl or Gp96 mo) infected with wt (low dose) or Δhly (high dose > 10,000 CFU) Lm and analysed at 0, 24, 48 hpi. Results are mean ± SEM (n≥3) (horizontal bars) and each circle represents 1 larvae. p-values were calculated using one-way-ANOVA with Tukey post hoc analyses **p<0.01.(E) Survival curves of zebrafish larvae (Ctrl-mo or Gp96-mo) infected with a wt (low dose) or Δhly (high dose > 10,000 CFU) Lm. Results are the mean ± SEM (n≥3). wt infection of Ctrl and Gp96 mo, n=28 larvae; Δhly infection of Gp96 mo n=13. p-values were calculated using Log Rank test, **p<0.01.(F) CFU counts per zebrafish larvae infected with a high dose of ∆hly Lm at different times post-infection. Results are mean ± SEM (n≥3) (horizontal bars); each point represents 1 larvae. See also Fig EV5.


Figure 1A ) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation ( 15 Gy ) followed by analysis by SDS - PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin . ( NT , no treatment ) . The lysate from the 24 hours time point was treated with phosphatase ( Ppase ) . B ) U2OS cells were left untreated or treated with ATM ( KU 55933 , 10 μM ) , ATR ( ETP - 464 , 1 μM ) , or DNA - PK ( NU7441 , 1 μM ) inhibitors 30 minutes prior to exposure to IR ( 15 Gy , 2h recovery ) . Immunoblots were performed with indicated antibodies . C ) U2OS cells were left untreated ( NT ) or exposed to HU ( 2 mM ) for the indicated times . The lysate from the 24 hour time point was treated with phosphatase ( Ppase ) . Immunoblots were performed with indicated antibodies . D ) Untreated U2OS cells or cells exposed to either HU ( 2 mM , 24h ) or IR ( 15 Gy , 2h recovery ) alone or together with ATM inhibitor ( KU55933 , 10 μM ) or inhibitors to ATR ( ATR # 1 : ETP - 464 , 1 μM ; ATR # 2 : AZ - 20 , 3 μM ) were harvested and lysates were prepared . The inhibitors were added 30 minutes prior to HU or IR treatment . Immunoblots were performed with indicated antibodies . E ) ATR kinase assay was performed with N - terminal PALB2 ( aa 1 - 560 ; GST - PALB2 - N ) as a substrate and HA - tagged ATR as kinase and visualized by Fujifilm Phosphoimager . 293T cells transfected with empty vector ( EV ) , GST , and ATR inhibition ( AZ - 20 , 30 μM ) are controls . HA - ATR and GST - PALB2 - N was controlled by immunoblotting with HA and GST antibodies .

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Figure 1A) U2OS cells were harvested at the indicated time points after treatment with ionizing radiation (15 Gy) followed by analysis by SDS-PAGE and immunoblotting with antibodies recognizing PALB2 and Vinculin. (NT, no treatment). The lysate from the 24 hours time point was treated with phosphatase (Ppase).B) U2OS cells were left untreated or treated with ATM (KU 55933, 10 μM), ATR (ETP-464, 1 μM), or DNA-PK (NU7441, 1 μM) inhibitors 30 minutes prior to exposure to IR (15 Gy, 2h recovery). Immunoblots were performed with indicated antibodies.C) U2OS cells were left untreated (NT) or exposed to HU (2 mM) for the indicated times. The lysate from the 24 hour time point was treated with phosphatase (Ppase). Immunoblots were performed with indicated antibodies.D) Untreated U2OS cells or cells exposed to either HU (2 mM, 24h) or IR (15 Gy, 2h recovery) alone or together with ATM inhibitor (KU55933, 10 μM) or inhibitors to ATR (ATR#1: ETP-464, 1 μM; ATR#2: AZ-20, 3 μM) were harvested and lysates were prepared. The inhibitors were added 30 minutes prior to HU or IR treatment. Immunoblots were performed with indicated antibodies.E) ATR kinase assay was performed with N-terminal PALB2 (aa 1-560; GST-PALB2-N) as a substrate and HA-tagged ATR as kinase and visualized by Fujifilm Phosphoimager. 293T cells transfected with empty vector (EV), GST, and ATR inhibition (AZ-20, 30 μM) are controls. HA-ATR and GST-PALB2-N was controlled by immunoblotting with HA and GST antibodies.


Figure 2B ) Characterization of the Doxycycline - inducible cell lines expressing siRNA resistant wild type ( WT ) or mutant ( TMA or TMD ) FLAG / HA - tagged PALB2 . The cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA for 24 hours prior to induction with Doxycycline . Immunoblots were performed with indicated antibodies . C ) Cell line characterization of FLAG / HA - tagged PALB2 localization 2 hours following exposure to 15 Gy of IR . Z - stack max intensity image projection and background subtraction was done using ImageJ / Fiji . Scale bar = 5μm . D ) Kinase assay was performed as described in Figure 1E except that the SDS - PAGE gel was Coomassie stained following electrophoresis . WT was used at two different concentrations ( 1x and 0 . 75x ) . E ) Doxycycline - inducible stable U2OS cell lines were left untreated or treated with Doxycycline and / or IR ( 15 Gy , 2h recovery ) . Following protein extraction the lysates were immunoprecipitated ( IP ) with IgG or antibody recognizing phosphorylated S / Q sites ( pS / Q ) and analyzed by SDS - PAGE . Immunoblots were performed with indicated antibodies .

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Figure 2B) Characterization of the Doxycycline-inducible cell lines expressing siRNA resistant wild type (WT) or mutant (TMA or TMD) FLAG/HA-tagged PALB2. The cell lines were transfected with UNC (Negative Control) or PALB2 siRNA for 24 hours prior to induction with Doxycycline. Immunoblots were performed with indicated antibodies.C) Cell line characterization of FLAG/HA-tagged PALB2 localization 2 hours following exposure to 15 Gy of IR. Z-stack max intensity image projection and background subtraction was done using ImageJ/Fiji. Scale bar = 5μm.D) Kinase assay was performed as described in Figure 1E except that the SDS-PAGE gel was Coomassie stained following electrophoresis. WT was used at two different concentrations (1x and 0.75x).E) Doxycycline-inducible stable U2OS cell lines were left untreated or treated with Doxycycline and/or IR (15 Gy, 2h recovery). Following protein extraction the lysates were immunoprecipitated (IP) with IgG or antibody recognizing phosphorylated S/Q sites (pS/Q) and analyzed by SDS-PAGE. Immunoblots were performed with indicated antibodies.


Figure 3A ) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later . Z - stack images were acquired with a 60X oil objective ( Deltavision ) . Z stack max intensity projection , background subtraction , and foci count was done using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm . B ) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU . 24 hours later the cells were fixed and processed for IF as in Figure 3A . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Scale bar = 5μm . C ) HeLa DR - GFP cell line was used to analyze homologous recombination activity . Cells were transfected with PALB2 siRNA followed by transfection of I - SceI together with empty vector ( EV ) , WT - , TMA - , or TMD - PALB2 the next day . 72 hours later cells were fixed and analyzed by FACS . The percentage of cells with GFP signal from the empty vector sample was set to one ( dotted line ) . The graph shows a representative image from three biological repeats ( error bars = SEM ) . D ) Phosphorylation of PALB2 supports RAD51 foci . Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1 / siBLM for 6 hours then induced for expression of PALB2 ( WT and TMA ) . 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B . Cells in S - phase ( EdU + ) at the time of HU treatment were Click - IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ / Fiji ( * p < 0 . 0001 , unpaired Student ' s t - test ) . Representative images of displaying RAD51 ( green ) and HA - PALB2 ( red ) localization in HU treated EdU positive ( not shown ) cells . Scale bar = 5μm .

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Figure 3A) PALB2 cell lines transfected with PALB2 siRNA followed by inducible expression of siRNA resistant PALB2 versions were exposed to 15 Gy of IR and fixed for IF 2 hours later. Z-stack images were acquired with a 60X oil objective (Deltavision). Z stack max intensity projection, background subtraction, and foci count was done using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar = 5μm.B) PALB2 cell lines were transfected and the exogenous PALB2 induced as in Figure 3A before they were pulsed with 10 μM EdU for 20 minutes prior to addition of 2 mM HU. 24 hours later the cells were fixed and processed for IF as in Figure 3A. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji. (* p<0.0001, unpaired Student's t-test). Scale bar=5μm.C) HeLa DR-GFP cell line was used to analyze homologous recombination activity. Cells were transfected with PALB2 siRNA followed by transfection of I-SceI together with empty vector (EV), WT-, TMA-, or TMD-PALB2 the next day. 72 hours later cells were fixed and analyzed by FACS. The percentage of cells with GFP signal from the empty vector sample was set to one (dotted line). The graph shows a representative image from three biological repeats (error bars=SEM).D) Phosphorylation of PALB2 supports RAD51 foci. Cells were transfected with siPALB2 overnight followed by forward transfection of siFBH1/siBLM for 6 hours then induced for expression of PALB2 (WT and TMA). 24 hours following induction cells were pulsed with EdU and exposed to HU as in Figure 3B. Cells in S-phase (EdU+) at the time of HU treatment were Click-IT labeled with an Alexa Fluor 647 azide and RAD51 foci in EdU positive cells were enumerated using ImageJ/Fiji (* p<0.0001, unpaired Student's t-test). Representative images of displaying RAD51 (green) and HA-PALB2 (red) localization in HU treated EdU positive (not shown) cells. Scale bar=5μm.


Figure 4A ) U2OS and PALB2 cell lines were transfected with UNC ( Negative Control ) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines . Left panel , cells were treated the following day with IR ( 5 Gy ) and 2 hours later Nocodazole was added for 6 hours , or treated with only Nocodazole for 6 hours . Right panel , cells were treated with IR ( 5 Gy ) and left to recover for 16 hours . Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry . The percentages of mitotic cells in the IR + Nocodazole samples were normalized to the Nocodazole samples ( left panel , n = 3 , error bars = SEM ) . For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT - PALB2 expressing cells was set to one ( right panel ) . ( n = 3 , * * p < 0 . 005 , * p < 0 . 05 , unpaired Student ' s t - test , error bars = SEM ) . B ) The relative intensity of phosphorylated H2AX ( pS139 ) was examined in the total population of WT - and TMA - PALB2 expressing cell lines 18 hours post exposure to IR ( 5 Gy ) . Cell lines were prepared as in above , fixed at the indicated time point , and stained for pH2AX . Cells were imaged with a 20X air objective on a Scan ^ R workstation ( Olympus ) , mean relative pH2AX intensity was calculated from background subtracted images using the Scan ^ R analysis software . ( * p < 0 . 0001 , unpaired Student ' s t - test ) . C ) Number of 53BP1 nuclear in TMA - PALB2 expressing G1 daughter cells . Cell lines were prepared as above and left untreated ( NT ) or exposed to IR ( 3 Gy ) , and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis . The irradiated bi - nucleated cells were scored for 53BP1 nuclear bodies . Scale bar = 10μm .

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 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 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

Figure 4A) U2OS and PALB2 cell lines were transfected with UNC (Negative Control) or PALB2 siRNA and 24 hours later Doxycycline was added to the PALB2 cell lines. Left panel, cells were treated the following day with IR (5 Gy) and 2 hours later Nocodazole was added for 6 hours, or treated with only Nocodazole for 6 hours. Right panel, cells were treated with IR (5 Gy) and left to recover for 16 hours. Cells were fixed and stained with the pMPM2 antibody to detect the mitotic cells by flow cytometry. The percentages of mitotic cells in the IR+Nocodazole samples were normalized to the Nocodazole samples (left panel, n=3, error bars=SEM). For the samples with the 16 hour recovery after IR the percentage of mitotic cells in the WT-PALB2 expressing cells was set to one (right panel). (n=3, **p<0.005, *p<0.05, unpaired Student's t-test, error bars=SEM).B) The relative intensity of phosphorylated H2AX (pS139) was examined in the total population of WT- and TMA-PALB2 expressing cell lines 18 hours post exposure to IR (5 Gy). Cell lines were prepared as in above, fixed at the indicated time point, and stained for pH2AX. Cells were imaged with a 20X air objective on a Scan^R workstation (Olympus), mean relative pH2AX intensity was calculated from background subtracted images using the Scan^R analysis software. (* p<0.0001, unpaired Student's t-test).C) Number of 53BP1 nuclear in TMA-PALB2 expressing G1 daughter cells. Cell lines were prepared as above and left untreated (NT) or exposed to IR (3 Gy), and 16 hours later Cytochalasin B was added for 8 hours to block cytokinesis. The irradiated bi-nucleated cells were scored for 53BP1 nuclear bodies. Scale bar = 10μm.


Figure 1B , Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope . C , Brightfield and fluorescence images of a cell migrating through a 2 µm constriction . Arrows indicate areas of accumulation of nesprin - 2 at the front of the nucleus as it is deformed through the constriction . E , Brightfield and fluorescence images of a cell migrating along a 15 µm control channel . F , Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel . The average intensity at the front and the back is normalized to the intensity at the sides , demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation . Error bars correspond to the standard deviation . Constriction : n = 17 cells over 4 biological replicates , 15 µm channels : n = 20 cells over 3 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point . G , GFP - nesprin - 2 cells deformed by the pressure gradient in a micropipette aspiration device . Note that the GFP signal does not increase at the tip but increases at sides of the nucleus .

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Figure 1B, Mouse embryonic fibroblasts genetically modified using CRISPR to introduce a GFP sequence immediately after the start codon of Syne2 display fluorescence at the nuclear envelope.C, Brightfield and fluorescence images of a cell migrating through a 2 µm constriction. Arrows indicate areas of accumulation of nesprin-2 at the front of the nucleus as it is deformed through the constriction.E, Brightfield and fluorescence images of a cell migrating along a 15 µm control channel.F, Quantification of the intensity of the fluorescent signal around the perimeter of the nucleus in a 2 µm constriction and a 15 µm control channel. The average intensity at the front and the back is normalized to the intensity at the sides, demonstrating an increase in the signal at the front of the nucleus as it is deformed and a reduction after deformation. Error bars correspond to the standard deviation. Constriction: n=17 cells over 4 biological replicates, 15 µm channels: n=20 cells over 3 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.G, GFP-nesprin-2 cells deformed by the pressure gradient in a micropipette aspiration device. Note that the GFP signal does not increase at the tip but increases at sides of the nucleus.


Figure 2A , CRISPR - modified cells ( Brightfield , left ) that express GFP - nesprin - 2 ( middle ) and mCh - LAC ( right ) show nesprin ( orange arrow ) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction . B , Quantification of intensity of GFP - nesprin - 2 and mCh - LAC , normalized to the intensity at the sides . Error bars correspond to the standard deviation . Constriction : n = 19 cells over 5 biological replicates , 15 µm : n = 27 over 6 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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

Figure 2A, CRISPR-modified cells (Brightfield, left) that express GFP-nesprin-2 (middle) and mCh-LAC (right) show nesprin (orange arrow) but not lamin accumulation at the front of the nucleus as it is deformed through a narrow constriction. B, Quantification of intensity of GFP-nesprin-2 and mCh-LAC, normalized to the intensity at the sides. Error bars correspond to the standard deviation. Constriction: n=19 cells over 5 biological replicates, 15 µm: n= 27 over 6 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars (*P < 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


Figure 3A , The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility . Inset are shown close - up views of the bleached areas at the front of the nucleus . B , Quantification of the recovery . Error bars correspond to the standard error of the mean , n = 24 over 3 biological replicates .

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Figure 3A, The deformed nucleus of cells migrating through narrow constrictions was bleached at the front and the side to determine mobility. Inset are shown close-up views of the bleached areas at the front of the nucleus. B, Quantification of the recovery. Error bars correspond to the standard error of the mean, n=24 over 3 biological replicates.


Figure 4A , Expression of the artificial mini - nesprin - 2G construct results in significant nesprin accumulation ( orange arrow ) in cells migrating through narrow constrictions . C , Quantification of the mini - nesprin - 2G accumulation of cells migrating through narrow constrictions . Note that the mutant construct does not accumulate at the front of the nucleus . Error bars correspond to the standard deviation . Mini - nesprin - 2G : n = 10 cells over 4 biological replicates ; Mutant : n = 11 cells over 4 biological replicates . A Wilcoxon signed - rank test is used to compare matched samples , stars ( * P < 0 . 05 , * * P < 0 . 01 ) denote comparisons to the measurement at the side , and pounds ( # P < 0 . 05 , # # P < 0 . 01 ) denote a comparison to the " before " time point .

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Figure 4A, Expression of the artificial mini-nesprin-2G construct results in significant nesprin accumulation (orange arrow) in cells migrating through narrow constrictions.C, Quantification of the mini-nesprin-2G accumulation of cells migrating through narrow constrictions. Note that the mutant construct does not accumulate at the front of the nucleus. Error bars correspond to the standard deviation. Mini-nesprin-2G: n=10 cells over 4 biological replicates; Mutant: n=11 cells over 4 biological replicates. A Wilcoxon signed-rank test is used to compare matched samples, stars(*P< 0.05, **P < 0.01) denote comparisons to the measurement at the side, and pounds (#P < 0.05, ##P < 0.01) denote a comparison to the "before" time point.


Figure 5A , Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces . Cross - sections along the direction of migration ( yellow ) and perpendicular to the direction of migration ( blue , 1 - 4 ) are shown below . B , A confocal slice on the surface of the nucleus indicates that GFP - nesprin - 2 co - localizes with actin cables at the surface of the nucleus . Dashed white lines represent the outlines of the constriction . C , Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope . Dashed white lines represent the outlines of the constriction . The distance between mCh - LAC and GFP - nesprin - 2 was measured at the back , at the constriction , at the front near the constriction , and at the center of the front . ( N = 17 cells , over 3 biological replicates . The alignment of the two channels was corrected using at least 28 fluorescent beads . ) D , Two - photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus , but not ablation at the back . The nuclei of DN - KASH , which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei . Bars represent the average value . Error bars represent the standard error on the mean . Back : N = 9 , Front : N = 30 , Cell Front : N = 16 , and DNKASH : N = 31 , 3 biological replicates .

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Figure 5A, Fixed cells labelled with phalloidin shows filamentous actin accumulation on the edges of the pillars and along the top and bottom surfaces. Cross-sections along the direction of migration (yellow) and perpendicular to the direction of migration (blue, 1-4) are shown below.B, A confocal slice on the surface of the nucleus indicates that GFP-nesprin-2 co-localizes with actin cables at the surface of the nucleus. Dashed white lines represent the outlines of the constriction.C, Spinning disk confocal microscopy on live cells reveals the elongation state of nesprins at the nuclear envelope. Dashed white lines represent the outlines of the constriction. The distance between mCh-LAC and GFP-nesprin-2 was measured at the back, at the constriction, at the front near the constriction, and at the center of the front. (N = 17 cells, over 3 biological replicates. The alignment of the two channels was corrected using at least 28 fluorescent beads.)D, Two-photon laser ablation reveals that ablation at the front of the nucleus results in a rearward movement of the nucleus, but not ablation at the back. The nuclei of DN-KASH, which show a decrease of endogenous nesprin at the nuclear envelope show a dampened rearward movement upon ablation at the front of nuclei. Bars represent the average value. Error bars represent the standard error on the mean. Back: N= 9, Front: N= 30, Cell Front: N = 16, and DNKASH: N=31, 3 biological replicates.


Figure 1HMRs express RNAs . Heat maps show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses . metaplots ( B ) show RNA expression levels in 16 . 5 dpc prospermatogonia . Only uniquely mapped RNA - seq reads were used for the analyses . Positions ( 1 ) - ( 4 ) in ( A and B ) ( green arrows ) indicate the same positions ( ± 2 kbp from the centers ) . The lower chart in ( B ) represents mappability defined as the percentage of unique sequences in all possible 50 - nt sequences ( see Appendix Figure S2B ) . TSS isoforms are included in 55798 TSSs . Low mappability at the centers of HMR _ - L1s is due to high occurrence of repeat ( retrotransposon ) sequences . RPM , reads per million mapped reads .

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Figure 1HMRs express RNAs. Heat maps show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses.metaplots (B) show RNA expression levels in 16.5 dpc prospermatogonia. Only uniquely mapped RNA-seq reads were used for the analyses. Positions (1)-(4) in (A and B) (green arrows) indicate the same positions (±2 kbp from the centers). The lower chart in (B) represents mappability defined as the percentage of unique sequences in all possible 50-nt sequences (see Appendix Figure S2B). TSS isoforms are included in 55798 TSSs. Low mappability at the centers of HMR_-L1s is due to high occurrence of repeat (retrotransposon) sequences. RPM, reads per million mapped reads.


Figure 2Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons ( HMR _ - L1s ) . Metaplots show the average ChIP - seq signals across HMR _ - L1s , transcription start sites ( TSSs ) , and randomly selected sites . The signals were normalized by either H3 or input signal . The results shown in heat maps were not normalized by H3 or input . The initiation form of Pol2 with an unphosphorylated carboxy terminal domain ( Pol2 null - P CTD ) is highly enriched in HMR _ - L1s .

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Figure 2Chromatin marks associated with promoters are observed in hypomethylated regions without young LINE1 retrotransposons (HMR_-L1s). Metaplots show the average ChIP-seq signals across HMR_-L1s, transcription start sites (TSSs), and randomly selected sites. The signals were normalized by either H3 or input signal. The results shown in heat maps were not normalized by H3 or input.The initiation form of Pol2 with an unphosphorylated carboxy terminal domain (Pol2 null-P CTD) is highly enriched in HMR_-L1s.


Figure 3RNA - immunoprecipitation ( RIP ) analyses of EGFP - MIWI2 and EGFP . EGFP - MIWI2 and EGFP were immunoprecipitated from 16 . 5 dpc prospermatogonia , and long RNAs bound to these proteins and input RNAs were sequenced . RNAs expressed from HMR _ - L1s and piRNA clusters , but not Ensembl and prospermatogonium - expressed mRNAs , are overall enriched by immunoprecipitation ( IP ) of EGFP - MIWI2 ( left ) . This enrichment is not observed when EGFP is immunoprecipitated ( right ) . The numbers of mRNAs , piRNA clusters , and HMR _ - L1s analyzed were shown in parentheses . Prospermatogonium - expressed mRNAs are those that are highly and specifically expressed in prospermatogonia . The bottom and top of boxes represent 25 % percentile and 75 % percentile , respectively . The line in the box represents the median . Circles represent outliers , and the bottom ( top ) of whiskers represents the minimum ( maximum ) values except for outliers . RIP analyses of EGFP - MIWI2 in newborn testes from WT and Mili KO mice . RNA bound to EGFP - MIWI2 and input RNA were sequenced . All RNAs derived from each class was counted for this analysis . HMR _ - L1s preferentially generate piRNAs . X - and Y - axes represent expression levels of long RNAs ( 16 . 5 dpc prospermatogonia ) as determined by RNA - seq and MIWI2 - bound piRNAs ( 18 . 5 dpc testes ) as determined by small RNA - seq , respectively . Circles in the charts represent individual mRNAs , piRNA clusters and HMR _ - L1s . More than half of the HMR _ - L1s ( right ) are located in the area of y > x ( upper part of the diagonal ) , whereas most mRNAs ( left ) are located in the area of y < x ( lower part of the diagonal ) . RPKM , reads per kilobase per million mapped reads . Frequent 10 - nt overlap between sense and antisense piRNAs derived from HMR _ - L1s . Top 100 HMR _ - L1s that generate the largest number of unique piRNAs were analyzed . Frequencies of 1 − 18 - nt overlaps were analyzed for each HMR _ - L1 . piRNAs derived from HMR _ - L1s are enriched in adenine at their 10th nucleotide . HMR _ - L1s generating more than 40 unique piRNAs were analyzed ( 678 HMR _ - L1s ) . The proportions of piRNAs with adenine ( left ) and uridine ( right ) at the indicated positions were analyzed for each HMR _ - L1 . See A for the explanation of box plots .

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

Figure 3RNA-immunoprecipitation (RIP) analyses of EGFP-MIWI2 and EGFP. EGFP-MIWI2 and EGFP were immunoprecipitated from 16.5 dpc prospermatogonia, and long RNAs bound to these proteins and input RNAs were sequenced. RNAs expressed from HMR_-L1s and piRNA clusters, but not Ensembl and prospermatogonium-expressed mRNAs, are overall enriched by immunoprecipitation (IP) of EGFP-MIWI2 (left). This enrichment is not observed when EGFP is immunoprecipitated (right). The numbers of mRNAs, piRNA clusters, and HMR_-L1s analyzed were shown in parentheses. Prospermatogonium-expressed mRNAs are those that are highly and specifically expressed in prospermatogonia. The bottom and top of boxes represent 25% percentile and 75% percentile, respectively. The line in the box represents the median. Circles represent outliers, and the bottom (top) of whiskers represents the minimum (maximum) values except for outliers.RIP analyses of EGFP-MIWI2 in newborn testes from WT and Mili KO mice. RNA bound to EGFP-MIWI2 and input RNA were sequenced. All RNAs derived from each class was counted for this analysis.HMR_-L1s preferentially generate piRNAs. X- and Y-axes represent expression levels of long RNAs (16.5 dpc prospermatogonia) as determined by RNA-seq and MIWI2-bound piRNAs (18.5 dpc testes) as determined by small RNA-seq, respectively. Circles in the charts represent individual mRNAs, piRNA clusters and HMR_-L1s. More than half of the HMR_-L1s (right) are located in the area of y > x (upper part of the diagonal), whereas most mRNAs (left) are located in the area of y < x (lower part of the diagonal). RPKM, reads per kilobase per million mapped reads.Frequent 10-nt overlap between sense and antisense piRNAs derived from HMR_-L1s. Top 100 HMR_-L1s that generate the largest number of unique piRNAs were analyzed. Frequencies of 1−18-nt overlaps were analyzed for each HMR_-L1.piRNAs derived from HMR_-L1s are enriched in adenine at their 10th nucleotide. HMR_-L1s generating more than 40 unique piRNAs were analyzed (678 HMR_-L1s). The proportions of piRNAs with adenine (left) and uridine (right) at the indicated positions were analyzed for each HMR_-L1. See A for the explanation of box plots.


Figure 4DNA methylation status of a portion of the Rasgrf1 differentially methylated region ( DMR ) in 0 − 2 - dpp Mili + / − and Mili − / − prospermatogonia . Regions corresponding to B , C , D , E , and F are demethylated in Mili − / − prospermatogonia . Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing ( see below ) . The top dark gray bar represents a portion of the Rasgrf1 gametic DMR ( Tomizawa et al , 2011 ) . The CpG cluster is shown . The deleted RMER4B sequence is indicated . DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B / ΔRMER4B and control spermatogonia are shown . The regions examined are shown in ( A ) . Black ( white ) circles represent methylated ( unmethylated ) CpGs . Rasgrf1 RMER4B functions in cis . Wild - type and mutant alleles of the Rasgrf1 DMR in Rasgrf1 + / ΔRMER4B spermatogonia were analyzed separately .

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Figure 4DNA methylation status of a portion of the Rasgrf1 differentially methylated region (DMR) in 0−2-dpp Mili+/− and Mili−/− prospermatogonia. Regions corresponding to B, C, D, E, and F are demethylated in Mili−/− prospermatogonia. Vertical bars represent DNA methylation levels at CpG sites determined by whole genome bisulfite sequencing (see below). The top dark gray bar represents a portion of the Rasgrf1 gametic DMR (Tomizawa et al, 2011). The CpG cluster is shown. The deleted RMER4B sequence is indicated.DNA methylation status of the Rasgrf1 DMR in Rasgrf1ΔRMER4B/ΔRMER4B and control spermatogonia are shown. The regions examined are shown in (A). Black (white) circles represent methylated (unmethylated) CpGs.Rasgrf1 RMER4B functions in cis. Wild-type and mutant alleles of the Rasgrf1 DMR in Rasgrf1+/ΔRMER4B spermatogonia were analyzed separately.


Figure 5Structure of the chr7 piRNA cluster . Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP - seq , RNA - seq , and small RNA - seq . Only uniquely mapped reads were used for displaying the sequencing result . For RNA - seq and small RNA - seq , only the results of minus strand are shown , because plus strand does not express appreciable level of RNA . A promoter region deleted is shown above the putative piRNA precursor . RPM , Read Per Million . Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter / Δpromoter testes . Unchanged expression of piRNAs from chr10 cluster ( bottom ) shows chr7 cluster is specifically affected . Only uniquely mapped piRNAs are shown . DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔpromoter / Δpromoter . See Figures 4A and B for information about the region examined .

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Figure 5Structure of the chr7 piRNA cluster. Top gray bars represent a putative piRNA precursor predicted by H3K4me3 ChIP-seq, RNA-seq, and small RNA-seq. Only uniquely mapped reads were used for displaying the sequencing result. For RNA-seq and small RNA-seq, only the results of minus strand are shown, because plus strand does not express appreciable level of RNA. A promoter region deleted is shown above the putative piRNA precursor. RPM, Read Per Million.Low expression level of piRNAs from chr7 cluster in chr7 piRNA clusterΔpromoter/Δpromoter testes. Unchanged expression of piRNAs from chr10 cluster (bottom) shows chr7 cluster is specifically affected. Only uniquely mapped piRNAs are shown.DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔpromoter/Δpromoter. See Figures 4A and B for information about the region examined.


Figure 6Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . Top panel shows overall view of chr7 piRNA cluster , and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster . Only uniquely mapped piRNAs are shown . DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatogonia from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . DNA methylation status of the Rasgrf1 differentially methylated region ( DMR ) in spermatocytes , spermatids , and sperms ( D ) from chr7 piRNA clusterΔRMER4B / ΔRMER4B mice . DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B / ΔRMER4B mice . DNA methylation status of the Rasgrf1 DMR in E12 . 5 whole embryos is shown . E12 . 5 whole embryos were obtained by the crosses shown in the panel . Two independent bisulfite - treated DNA sample were examined for Homo13 embryo .

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Figure 6Depletion of piRNAs from chr7 piRNA cluster RMER4B sequence in chr7 piRNA clusterΔRMER4B/ΔRMER4B mice. Top panel shows overall view of chr7 piRNA cluster, and lower panel shows magnified view of RMER4B region in chr7 piRNA cluster. Only uniquely mapped piRNAs are shown.DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatogonia from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.DNA methylation status of the Rasgrf1 differentially methylated region (DMR) in spermatocytes, spermatids, and sperms (D) from chr7 piRNA clusterΔRMER4B/ΔRMER4B mice.DNA methylation pattern in the progeny of male Rasgrf1ΔRMER4B/ΔRMER4B mice. DNA methylation status of the Rasgrf1 DMR in E12.5 whole embryos is shown. E12.5 whole embryos were obtained by the crosses shown in the panel. Two independent bisulfite-treated DNA sample were examined for Homo13 embryo.


Figure 1Conditioned medium was collected from pericytes 48 h after adenoviral transduction . Western blotting verified the secretion of Sema3CΔ13 ( 81 kDa ) and Sema3Cp60 ( 60 kDa ) isoforms . HUVECs were suspended in conditioned medium with VEGF ( 25 ng / ml ) and plated on Matrigel . Branch points of the capillary - like network were counted after 18 h . n = 4 independent experiments ; scale bar , 200 μm . Control vs . Sema3C , * P = 0 . 0375 and control vs . Sema3CΔ13 , * P = 0 . 0162 . HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF ( 25 ng / ml ) . After 24 h , the cumulative sprout length per spheroid was quantified . Data are representative of three independent experiments with ten spheroids per group . Scale bar , 100 μm . Control vs . Sema3C ( − VEGF ) , * P = 0 . 0276 ; control vs . Sema3CΔ13 ( − VEGF ) , * P = 0 . 0443 ; control vs . Sema3C ( + VEGF ) , * P = 0 . 028 and control vs . Sema3CΔ13 ( + VEGF ) , * P = 0 . 032 .

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Figure 1Conditioned medium was collected from pericytes 48 h after adenoviral transduction. Western blotting verified the secretion of Sema3CΔ13 (81 kDa) and Sema3Cp60 (60 kDa) isoforms.HUVECs were suspended in conditioned medium with VEGF (25 ng/ml) and plated on Matrigel. Branch points of the capillary-like network were counted after 18 h. n = 4 independent experiments; scale bar, 200 μm. Control vs. Sema3C, *P = 0.0375 and control vs. Sema3CΔ13, *P = 0.0162.HUVEC spheroids in collagen were treated with conditioned medium with or without VEGF (25 ng/ml). After 24 h, the cumulative sprout length per spheroid was quantified. Data are representative of three independent experiments with ten spheroids per group. Scale bar, 100 μm. Control vs. Sema3C (− VEGF), *P = 0.0276; control vs. Sema3CΔ13 (− VEGF), *P = 0.0443; control vs. Sema3C (+ VEGF), *P = 0.028 and control vs. Sema3CΔ13 (+ VEGF), *P = 0.032.


Figure 2Microvessels within the plugs stained against human - specific CD34 . Cell nuclei were stained with DAPI . Scale bar , 100 μm . The area of CD34 - positive microvessels was quantified and normalized to the total area of plug . Results represent the means ± s . e . m of 5 plugs ( control ) and 7 plugs ( Sema3C ) , * P = 0 . 018 . Vessel coverage was assessed by αSMA staining . Scale bar , 100 μm . Vessel coverage was quantified as the ratio of endothelial cell - associated mural cells to total amount of vessels ( n = 5 ( control ) and n = 7 ( Sema3C ) ) . Vessel perfusion was evaluated by i . v . injection of FITC - lectin ( 0 . 15 mg ) 30 min before mice were sacrificed . The data show the percentage of perfused vessels in the plug and normalized to total vessel area ( n = 5 ( control ) and n = 7 ( Sema3C ) ) .

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Figure 2Microvessels within the plugs stained against human-specific CD34. Cell nuclei were stained with DAPI. Scale bar, 100 μm.The area of CD34-positive microvessels was quantified and normalized to the total area of plug. Results represent the means ± s.e.m of 5 plugs (control) and 7 plugs (Sema3C), *P = 0.018.Vessel coverage was assessed by αSMA staining. Scale bar, 100 μm.Vessel coverage was quantified as the ratio of endothelial cell-associated mural cells to total amount of vessels (n = 5 (control) and n = 7 (Sema3C)).Vessel perfusion was evaluated by i.v. injection of FITC-lectin (0.15 mg) 30 min before mice were sacrificed. The data show the percentage of perfused vessels in the plug and normalized to total vessel area (n = 5 (control) and n = 7 (Sema3C)).