Figure 4 ( A ) Dose - dependent effects of IPZ on MI spindle assembly are shown by maximum z - projection images from confocal time - lapse imaging of oocytes obtained from transgenic histone H2B - EGFP mice stained with SiR - tubulin . Dashed circles indicate oocyte edges ; scale bars , 10 µm . ( B - C ) Live - cell imaging of oocytes was used to quantify the effects of IPZ on GVBD ( B ) and anaphase I entry ( C ) . Anaphase I was defined as the first time point when chromosome segregation was visible . Fisher ' s exact test , oocyte numbers are indicated in brackets . ( D ) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars . ( E ) MI spindle formation in oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin matured in the presence of 15 µM IPZ . Representative still images of maximum z - projections are shown . Time in hh : mm is relative to GVBD . Scale bar , 10 µm . ( F ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group ( means ± SDs , Mann - Whitney - test ) . Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ - treated oocytes were analyzed . ( G ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group ( means ± SDs ) . Oocyte numbers are indicated in brackets . ( H ) Oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin were treated with 15 µM IPZ after being cultured for 5h in control media . Maximum intensity z - projections from confocal live - cell imaging are shown . Time in hh : mm is relative to GVBD , DMSO or 15 uM IPZ was added at time 5 : 00 . Scale bars , 10 µm . ( I ) . Spindle volume at 1h and metaphase I after IPZ addition . The metaphase time was defined as the last time point before anaphase or , in the IPZ - treated oocytes that failed anaphase entry , as the average control metaphase time . Means ± SDs , Mann - Whitney test , oocyte numbers are indicated in brackets . ( J ) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media . Oocyte numbers are indicated in brackets .

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

Figure 4(A) Dose-dependent effects of IPZ on MI spindle assembly are shown by maximum z-projection images from confocal time-lapse imaging of oocytes obtained from transgenic histone H2B-EGFP mice stained with SiR-tubulin. Dashed circles indicate oocyte edges; scale bars, 10 µm.(B-C) Live-cell imaging of oocytes was used to quantify the effects of IPZ on GVBD (B) and anaphase I entry (C). Anaphase I was defined as the first time point when chromosome segregation was visible. Fisher's exact test, oocyte numbers are indicated in brackets.(D) Frequency and severity of MI spindle disruption induced by increasing doses of IPZ Oocyte numbers are shown at the top of the bars.(E) MI spindle formation in oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin matured in the presence of 15 µM IPZ. Representative still images of maximum z-projections are shown. Time in hh:mm is relative to GVBD. Scale bar, 10 µm.(F) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group (means ± SDs, Mann-Whitney-test). Total of 184 MTOC in 17 control oocytes and a total of 170 MTOCs in 17 IPZ-treated oocytes were analyzed. (G) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group (means ± SDs). Oocyte numbers are indicated in brackets. (H) Oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin were treated with 15 µM IPZ after being cultured for 5h in control media. Maximum intensity z-projections from confocal live-cell imaging are shown. Time in hh:mm is relative to GVBD, DMSO or 15 uM IPZ was added at time 5:00. Scale bars, 10 µm.(I). Spindle volume at 1h and metaphase I after IPZ addition. The metaphase time was defined as the last time point before anaphase or, in the IPZ-treated oocytes that failed anaphase entry, as the average control metaphase time. Means ± SDs, Mann-Whitney test, oocyte numbers are indicated in brackets.(J) Time of anaphase I entry in oocytes that were treated with 15 µM IPZ after being cultured for 5h in control media. Oocyte numbers are indicated in brackets.


Figure 5 ( A ) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B - mCherry from microinjected mRNA , co - stained with SiR - tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2 . Maximum intensity z - stack projections from confocal live - cell movies 30 min after GVBD , at metaphase I and anaphase I . Time in hh : mm is relative to GVBD . Scale bars , 10 µm . ( B - C ) The frequency of GVBD ( B ) and anaphase I entry ( C ) quantified by live confocal imaging of oocytes stained and treated Fisher ' s exact test , oocyte numbers are shown in brackets . ( D ) Changes in spindle volume over time after GVBD . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets .

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

Figure 5(A) The rescue of the effects of 15 µM IPZ on MI spindles was examined in mouse oocytes expressing histone H2B-mCherry from microinjected mRNA, co-stained with SiR-tubulin and noninjected or injected with mRNAs coding for RanQ69L or hTPX2. Maximum intensity z-stack projections from confocal live-cell movies 30 min after GVBD, at metaphase I and anaphase I. Time in hh: mm is relative to GVBD. Scale bars, 10 µm.(B-C) The frequency of GVBD (B) and anaphase I entry (C) quantified by live confocal imaging of oocytes stained and treated Fisher's exact test, oocyte numbers are shown in brackets. (D) Changes in spindle volume over time after GVBD. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.


Figure 6 ( A ) The effects of RanT24N , T42A , and RanT24N on MI spindle assembly observed in maximum z - projection images from confocal time - lapse imaging of oocytes expressing CDK5RAP2 - EGFP , histone H2B - mCherry and stained with SiR - tubulin . Time in hh : mm is relative to GVBD . Scale bar , 10 µm . ( B , C ) Live - cell imaging of oocytes as in ( A ) was used to quantify the effects of Ran mutant . Means ± SDs . , Mann - Whitney test , oocyte numbers are indicated in brackets . ( D ) MT intensity at MTOCs at the time of GVBD quantified from SiR - tubulin signal that was normalized to mean cytoplasmic SiR - tubulin signal . Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group . Means ± SDs , Kruskal - Wallis , and Dunn ' s tests . 25 control oocytes with 264 MTOCs in total , 10 RanT24N - expression oocytes with 70 MTOCs in total and 19 RanT24N , T42A - expressing oocytes with 96 MTOCs in total were analyzed . ( E ) Time course of the MT intensity in the area of spindle after GVBD . Mean of MT intensity was arbitrarily centered to 1 in the control group . Means ± SDs , mixed - design ANOVA , oocyte numbers are shown in brackets . ( F ) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N , T42A . Oocyte numbers are shown in brackets . ( G , H ) Quantification of the percentage of oocytes ( G ) with chromosome segregation errors in anaphase I and ( H ) with defective MTOC coalescence during metaphase I . Means ± SDs , oocyte numbers are shown in brackets . ( I ) Detection of MTs and pericentrin - containing MTOCs by IF in control and RanT24N , T42A - expressing oocytes . Arrow indicates unaligned chromosome . ( J ) Quantification of chromosomal spread area in oocytes depicted in ( I ) , Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets .

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

Figure 6(A) The effects of RanT24N, T42A, and RanT24N on MI spindle assembly observed in maximum z-projection images from confocal time-lapse imaging of oocytes expressing CDK5RAP2-EGFP, histone H2B-mCherry and stained with SiR-tubulin. Time in hh:mm is relative to GVBD. Scale bar, 10 µm. (B, C) Live-cell imaging of oocytes as in (A) was used to quantify the effects of Ran mutant. Means ± SDs., Mann-Whitney test, oocyte numbers are indicated in brackets. (D) MT intensity at MTOCs at the time of GVBD quantified from SiR-tubulin signal that was normalized to mean cytoplasmic SiR-tubulin signal. Mean of MT intensity on MTOCs was arbitrarily centered to 1 in the control group. Means ± SDs, Kruskal-Wallis, and Dunn's tests. 25 control oocytes with 264 MTOCs in total, 10 RanT24N-expression oocytes with 70 MTOCs in total and 19 RanT24N, T42A-expressing oocytes with 96 MTOCs in total were analyzed.(E) Time course of the MT intensity in the area of spindle after GVBD. Mean of MT intensity was arbitrarily centered to 1 in the control group. Means ± SDs, mixed-design ANOVA, oocyte numbers are shown in brackets.(F) Frequency and severity of MI spindle phenotypes in the oocytes expressing RanT24N or RanT24N, T42A. Oocyte numbers are shown in brackets.(G, H) Quantification of the percentage of oocytes (G) with chromosome segregation errors in anaphase I and (H) with defective MTOC coalescence during metaphase I. Means ± SDs, oocyte numbers are shown in brackets.(I) Detection of MTs and pericentrin-containing MTOCs by IF in control and RanT24N, T42A - expressing oocytes. Arrow indicates unaligned chromosome. (J) Quantification of chromosomal spread area in oocytes depicted in (I), Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.


Figure 7 ( A ) . Maximum intensity z - stack projections of confocal IF images ( DNA stain , indirect IF for tubulin and TPX2 ) in untreated oocytes ( control ) , oocytes expressing RanT24N or injected with importin β ( 71 - 876 ) . The representative of at least 5 experiments is shown . ( B - D ) Maximum intensity z - stack projections of IF images in control and importin β ( 71 - 876 ) - injected cells were used to quantify the spindle vs . whole - cell area fraction ( B ) and the ratios of the average signal in the spindle vs . cytoplasm for the tubulin ( C ) and TPX2 ( D ) staining . Means ± SDs , with t - test in ( B , C ) and Mann - Whitney test in ( D ) , oocyte numbers indicated in brackets . ( E ) Ratios of the spindle - associated fractions of TPX2 vs . tubulin were calculated in maximum intensity z - stack projections of IF images . Means ± SDs , Mann - Whitney test , oocyte numbers indicated in brackets . ( F ) Maximum intensity z - stack projections of IF images were used to quantify the number of ectopic MT asters . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests , oocyte numbers indicated in brackets . ( G ) Maximum intensity z - stack projections of confocal IF images stained for DNA , pericentrin , and tubulin ( directly labeled primary antibody ) . A representative example of at least 2 experiments is shown . Scale bar , 20 µm . ( H - I ) The number and size of pericentrin foci were quantified in maximum - intensity z - stack projections of IF images stained as in ( G ) in at least 2 separate experiments . Means ± SDs , ANOVA with Kruskal - Wallis and Dunn ' s tests .

O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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). Maximum intensity z-stack projections of confocal IF images (DNA stain, indirect IF for tubulin and TPX2) in untreated oocytes (control), oocytes expressing RanT24N or injected with importin β(71-876). The representative of at least 5 experiments is shown.(B-D) Maximum intensity z-stack projections of IF images in control and importin β(71-876)-injected cells were used to quantify the spindle vs. whole-cell area fraction (B) and the ratios of the average signal in the spindle vs. cytoplasm for the tubulin (C) and TPX2 (D) staining. Means ± SDs, with t-test in (B, C) and Mann-Whitney test in (D), oocyte numbers indicated in brackets.(E) Ratios of the spindle-associated fractions of TPX2 vs. tubulin were calculated in maximum intensity z-stack projections of IF images. Means ± SDs, Mann-Whitney test, oocyte numbers indicated in brackets.(F) Maximum intensity z-stack projections of IF images were used to quantify the number of ectopic MT asters. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests, oocyte numbers indicated in brackets.(G) Maximum intensity z-stack projections of confocal IF images stained for DNA, pericentrin, and tubulin (directly labeled primary antibody). A representative example of at least 2 experiments is shown. Scale bar, 20 µm. (H-I) The number and size of pericentrin foci were quantified in maximum-intensity z-stack projections of IF images stained as in (G) in at least 2 separate experiments. Means ± SDs, ANOVA with Kruskal-Wallis and Dunn's tests.


figf1 ( a ) Confocal microscopy of synchronized HeLa cells stained for VHR ( green ) and BrdU ( red ) to indicate S - phase entry , and DNA ( DAPI ; blue ) . The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times . The cells shown are representative of the majority of cells in each sample . b ) Anti - VHR , anti - actin and anti - Prc1immunoblots of lysates of the same cells . ( c ) DNA histograms of the same cells at the indicated time points . ( d ) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin ( green ) , VHR ( red ) and DNA ( DAPI ; blue ) . ( e ) Anti - VHR immunoblot of synchronized cells from G1 , S , G2 and M phase treated for 30 min with 50 μg ml − 1 of the protein synthesis inhibitor cycloheximide . Note that only cells in G1 showed a rapid loss of VHR protein . The scale bars represent 5 μm in a and 2 μm in b .

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

figf1(a) Confocal microscopy of synchronized HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI; blue). The cells were synchronized using a double thymidine block and then washed and placed in culture for the indicated times. The cells shown are representative of the majority of cells in each sample.b) Anti-VHR, anti-actin and anti-Prc1immunoblots of lysates of the same cells.(c) DNA histograms of the same cells at the indicated time points.(d) Confocal microscopy of HeLa cells at various stages of mitosis stained for tubulin (green), VHR (red) and DNA (DAPI; blue).(e) Anti-VHR immunoblot of synchronized cells from G1, S, G2 and M phase treated for 30 min with 50 μg ml−1 of the protein synthesis inhibitor cycloheximide. Note that only cells in G1 showed a rapid loss of VHR protein. The scale bars represent 5 μm in a and 2 μm in b.


figf2 ( a ) Anti - VHR and anti - actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days . ( b ) Number of cells at the start of the experiment ( 200 , 000 ) and on day 3 for HeLa cells transfected with a control luciferase siRNA ( black bars ) or with VHR siRNA ( white bars ) . ( c ) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days . ( d ) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA . The data represent the mean ± sd . ( n = 3 ) . ( e ) β - galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days . Note the increased number of blue - staining granules in many cells . ( ( f ) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA . Each sample was analysed in triplicate , with RNAse added to a fourth sample . ( g ) Anti - VHR immunoblot of the same cells as in f . ( h ) Anti - actin control immunoblot of the same cells as in f . The scale bars represent 10 μm in c and 25 μm in e .

O B-PANEL_START O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O 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) Anti-VHR and anti-actin immunoblots of lysates of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for the indicated number of days.(b) Number of cells at the start of the experiment (200,000) and on day 3 for HeLa cells transfected with a control luciferase siRNA (black bars) or with VHR siRNA (white bars).(c) Light microscopy of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days.(d) Thymidine incorporation by HeLa cells transfected with a control luciferase siRNA or with VHR siRNA. The data represent the mean ± sd. (n = 3).(e) β-galactosidase staining of HeLa cells transfected with a control luciferase siRNA or VHR siRNA and cultured for three days. Note the increased number of blue-staining granules in many cells. ((f) Telomerase activity of cells treated with the indicated concentrations of VHR siRNA. Each sample was analysed in triplicate, with RNAse added to a fourth sample.(g) Anti-VHR immunoblot of the same cells as in f. (h) Anti-actin control immunoblot of the same cells as in f. The scale bars represent 10 μm in c and 25 μm in e.


figf3 ( a ) Cell - cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4 - 5 days . The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture . ( b ) Confocal microscopy of the same HeLa cells stained for VHR ( green ) and BrdU ( red ) to indicate S - phase entry , and DNA ( DAPI , blue ) . Control siRNA - treated cells and cells treated with VHR are shown . The bottom panels represent merges of the 3 images above ( c ) Quantification of the BrdU positive cells in b . The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number . d ) Quantification of cells staining for phosphoH3 . The indicated number of cells were counted and the data are given as percentage of phosphoH3 - positive cells of this total number . ( e ) Confocal microscopy of control or VHR siRNA - transfected HeLa cells stained for VHR ( green ) and phospho H3 ( red ) to indicate M - phase entry , and DNA ( DAPI , blue ) . The bottom panels represent merges of the images above . ( f ) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies . Note that VHR was downregulated by approximately 80 % , wheras actin levels were unchanged . ( g ) Immunoblot with anti - p21Cip1 - WAF1 of total lysates of cells transfected with control siRNA ( lanes 1 - 2 ) , two different VHR siRNAs cells ( lanes 3 - 6 ) or another control siRNA ( lanes 7 - 8 ) . The scale bars represent 10 μm in b and e .

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

figf3(a) Cell-cycle distribution and cell number count of cells transfected with control or VHR siRNA and observed for 4-5 days. The cells were stained with propidium iodide for DNA content and analysed by FACS after the indicated number of days in culture.(b) Confocal microscopy of the same HeLa cells stained for VHR (green) and BrdU (red) to indicate S-phase entry, and DNA (DAPI, blue). Control siRNA-treated cells and cells treated with VHR are shown. The bottom panels represent merges of the 3 images above(c) Quantification of the BrdU positive cells in b. The indicated numbers of cells were counted and the data are given as percentage of BrdU positive cells of this total number.d) Quantification of cells staining for phosphoH3. The indicated number of cells were counted and the data are given as percentage of phosphoH3-positive cells of this total number.(e) Confocal microscopy of control or VHR siRNA-transfected HeLa cells stained for VHR (green) and phospho H3 (red) to indicate M-phase entry, and DNA (DAPI, blue). The bottom panels represent merges of the images above.(f) Immunoblots of total lysates of control siRNA transfected cells and VHR siRNA cells with the indicated antibodies. Note that VHR was downregulated by approximately 80%, wheras actin levels were unchanged.(g) Immunoblot with anti-p21Cip1-WAF1 of total lysates of cells transfected with control siRNA (lanes 1-2), two different VHR siRNAs cells (lanes 3-6) or another control siRNA (lanes 7-8). The scale bars represent 10 μm in b and e.


figf4 ( a ) Anti - VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b - e . The cells were stimulated with 10 % serum for 0 , 1 , 5 , 15 , 30 and 60 min , as indicated at the bottom of the figure . Equal amounts of total protein were loaded into each lane . ( b - e ) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation .

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figf4(a) Anti-VHR immunoblots of the same cell lysates used for assessment of MAP kinase activation in b-e. The cells were stimulated with 10% serum for 0, 1, 5, 15, 30 and 60 min, as indicated at the bottom of the figure. Equal amounts of total protein were loaded into each lane. (b-e) Immunoblots of the same lysates with the indicated antibodies for assement of MAP kinase activation.


figf5 ( a ) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek , Jnk or Mek plus Jnk siRNAs . The data are calculated as percentage of control ( control siRNA alone ) and represent the mean ± s . d . from three independent experiments . The starting cell numbers were 150 , 000 or 200 , 000 and the control cells had increased by 5 . 3 , 5 . 0 and 13 - fold in the three experiments . ( b ) Immunoblots of the same cells with the indicated antibodies .

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figf5(a) Proliferation of cells transfected 3 days earlier with a combination of control siRNA or VHR siRNA together with Mek, Jnk or Mek plus Jnk siRNAs. The data are calculated as percentage of control (control siRNA alone) and represent the mean ± s.d. from three independent experiments. The starting cell numbers were 150,000 or 200,000 and the control cells had increased by 5.3, 5.0 and 13-fold in the three experiments.(b) Immunoblots of the same cells with the indicated antibodies.


Figure 1 ( A and B ) One hundred human keratinocytes ( YF29 , passage V ) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature set at 35oC , 36oC , 37oC , or 38oC . Temperature of each incubator was monitored with independent thermo probes In parallel , the temperature of the culture medium was recorded every ten seconds using immerged RFID temperature sensors Sharp spikes resulted from wireless communication artifacts . ( B ) Cells were fixed and stained with 1 % Rhodamine B . Temperature did not affect colony - forming efficiency ( CFE ) but impacted colony size ; colonies were smaller when temperatures were below 37oC and larger at 38oC . Asterisks indicate thermo probes for monitoring the temperature . ( C and D ) Human keratinocytes ( YF29 ) were cultured in mass for seven days at different temperatures before the cultures were dissociated . ( C ) Colony size was quantified using an image processing software ( ImageJ ) . ( D ) The number and the size of the cells were determined using a TaliTM cytometer . Temperature impacted both cell number and size . Data information : In C and D , data are presented as the mean ± SD ( n = 4 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 ( Two - tailed Student ' s t - test ) . The data shown were confirmed in two technical replicates .

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Figure 1(A and B) One hundred human keratinocytes (YF29, passage V) were cultured in duplicate for twelve days in individual Thermo Scientific Heracell 150 incubators with the temperature set at 35oC, 36oC, 37oC, or 38oC. Temperature of each incubator was monitored with independent thermo probes In parallel, the temperature of the culture medium was recorded every ten seconds using immerged RFID temperature sensors Sharp spikes resulted from wireless communication artifacts. (B) Cells were fixed and stained with 1% Rhodamine B. Temperature did not affect colony-forming efficiency (CFE) but impacted colony size; colonies were smaller when temperatures were below 37oC and larger at 38oC. Asterisks indicate thermo probes for monitoring the temperature.(C and D) Human keratinocytes (YF29) were cultured in mass for seven days at different temperatures before the cultures were dissociated. (C) Colony size was quantified using an image processing software (ImageJ). (D) The number and the size of the cells were determined using a TaliTM cytometer. Temperature impacted both cell number and size. Data information: In C and D, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05, ** p < 0.01 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.


Figure 2 ( A ) The transcripts of eight thermoTRP channels were quantified by qPCR in two different strains of human diploid keratinocytes ( YF29 and OR - CA ) . Data are presented as the mean ± SEM ( n = 3 biological replicates ) . ( F and G ) YF29 cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator . Cells were then maintained at 37oC or switched to an adjacent incubator with temperature set at 32oC . Temperature was monitored with independent thermo probes in the presence or absence of rapamycin ( 100 nM ) , or a cold agonist ( menthol - 500 μM ) , a warm agonist ( 2 - APB - 200 μM ) and the vehicle ( control - ethanol 70 % ) for 30 and 60 minutes . ( F ) mTORC1 kinase activity decreases in the presence of rapamycin and when the temperature is switched from 37oC to 32oC . ( G ) mTORC1 kinase activity decreases in the presence of menthol and increases in presence of 2 - APB , indicating that ThermoTRP and mTOR signaling are connected . Rapamycin ( Rapa ) , menthol , and 2 - APB were added in the keratinocyte cultures 1 h before starting the temperature changes .

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Figure 2(A) The transcripts of eight thermoTRP channels were quantified by qPCR in two different strains of human diploid keratinocytes (YF29 and OR-CA). Data are presented as the mean ± SEM (n = 3 biological replicates).(F and G) YF29 cells were cultured for seven days at 37oC in a Nuaire 8700E culture incubator. Cells were then maintained at 37oC or switched to an adjacent incubator with temperature set at 32oC. Temperature was monitored with independent thermo probes in the presence or absence of rapamycin (100 nM), or a cold agonist (menthol - 500 μM), a warm agonist (2-APB - 200 μM) and the vehicle (control - ethanol 70%) for 30 and 60 minutes. (F) mTORC1 kinase activity decreases in the presence of rapamycin and when the temperature is switched from 37oC to 32oC. (G) mTORC1 kinase activity decreases in the presence of menthol and increases in presence of 2-APB, indicating that ThermoTRP and mTOR signaling are connected. Rapamycin (Rapa), menthol, and 2-APB were added in the keratinocyte cultures 1 h before starting the temperature changes.


Figure 3 ( A ) Colony - forming efficiency ( CFE ) of keratinocytes was unaltered when keratinocytes were cultured at 35oC or in the presence of 100 nM rapamycin . However , the mean area of colonies was smaller when compared to cells cultured at 37oC . ( B ) Culture at 35oC and treatment with rapamycin decreased keratinocyte proliferation as shown by the lower total number of cells after 7 days of culture compared with in the control condition . ( C ) Culture at 35oC and treatment with rapamycin also reduced cell growth as shown by a smaller mean diameter of keratinocytes compared with that in the control condition after 7 days of culture . Data information : In B and C , data are presented as the mean ± SD ( n = 4 biological replicates ) . * p < 0 . 05 ( Two - tailed Student ' s t - test ) . The data shown were confirmed in two technical replicates . ( D ) Long - term exposure to temperatures below 37oC decreased mTORC1 kinase activity .

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Figure 3(A) Colony-forming efficiency (CFE) of keratinocytes was unaltered when keratinocytes were cultured at 35oC or in the presence of 100 nM rapamycin. However, the mean area of colonies was smaller when compared to cells cultured at 37oC. (B) Culture at 35oC and treatment with rapamycin decreased keratinocyte proliferation as shown by the lower total number of cells after 7 days of culture compared with in the control condition. (C) Culture at 35oC and treatment with rapamycin also reduced cell growth as shown by a smaller mean diameter of keratinocytes compared with that in the control condition after 7 days of culture. Data information: In B and C, data are presented as the mean ± SD (n = 4 biological replicates). * p < 0.05 (Two-tailed Student's t-test). The data shown were confirmed in two technical replicates.(D) Long-term exposure to temperatures below 37oC decreased mTORC1 kinase activity.


Figure 4 ( B ) Proteins were extracted from different YF29 subcellular components with ProteoExtractTM Subcellular Proteome Extraction kit ( Calbiochem ) . The purity of each subcellular fraction was confirmed using specific markers , GAPDH for the cytosolic fraction , calnexin ( CANX ) for the membrane and organelles fraction , keratin 5 ( KRT5 ) for the cytoskeletal fraction and HDAC2 for the nuclear fraction . mTOR , p - mTOR , RICTOR and RAPTOR were identified both in the membranes / organelles and nuclear fractions by western blotting . RICTOR and RAPTOR were also identified in the cytosolic fraction . ( D ) CHO cells engineered to transiently express SNAP - mTOR , SNAP - RAPTOR , and SNAP - RICTOR ( Gautier et al . , 2008 ) were incubated with the nuclear export inhibitor leptomycin B ( LMB ) ( 6 . 4 ng / mL ) for six hours at 37 ° C . Cells were labeled with TMR - Star ( 1 μM ) for 30 min , washed and then imaged . This experiment demonstrated that mTOR , RAPTOR , and RICTOR accumulated in the nucleus after LMB treatment . Scale bars , 10μm .

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Figure 4(B) Proteins were extracted from different YF29 subcellular components with ProteoExtractTM Subcellular Proteome Extraction kit (Calbiochem). The purity of each subcellular fraction was confirmed using specific markers, GAPDH for the cytosolic fraction, calnexin (CANX) for the membrane and organelles fraction, keratin 5 (KRT5) for the cytoskeletal fraction and HDAC2 for the nuclear fraction. mTOR, p-mTOR, RICTOR and RAPTOR were identified both in the membranes/organelles and nuclear fractions by western blotting. RICTOR and RAPTOR were also identified in the cytosolic fraction.(D) CHO cells engineered to transiently express SNAP-mTOR, SNAP-RAPTOR, and SNAP-RICTOR (Gautier et al., 2008) were incubated with the nuclear export inhibitor leptomycin B (LMB) (6.4 ng/mL) for six hours at 37°C. Cells were labeled with TMR-Star (1 μM) for 30 min, washed and then imaged. This experiment demonstrated that mTOR, RAPTOR, and RICTOR accumulated in the nucleus after LMB treatment. Scale bars, 10μm.


Figure 5A genome - wide expression screen was performed in parallel on normal human epidermal keratinocytes ( YF29 ) cultured for 10 days at 32oC , 35oC , 36oC , 37oC , 38oC and at 37oC in the presence of rapamycin ( 100 nM ) . ( C ) Quantitative PCR analysis of epidermal differentiation - associated genes . Data are presented as the mean ± SD ( n = 3 biological replicates ) . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 ( one - way ANOVA with Dunnett ' s multiple comparisons test vs 37oC ) .

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Figure 5A genome-wide expression screen was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (C) Quantitative PCR analysis of epidermal differentiation-associated genes. Data are presented as the mean ± SD (n = 3 biological replicates). * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA with Dunnett's multiple comparisons test vs 37oC).


Figure 6Comprehensive miRNA expression was performed in parallel on normal human epidermal keratinocytes ( YF29 ) cultured for 10 days at 32oC , 35oC , 36oC , 37oC , 38oC and at 37oC in the presence of rapamycin ( 100 nM ) . ( B ) The MA plot showing all miRNAs without low expression or log2FC = 0 . miRNAs indicated in pink : upregulated ( ≥ 1 log2 ( FC ) ) , blue : downregulated ( ≤ - 1 log2 ( FC ) ) , grey : others ( - 1 < log2 ( FC ) < 1 ) and black : miRNAs related to epidermal differentiation . log2 ( FC ) values are calculated using the edgeR package .

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Figure 6Comprehensive miRNA expression was performed in parallel on normal human epidermal keratinocytes (YF29) cultured for 10 days at 32oC, 35oC, 36oC, 37oC, 38oC and at 37oC in the presence of rapamycin (100 nM). (B) The MA plot showing all miRNAs without low expression or log2FC = 0. miRNAs indicated in pink: upregulated (≥ 1 log2(FC)), blue: downregulated (≤ -1 log2(FC)), grey: others (-1 < log2(FC) < 1) and black: miRNAs related to epidermal differentiation. log2(FC) values are calculated using the edgeR package.


Figure 7 ( B ) Cultured human keratinocytes ( YF29 passage III ) treated with rapamycin maintained a basal phenotype as shown by the expression of several basal ( KRT14 , BNC1 ) and terminal differentiation markers ( SPINK5 , IVL , KRT1 , KRT10 ) using quantitative PCR ( left panel ) and Western blotting ( right panel ) . Data are presented as the mean ( n = 2 biological replicates ) . ( E ) Rapamycin - treated keratinocytes ( YF29 passage IV ) can fully regenerate an epidermis when transplanted onto SCID mice ; grafts were harvested after 71 days and immunostained with an HLA - 1 antibody to identify the human cells ( white dotted line indicates the boundary between mouse and human epidermis ) . Scale bar , 100 μm .

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Figure 7(B) Cultured human keratinocytes (YF29 passage III) treated with rapamycin maintained a basal phenotype as shown by the expression of several basal (KRT14, BNC1) and terminal differentiation markers (SPINK5, IVL, KRT1, KRT10) using quantitative PCR (left panel) and Western blotting (right panel). Data are presented as the mean (n = 2 biological replicates).(E) Rapamycin-treated keratinocytes (YF29 passage IV) can fully regenerate an epidermis when transplanted onto SCID mice; grafts were harvested after 71 days and immunostained with an HLA-1 antibody to identify the human cells (white dotted line indicates the boundary between mouse and human epidermis). Scale bar, 100 μm.


Figure 1B Hops − / − mouse genotyping and protein expression . Genotyping by PCR using genomic DNA showed a 242bp band for wild type ( Hops + / + ) allele and a 331bp band for mutant ( Hops − / − ) allele . NTC is Null Template Control ( left panel ) . Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops − / − mice ( middle panel ) . Protein extracts from Hops + / + and Hops − / − MEFs were analysed by Western Blot using anti - HOPS antibody and anti - α - tubulin antibody as loading control ( right panel ) . Data information : in B data are presented as mean ± SD . C Offspring distribution . Different HOPS genotypes at weaning age ( approximately postnatal day 21 ) from crosses between homozygous knockout mice males and heterozygous females ( n = 1525 pups ) . Data information : In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops − / − to Hops + / − mice . P < 0 . 001 , by two tailed Student ' s t - test .

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Figure 1B Hops−/− mouse genotyping and protein expression. Genotyping by PCR using genomic DNA showed a 242bp band for wild type (Hops+/+) allele and a 331bp band for mutant (Hops−/−) allele. NTC is Null Template Control (left panel). Genotyping by real time PCR confirmed the absence of Hops mRNA in Hops−/− mice (middle panel). Protein extracts from Hops+/+ and Hops−/− MEFs were analysed by Western Blot using anti-HOPS antibody and anti-α-tubulin antibody as loading control (right panel). Data information: in B data are presented as mean ±SD.C Offspring distribution. Different HOPS genotypes at weaning age (approximately postnatal day 21) from crosses between homozygous knockout mice males and heterozygous females (n=1525 pups). Data information: In C the χ2 test was performed to determine significant differences between the expected and observed ratios of Hops−/− to Hops+/− mice. P < 0.001, by two tailed Student's t-test.


Figure 2A Reduced caspase - 3 activation in Hops − / − mice upon apoptotic stimulus . Hops + / + and Hops − / − mice were treated with etoposide for the indicated times and thymus , spleen , testis and liver were harvested and Tissue - Tek ® O . C . T . embedded . Sections were immunostained with anti - cleaved caspase - 3 antibody followed by appropriate secondary antibody labelling ( green ) . The graphs display the relative positive cells number . Scale bars , 10µm . Data information : all the experiments were performed three times in three different mice per condition and a representative example is shown . In A data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . B HOPS affects p53 increase after stress in vivo . Whole - cell homogenates from organs , were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH . The p53 levels were semi - quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1 . Data information : all the experiments were performed three times in three different mice per condition and a representative example is shown . In B data are presented as mean ± SD . * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test .

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Figure 2A Reduced caspase-3 activation in Hops−/− mice upon apoptotic stimulus. Hops+/+ and Hops−/− mice were treated with etoposide for the indicated times and thymus, spleen, testis and liver were harvested and Tissue-Tek® O.C.T. embedded. Sections were immunostained with anti-cleaved caspase-3 antibody followed by appropriate secondary antibody labelling (green). The graphs display the relative positive cells number. Scale bars, 10µm. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In A data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.B HOPS affects p53 increase after stress in vivo. Whole-cell homogenates from organs, were immediately prepared and immunoblotted with antibodies to detect p53 and GAPDH. The p53 levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 1. Data information: all the experiments were performed three times in three different mice per condition and a representative example is shown. In B data are presented as mean ±SD. ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.


Figure 3A Thymocytes from Hops + / + and Hops − / − mice were treated with dexamethasone ( Dex ) and the apoptotic cells were measured by flow cytometry . The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right . Data information : the experiments were performed three times in primary cells from three different mice per condition . In A data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . B HOPS was pivotal for apoptosis induction . Hops + / + and Hops − / − MEFs were subjected to etoposide ( Eto ) and apoptosis was analysed by flow cytometry ( left ) and showed as a graph ( right ) . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown . In data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . C HOPS overexpression in MEFs promotes apoptosis . Hops + / + and Hops − / − MEFs were transiently transfected with an empty vector ( pEGFP - N1 ) or with a Hops encoding vector ( pEGFP - N1 - Hops ) . The apoptotic cells were measured by flow cytometry ( left ) on the GFP positive and negative populations and showed as a graph ( right ) . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown . In C data are presented as mean ± SD . * P < 0 . 05 ; * * P < 0 . 01 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . D HOPS affects p53 levels following stress in vitro . Hops + / + and Hops − / − MEFs were treated with etoposide and harvested at the indicated time points . The p53 protein levels were semi - quantified using α - tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1 . Data information : the experiments were performed three times in primary cells from three different mice per condition . The experiments in MEFs were performed three times . Representative panels are shown .

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Figure 3A Thymocytes from Hops+/+ and Hops−/− mice were treated with dexamethasone (Dex) and the apoptotic cells were measured by flow cytometry. The flow cytometry analysis plots are shown on the left and the data represented as a histogram on the right. Data information: the experiments were performed three times in primary cells from three different mice per condition. In A data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.B HOPS was pivotal for apoptosis induction. Hops+/+ and Hops−/− MEFs were subjected to etoposide (Eto) and apoptosis was analysed by flow cytometry (left) and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.C HOPS overexpression in MEFs promotes apoptosis. Hops+/+ and Hops−/− MEFs were transiently transfected with an empty vector (pEGFP-N1) or with a Hops encoding vector (pEGFP-N1-Hops). The apoptotic cells were measured by flow cytometry (left) on the GFP positive and negative populations and showed as a graph (right). Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown. In C data are presented as mean ±SD. * P < 0.05; ** P < 0.01; *** P < 0.001, by two tailed Student's t-test.D HOPS affects p53 levels following stress in vitro. Hops+/+ and Hops−/− MEFs were treated with etoposide and harvested at the indicated time points. The p53 protein levels were semi-quantified using α-tubulin as loading control and relative p53 levels at 0 µM dosage were assumed as 1. Data information: the experiments were performed three times in primary cells from three different mice per condition. The experiments in MEFs were performed three times. Representative panels are shown.


Figure 4A HOPS loss and p53 half - life . Hops + / + and Hops − / − MEFs untransfected or transfected with HOPS , were analysed by immunoblotting following cycloheximide treatment for the indicated time ( left panel ) . The p53 protein levels were semi - quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100 % ( right panel ) . Data information : all the experiments in MEFs were performed three times . Representative panels are shown . In A data are presented as mean ± SD . B p19Arf - independent HOPS control of p53 half - life . p19Arf − / − MEFs untransfected ( CTRL ) , transfected with empty vector ( pSGV ) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti - p53 , anti - HOPS and anti - GAPDH antibodies ( left panel ) . The p53 protein levels were semi - quantified as above ( right panel ) . Data information : all the experiments in MEFs were performed three times . Representative panels are shown . In B data are presented as mean ± SD . C HOPS and p53 ubiquitination . p53 , HDM2 , His - tagged ubiquitin were co - expressed in H1299 cells with HOPS or HOPS - G176A or HOPS - K126A mutants and treated with MG132 . p53 ubiquitination rate was evaluated by elution using Ni2 + - NTA His - bind resin followed by immunoblotting with anti - p53 and the indicated antibodies . D HOPS depletion and HOPS - G176A mutant affect p53 ubiquitination . Hops + / + and Hops − / − MEFs untransfected and Hops − / − MEFs transfected with HOPS or HOPS - G176A mutant were co - expressed with p53 , HDM2 and His - tagged ubiquitin . Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above . Immunodetection of ubiquitinated p53 was performed using anti - p53 antibody and the samples were tested with the indicated antibodies . Data information : all the experiments in MEFs were performed three times . Representative panels are shown . E HOPS - G176A mutant ability to stabilize p53 . Hops − / − MEFs were transfected with HOPS - G176A , subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies . Data information : all the experiments in MEFs were performed three times . Representative panels are shown .

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

Figure 4A HOPS loss and p53 half-life. Hops+/+ and Hops−/− MEFs untransfected or transfected with HOPS, were analysed by immunoblotting following cycloheximide treatment for the indicated time (left panel). The p53 protein levels were semi-quantified using GAPDH as loading control and relative p53 levels at time 0 were assumed as 100% (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In A data are presented as mean ±SD.B p19Arf-independent HOPS control of p53 half-life. p19Arf−/− MEFs untransfected (CTRL), transfected with empty vector (pSGV) or HOPS were treated with cycloheximide for the indicated times and subjected to immunoblot analysis with anti-p53, anti-HOPS and anti-GAPDH antibodies (left panel). The p53 protein levels were semi-quantified as above (right panel). Data information: all the experiments in MEFs were performed three times. Representative panels are shown. In B data are presented as mean ±SD.C HOPS and p53 ubiquitination. p53, HDM2, His-tagged ubiquitin were co-expressed in H1299 cells with HOPS or HOPS-G176A or HOPS-K126A mutants and treated with MG132. p53 ubiquitination rate was evaluated by elution using Ni2+-NTA His-bind resin followed by immunoblotting with anti-p53 and the indicated antibodies.D HOPS depletion and HOPS-G176A mutant affect p53 ubiquitination. Hops+/+ and Hops−/− MEFs untransfected and Hops−/− MEFs transfected with HOPS or HOPS-G176A mutant were co-expressed with p53, HDM2 and His-tagged ubiquitin. Cells were treated with MG132 and the ubiquitinated p53 fraction was recovered as above. Immunodetection of ubiquitinated p53 was performed using anti-p53 antibody and the samples were tested with the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.E HOPS-G176A mutant ability to stabilize p53. Hops−/− MEFs were transfected with HOPS-G176A, subjected to cycloheximide treatment for the indicated times and analysed by immunoblotting using the indicated antibodies. Data information: all the experiments in MEFs were performed three times. Representative panels are shown.


Figure 5A Protein binding in RKO cells . PLA showed HOPS / p53 interaction with or without etoposide treatment at the indicated time . Red spots showed a single interaction . DNA counterstaining was performed with DAPI . α - Tubulin - FITC antibody was used to identify the cellular edge . Background control was performed according to the manufacturer ' s instruction . Scale bars , 10µm . The graph represents the binding increase . Non treated cells binding was assumed as 1 . Data information : all the experiments were performed three times , and representative examples are shown . In A data are presented as mean ± SD . B Biochemical analysis of nuclear - cytoplasmic protein distribution . RKO cells were treated with etoposide , collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions . Aliquots relative to the same cell number per fraction were immunoblotted using anti - HOPS and anti - p53 antibodies . GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively . C HOPS and p53 binding in cytoplasm following apoptosis induction . RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions . WCL and cytoplasmic fractions were immunoprecipitated with anti - HOPS antibody and subsequently immunoblotted using anti - p53 antibody .

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

Figure 5A Protein binding in RKO cells. PLA showed HOPS/p53 interaction with or without etoposide treatment at the indicated time. Red spots showed a single interaction. DNA counterstaining was performed with DAPI. α-Tubulin-FITC antibody was used to identify the cellular edge. Background control was performed according to the manufacturer's instruction. Scale bars, 10µm. The graph represents the binding increase. Non treated cells binding was assumed as 1. Data information: all the experiments were performed three times, and representative examples are shown. In A data are presented as mean ±SD.B Biochemical analysis of nuclear-cytoplasmic protein distribution. RKO cells were treated with etoposide, collected after 2 and 4 hours and processed to obtain nuclear and cytoplasmic fractions. Aliquots relative to the same cell number per fraction were immunoblotted using anti-HOPS and anti-p53 antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively.C HOPS and p53 binding in cytoplasm following apoptosis induction. RKO cells untreated or treated with etoposide as above were used to isolate cytoplasmic fractions. WCL and cytoplasmic fractions were immunoprecipitated with anti-HOPS antibody and subsequently immunoblotted using anti-p53 antibody.


Figure 6A HOPS localization at mitochondria . RKO cells were stained by MitoTracker ™ ( red ) , immediately fixed and stained to visualize HOPS ( green ) . Nuclei were DAPI stained ( blue ) . Samples were analysed with fluorescent microscope and representative images were shown . Scale bars , 10µm . B RKO cells were lysed and processed for mitochondria isolation . WCL , cytoplasm and mitochondria were analysed using anti - HOPS antibody . GAPDH , Lamin B1 and Tom20 are purity controls of mitochondrial fraction . C HOPS and p53 translocation to mitochondria . RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53 , HOPS and the reported extraction purity controls . D HOPS / p53 binding at mitochondria . RKO cells were treated with etoposide and subjected to mitochondrial purification , and subsequent immunoprecipitation using anti - HOPS antibody . Samples were immunoblotted to detect p53 , HOPS and the reported controls . E HOPS and p53 mitochondrial translocation in vivo . Hops + / + and Hops − / − mice were treated with etoposide and thymus , spleen and testis were harvested . Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53 . GAPDH and Tom20 are purity controls . p53 mitochondrial protein levels were semi - quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1 . Data information : assays involving animals were performed three times in three different mice per condition . In E data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . F p53 - dependent mitochondrial membrane potential ( Δψm ) induction in MEFs . Hops + / + and Hops − / − MEFs were treated with etoposide and then incubated with JC - 1 vital dye . The images were collected by fluorescent microscopy and representative images are shown ( left panel ) . Scale bars , 10µm . Fluorescence was analysed by TECAN plate reader and plotted in the graph ( right panel ) . Data information : assays involving animals were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times . In F data are presented as mean ± SD . * P < 0 . 05 ; * * * P < 0 . 001 , by two tailed Student ' s t - test . G p53 MOMP induction in HOPS deficiency . Mitochondria from Hops + / + and Hops − / − MEFs , were purified and incubated with p53 recombinant protein . Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting . Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker . Data information : assays involving animals were performed three times in three different mice per condition . Experiments on mitochondria from MEFs were performed five times .

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Figure 6A HOPS localization at mitochondria. RKO cells were stained by MitoTracker™ (red), immediately fixed and stained to visualize HOPS (green). Nuclei were DAPI stained (blue). Samples were analysed with fluorescent microscope and representative images were shown. Scale bars, 10µm.B RKO cells were lysed and processed for mitochondria isolation. WCL, cytoplasm and mitochondria were analysed using anti-HOPS antibody. GAPDH, Lamin B1 and Tom20 are purity controls of mitochondrial fraction.C HOPS and p53 translocation to mitochondria. RKO cells were treated with etoposide as indicated and processed for mitochondrial extraction and subsequent immunoblotting for p53, HOPS and the reported extraction purity controls.D HOPS/p53 binding at mitochondria. RKO cells were treated with etoposide and subjected to mitochondrial purification, and subsequent immunoprecipitation using anti-HOPS antibody. Samples were immunoblotted to detect p53, HOPS and the reported controls.E HOPS and p53 mitochondrial translocation in vivo. Hops+/+ and Hops−/− mice were treated with etoposide and thymus, spleen and testis were harvested. Organs were homogenized and mitochondrial fraction were isolated and analysed by immunoblotting versus p53. GAPDH and Tom20 are purity controls. p53 mitochondrial protein levels were semi-quantified using Tom20 as loading control and p53 levels at time 0 were assumed as 1. Data information: assays involving animals were performed three times in three different mice per condition. In E data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.F p53-dependent mitochondrial membrane potential (Δψm) induction in MEFs. Hops+/+ and Hops−/− MEFs were treated with etoposide and then incubated with JC-1 vital dye. The images were collected by fluorescent microscopy and representative images are shown (left panel). Scale bars, 10µm. Fluorescence was analysed by TECAN plate reader and plotted in the graph (right panel). Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times. In F data are presented as mean ±SD. * P < 0.05; *** P < 0.001, by two tailed Student's t-test.G p53 MOMP induction in HOPS deficiency. Mitochondria from Hops+/+ and Hops−/− MEFs, were purified and incubated with p53 recombinant protein. Cytochrome C release from mitochondria as metabolic marker was assayed by immunoblotting. Samples were tested to detect p53 as control and Tom20 as mitochondria structural marker. Data information: assays involving animals were performed three times in three different mice per condition. Experiments on mitochondria from MEFs were performed five times.


Figure 7A HOPS expression and p53 nuclear accumulation . HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 ( green ) . Nuclei were stained with DAPI ( blue ) . Samples were analysed with fluorescent microscope and merged images are shown ( right panel ) . Scale bars , 10µm . Cells were scored for p53 localization . The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells ( left panel ) . Data information : all the experiments detailed above were performed three times , and representative panels are shown . In A data are presented as mean ± SD . B Biochemical analysis of HOPS intracellular localization . RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component . Aliquots relative to the same cell number per fraction were immunoblotted using anti - p53 and anti - HOPS antibodies . GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively . Data information : all the experiments detailed above were performed three times , and representative panels are shown . HOPS and p53 binding to importin α . Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti - p53 and analysed by immunoblotting using anti - importin α antibody . Arrowhead indicates the importin α specific signal . Data information : all the experiments detailed above were performed three times , and representative panels are shown . D PCAF - mediated p53 acetylation . p53 and PCAF Flag - tagged were co - expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide . p53 acetylation levels were evaluated by immunoprecipitation with anti - p53 antibody followed by immunoblotting with anti - acetylated - Lysine ( AcK ) , anti - p53 , anti - Flag , anti - HOPS and anti - α - tubulin antibodies . Data information : all the experiments detailed above were performed three times , and representative panels are shown .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 HOPS expression and p53 nuclear accumulation. HOPS was overexpressed in RKO and cells were fixed and stained with antibody against p53 (green). Nuclei were stained with DAPI (blue). Samples were analysed with fluorescent microscope and merged images are shown (right panel). Scale bars, 10µm. Cells were scored for p53 localization. The results are shown as percentage of nuclear versus cytoplasmic p53 localization in untransfected and HOPS transfected cells (left panel). Data information: all the experiments detailed above were performed three times, and representative panels are shown. In A data are presented as mean ±SD.B Biochemical analysis of HOPS intracellular localization. RKO cells were transfected or not with HOPS and fractionated into nuclear and cytoplasmic component. Aliquots relative to the same cell number per fraction were immunoblotted using anti-p53 and anti-HOPS antibodies. GAPDH and Lamin B1 are purity controls of cytoplasmic and nuclear fraction respectively. Data information: all the experiments detailed above were performed three times, and representative panels are shown.HOPS and p53 binding to importin α. Untransfected or HOPS transfected RKO cells were treated with etoposide and each sample was evaluated by immunoprecipitation with anti-p53 and analysed by immunoblotting using anti-importin α antibody. Arrowhead indicates the importin α specific signal. Data information: all the experiments detailed above were performed three times, and representative panels are shown.D PCAF-mediated p53 acetylation. p53 and PCAF Flag-tagged were co-expressed in H1299 cells with or without HOPS and treated with trichostatin A and nicotinamide. p53 acetylation levels were evaluated by immunoprecipitation with anti-p53 antibody followed by immunoblotting with anti-acetylated-Lysine (AcK), anti-p53, anti-Flag, anti-HOPS and anti-α-tubulin antibodies. Data information: all the experiments detailed above were performed three times, and representative panels are shown.


Figure 1 ( A - B ) Live third instar discs incubated in DiBAC . DiBAC fluorescence is observed in the wing imaginal disc in both apical ( A - A ' ) , and basal ( B - B ' ) optical sections . Increased fluorescence is observed in the center of the pouch ( white arrow , B ' ) , and at the dorsoventral ( D - V ) compartment boundary in the anterior compartment ( yellow arrow , B ' , and inset , D ) . ( C - E ) Comparison of DiBAC with the voltage - insensitive dye FM4 - 64 . Incubation of live discs in FM4 - 64 ( E ) shows more uniform fluorescence when compared to DiBAC ( D ) . Quantitative comparison of fluorescence in the two white boxes in each panel is shown in ( C ) . N = 7 discs for each treatment , data compared using an unpaired t test , * * indicates p < 0 . 001 , error bars are standard deviations . The region boxed in yellow in ( D ) is shown at higher magnification to show DiBAC fluorescence at the D - V compartment boundary . ( F , G ) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence . ( H - H ' ' ) Live wing discs expressing UAS - RFP anterior to the compartment boundary , under the control of ptc - Gal4 , were incubated in DiBAC , showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression . White arrowhead in ( H ) indicates the stripe in the wing pouch ; yellow arrowheads indicate the stripe in the dorsal and ventral hinge . ( I , I ' ) Early L3 discs incubated in DiBAC . Patterned depolarization is evident throughout the third larval instar , with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time ( Compare I with A , bracket in I indicates width of increased DiBAC fluorescence ) .

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Figure 1(A-B) Live third instar discs incubated in DiBAC. DiBAC fluorescence is observed in the wing imaginal disc in both apical (A-A'), and basal (B-B') optical sections. Increased fluorescence is observed in the center of the pouch (white arrow, B'), and at the dorsoventral (D-V) compartment boundary in the anterior compartment (yellow arrow, B', and inset, D).(C-E) Comparison of DiBAC with the voltage-insensitive dye FM4-64. Incubation of live discs in FM4-64 (E) shows more uniform fluorescence when compared to DiBAC (D). Quantitative comparison of fluorescence in the two white boxes in each panel is shown in (C). N=7 discs for each treatment, data compared using an unpaired t test, ** indicates p<0.001, error bars are standard deviations. The region boxed in yellow in (D) is shown at higher magnification to show DiBAC fluorescence at the D-V compartment boundary.(F, G) Incubation in ouabain results in brighter and more uniform DiBAC fluorescence.(H-H'') Live wing discs expressing UAS-RFP anterior to the compartment boundary, under the control of ptc-Gal4, were incubated in DiBAC, showing that the stripe of increased DiBAC fluorescence coincides with the posterior edge of ptc expression. White arrowhead in (H) indicates the stripe in the wing pouch; yellow arrowheads indicate the stripe in the dorsal and ventral hinge.(I, I') Early L3 discs incubated in DiBAC. Patterned depolarization is evident throughout the third larval instar, with the stripe of increased fluorescence becoming narrower in more mature discs with developmental time (Compare I with A, bracket in I indicates width of increased DiBAC fluorescence).


Figure 2Expression of Rpk is increased anterior to the A - P compartment boundary , and in two rows of cells flanking the D - V compartment boundary in the A compartment . ( B - B ' ' ) Blockade of DEG / ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A - P compartment boundary ( white arrowhead ) . White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0 . 98 , standard deviation = 0 . 05 , n = 5 discs . Increased fluorescence at the D - V boundary in the A compartment is still observable ( yellow arrowhead ) . ( C , C ' ) Amiloride incubation does not diminish Rpk expression . White arrowhead indicates approximate position of the A - P compartment boundary , yellow arrowhead indicates approximate position of the D - V compartment boundary . ( D , E ) Expression of rpk RNAi using dpp - Gal4 results in diminished DiBAC fluorescence along the A - P compartment boundary ( white arrowhead ) , while increased fluorescence at the D - V boundary in the A compartment is still observable ( yellow arrowhead ) .

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Figure 2Expression of Rpk is increased anterior to the A-P compartment boundary, and in two rows of cells flanking the D-V compartment boundary in the A compartment.(B-B'') Blockade of DEG/ENaC channels by incubation in amiloride abolishes the stripe of increased DiBAC fluorescence along the A-P compartment boundary (white arrowhead). White boxes indicate regions used to calculate the average DiBAC fluorescence intensity ratio across the compartment boundary = 0.98, standard deviation = 0.05, n = 5 discs. Increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).(C, C') Amiloride incubation does not diminish Rpk expression. White arrowhead indicates approximate position of the A-P compartment boundary, yellow arrowhead indicates approximate position of the D-V compartment boundary.(D, E) Expression of rpk RNAi using dpp-Gal4 results in diminished DiBAC fluorescence along the A-P compartment boundary (white arrowhead), while increased fluorescence at the D-V boundary in the A compartment is still observable (yellow arrowhead).


Figure 3 ( A - A ' ' ' ) Immunostaining of discs expressing ptc > RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary . White arrowheads indicate approximate position of the A - P compartment boundary . ( B - C ' ) L3 discs that are temperature - sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk ( B , B ' ) and ATPα ( C , C ' ) anterior to the compartment boundary . Controls shown in Figure EV2 . White arrowheads indicate approximate position of the A - P compartment boundary . ( D - E ' ' ' ) Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment ( white box ) as well as the posterior compartment ( yellow arrowhead ) . ( E - E ' ' ' ) A single clone in the A compartment is shown at higher magnification . ( F - F ' ) A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC . ( G - G ' ) A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC . The clones also express RFP .

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Figure 3(A-A''') Immunostaining of discs expressing ptc>RFP with antibodies to Rpk and ATPα showing elevated levels of both proteins anterior to the compartment boundary. White arrowheads indicate approximate position of the A-P compartment boundary.(B-C') L3 discs that are temperature-sensitive for hh following shift to the restrictive temperature for 12 h show loss of increased expression of Rpk (B, B') and ATPα (C, C') anterior to the compartment boundary. Controls shown in Figure EV2. White arrowheads indicate approximate position of the A-P compartment boundary.(D-E''') Clones of cells expressing a constitutively activated form of the transcription factor Ci show elevated Rpk and ATPα expression in the anterior compartment (white box) as well as the posterior compartment (yellow arrowhead). (E-E''') A single clone in the A compartment is shown at higher magnification.(F-F') A clone of cells in the anterior compartment expressing ci3M following incubation in DiBAC.(G-G') A clone of cells in the anterior compartment expressing ptc RNAi following incubation in DiBAC. The clones also express RFP.


Figure 4 ( A - A ' ' ' ) Immunostaining of Smo protein ( red ) and full - length Ci ( light blue ) in discs expressing en - Venus . Smo is expressed at higher levels in the P compartment and in a stripe 5 - 10 cells wide just anterior to the A - P compartment boundary . White arrowhead indicates cells just anterior to the A - P compartment boundary . ( B - C ) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap - Gal4 . Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo ( B , B ' ) , and the activator form of Ci ( B , B ' ' ) . The expression of ptc , visualized using an anti - Ptc antibody ( C ) , a downstream target gene of Hh signaling , is also diminished . The Rpk RNAi line BL : 39053 is used in all panels shown , but the phenotype was validated with a second RNAi line ( BL : 25847 ) .

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

Figure 4(A-A''') Immunostaining of Smo protein (red) and full-length Ci (light blue) in discs expressing en-Venus. Smo is expressed at higher levels in the P compartment and in a stripe 5-10 cells wide just anterior to the A-P compartment boundary. White arrowhead indicates cells just anterior to the A-P compartment boundary.(B-C) Effect of expressing an RNAi against the ENaC channel rpk in the dorsal compartment of the disc using ap-Gal4. Knockdown of Rpk in the dorsal compartment results in decreased accumulation of the Hh signal transducer Smo (B, B'), and the activator form of Ci (B, B''). The expression of ptc, visualized using an anti-Ptc antibody (C), a downstream target gene of Hh signaling, is also diminished. The Rpk RNAi line BL:39053 is used in all panels shown, but the phenotype was validated with a second RNAi line (BL:25847).


Figure 5 ( A - C ) Localization of Smo protein ( blue ) following expression of 71B - Gal4 ( A ) , 71B - Gal4 and UAS - NaChBac ( B ) or 71B - Gal4 and UAS - Kir2 . 1 ( C ) . Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2 . 1 reduces membrane localization , and increases cytosolic Smo ( B , C ) . ( D ) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence . N = 7 salivary glands per genotype . Data were compared using ANOVA followed by Tukey test for significance ( * * indicates p < 0 . 01 , * * * indicates p < 0 . 001 ) , error bars are standard deviations . ( E , F ) Expression of the Hh target gene ptc is visualized using anti - Ptc immunostaining . Compared to 71B - Gal4 ( E ) , Ptc expression is increased in glands expressing 71B - Gal4 and UAS - NaChBac ( F ) . ( G - I ) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance . After dissection , glands were kept in either dark conditions ( G ) or exposed to 15 minutes of blue light ( H ) . ( I ) Quantification of membrane fluorescence ; N = 13 glands , data compared using an unpaired t test ( * * indicates p < 0 . 01 ) , error bars are standard deviations . ( J - L ) Effect of expression and activation of the Cl - - selective channelrhodopsin ChloC . An overall reduction in Smo immunostaining was observed following channel activation ( K ) , as compared to control glands kept in the dark ( J ) . ( L ) Quantification of membrane fluorescence ; N = 10 glands , data were compared using an unpaired t test , ( * * * indicates p < 0 . 001 ) , error bars are standard deviations . ( M - Q ) Time course of change in Smo localization . Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light , then fixed and stained for Smo protein ( M - P ' ' ) . ( Q ) Quantification of membrane - associated fluorescence ; N = 7 glands / timepoint , data were compared using ANOVA followed by Tukey test for significance ( * * indicates p < 0 . 01 ) , error bars are standard deviations .

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

Figure 5(A-C) Localization of Smo protein (blue) following expression of 71B-Gal4 (A), 71B-Gal4 and UAS-NaChBac (B) or 71B-Gal4 and UAS-Kir2.1 (C). Expression of the sodium channel NaChBac increases membrane localization of Smo while expression of the potassium channel Kir2.1 reduces membrane localization, and increases cytosolic Smo (B, C).(D) Quantification of the ratio of membrane Smo fluorescence to cytosol fluorescence. N=7 salivary glands per genotype. Data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01, *** indicates p<0.001), error bars are standard deviations.(E, F) Expression of the Hh target gene ptc is visualized using anti-Ptc immunostaining. Compared to 71B-Gal4 (E), Ptc expression is increased in glands expressing 71B-Gal4 and UAS-NaChBac (F).(G-I) Effect of expression and activation of the depolarizing channelrhodopsin ChR2 on Smo protein membrane abundance. After dissection, glands were kept in either dark conditions (G) or exposed to 15 minutes of blue light (H). (I) Quantification of membrane fluorescence; N=13 glands, data compared using an unpaired t test (** indicates p<0.01), error bars are standard deviations.(J-L) Effect of expression and activation of the Cl--selective channelrhodopsin ChloC. An overall reduction in Smo immunostaining was observed following channel activation (K), as compared to control glands kept in the dark (J). (L) Quantification of membrane fluorescence; N=10 glands, data were compared using an unpaired t test, (*** indicates p<0.001), error bars are standard deviations.(M-Q) Time course of change in Smo localization. Salivary glands expressing the channelrhodopsin ChR2 were subjected to increasing intervals of activating light, then fixed and stained for Smo protein (M-P''). (Q) Quantification of membrane-associated fluorescence; N=7 glands/timepoint, data were compared using ANOVA followed by Tukey test for significance (** indicates p<0.01), error bars are standard deviations.


Figure 6 ( A - B ' ' ) Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture , then fixed and stained for Smo protein . In ( B ' ' ) , the yellow arrowhead indicates dorsoventral compartment boundary , and the white arrowhead indicates anteroposterior compartment boundary . ( C ) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment . N = 5 for 0min discs , n = 9 for 25min discs , data were compared using an unpaired t test ( * * * p < 0 . 001 ) , error bars are standard deviations . Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence ( p = 0 . 132 ) , dorsal posterior fluorescence to dorsal anterior fluorescence ( p = 0 . 12 ) , and ventral posterior fluorescence to ventral anterior fluorescence ( p = 0 . 36 ) . ( D - E ' ) Discs expressing the red - light activated channelrhodopsin ReaChR under control of ap - Gal4 were raised on a 12hr light / dark cycle , dissected and stained for Smo protein ( D , D ' ) or Ptc protein ( E , E ' ) .

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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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'') Wing discs expressing the channelrhodopsin ChR2 were subjected to 0 minutes or 25 minutes of activating light in culture, then fixed and stained for Smo protein. In (B''), the yellow arrowhead indicates dorsoventral compartment boundary, and the white arrowhead indicates anteroposterior compartment boundary. (C) Quantification of the ratio of mean Smo fluorescence intensity in 25µm2 regions in the dorsal anterior compartment compared to the ventral posterior compartment. N=5 for 0min discs, n=9 for 25min discs, data were compared using an unpaired t test (***p<0.001), error bars are standard deviations. Identical analyses were carried out comparing dorsal posterior fluorescence to ventral posterior fluorescence (p=0.132), dorsal posterior fluorescence to dorsal anterior fluorescence (p=0.12), and ventral posterior fluorescence to ventral anterior fluorescence (p=0.36). (D-E') Discs expressing the red-light activated channelrhodopsin ReaChR under control of ap-Gal4 were raised on a 12hr light/dark cycle, dissected and stained for Smo protein (D, D') or Ptc protein (E, E').


Figure 1A ) Volcano plot of the fold - change ( x - axis ) plotted against significance ( y - axis ) of lipids changing upon HF / HS feeding . Lipids are colored according to fold - change ( log2 , absolute ) > 1 ( orange ) , p - value < 0 . 05 ( red ) or both ( green ) . Pvalues calculated from differential expression using limmaB ) Heatmap of the fold - change ( log2 ) of each lipid in HF / HS compared to chow diet . Only lipid species detected in all mice are shown . C ) Examples of different hepatic lipids within one class that are regulated in different directions in HF / HS fed as compared chow fed mice .

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Figure 1A) Volcano plot of the fold-change (x-axis) plotted against significance (y-axis) of lipids changing upon HF/HS feeding. Lipids are colored according to fold-change (log2, absolute) > 1 (orange), p-value <0.05 (red) or both (green). Pvalues calculated from differential expression using limmaB) Heatmap of the fold-change (log2) of each lipid in HF/HS compared to chow diet. Only lipid species detected in all mice are shown.C) Examples of different hepatic lipids within one class that are regulated in different directions in HF/HS fed as compared chow fed mice.


Figure 2A ) The relative genetic variation of hepatic lipidome composition ; all lipids were quantified in proportion to the total lipidome . Each lipid class is shown in a different color where differences can be observed across the strains . B ) Heatmap showing correlations between different lipid species ( x - axis ) and the abundance of gut microbes ( y - axis ) . Microbes were summarized at the levels of order ( o _ ) , genus ( g _ ) or family ( f _ ) . * = p < 0 . 05 , * * = p < 0 . 01 Pvalues were calculated based on significance of regression ( students test ) and adjusted for multiple comparisons ( FDR = 0 . 05 ) .

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Figure 2A) The relative genetic variation of hepatic lipidome composition; all lipids were quantified in proportion to the total lipidome. Each lipid class is shown in a different color where differences can be observed across the strains.B) Heatmap showing correlations between different lipid species (x-axis) and the abundance of gut microbes (y-axis). Microbes were summarized at the levels of order (o_), genus (g_) or family (f_). *=p<0.05, **=p<0.01 Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05).


Figure 3A ) Heatmap showing correlations among hepatic lipids . B ) Heatmap showing concordance between different lipid species ( class listed on y - axis ) and certain phenotypic traits on the x - axis .

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Figure 3A) Heatmap showing correlations among hepatic lipids.B) Heatmap showing concordance between different lipid species (class listed on y-axis) and certain phenotypic traits on the x-axis.


Figure 4A ) Manhattan plot of genome - wide association for the levels of hepatic Pex16 transcript , where the only significant locus appears directly surrounding the genomic location ( red arrow ) . Significant cut - offs are shown for FDR ( blue ) and Bonferroni ( red ) . The peak SNP ( rs27364570 ) is highlighted with a dark red box . Y - axis shows the - log10 ( pvalue ) vs x - axis showing each SNP measured . P - values for GWAS associations were calculated using FaST - LMM . B ) Allelic distribution comparing GG vs TT ( x - axis ) for the peak SNP of the Pex16 association ( rs27364570 ) , where the abundance of each LPC species ( y - axis ) showed significantly different levels depending on the allele . P - values for GWAS associations were calculated using FaST - LMM . Boxplots show mean ( middle line ) , 25 - 75 % quantiles ( colored box ) and 5 - 95 % quantiles ( vertical lines ) C ) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits . Box color indicates bicor value , where all relationships are positive and number in each box shows p - value for each correlation . Pvalues were calculated based on significance of regression ( students test ) and adjusted for multiple comparisons ( FDR = 0 . 05 )

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

Figure 4A) Manhattan plot of genome-wide association for the levels of hepatic Pex16 transcript, where the only significant locus appears directly surrounding the genomic location (red arrow). Significant cut-offs are shown for FDR (blue) and Bonferroni (red). The peak SNP (rs27364570) is highlighted with a dark red box. Y-axis shows the -log10(pvalue) vs x-axis showing each SNP measured. P-values for GWAS associations were calculated using FaST-LMM.B) Allelic distribution comparing GG vs TT (x-axis) for the peak SNP of the Pex16 association (rs27364570), where the abundance of each LPC species (y-axis) showed significantly different levels depending on the allele. P-values for GWAS associations were calculated using FaST-LMM. Boxplots show mean (middle line), 25-75% quantiles (colored box) and 5-95% quantiles (vertical lines)C) Correlations between expression of hepatic Pex16 with LPC species and phenotypic traits. Box color indicates bicor value, where all relationships are positive and number in each box shows p-value for each correlation. Pvalues were calculated based on significance of regression (students test) and adjusted for multiple comparisons (FDR = 0.05)