Figure 6 ( A ) Western blotting shows that Vasa , Piwi , and Aub were ectopically expressed in Lint - OKO and L ( 3 ) mbtts1 larval brains at 29ºC . Anti - Lint - O , anti - L ( 3 ) mbt , anti - Vasa , andi - Piwi , anti - Aub , and anti - Tubulin antibodies were used . ( B ) Confocal images of y w , Lint - OKO , and L ( 3 ) mbtts1 immunostained for Vasa ( magenta ) , MIRA ( green ) , and ELAV ( green ) . Scale bar : 100 μm . ( C ) Quantification of brain lobe volume for the following genotypes : Lint - OKO - / + ( n = 42 biological replicates ) , Lint - OKO - / - ( n = 36 biological replicates ) , and L ( 3 ) mbtts1 - / - ( n = 34 biological replicates ) . Boxplot central bands , upper edges of boxes , lower edges of boxes , upper whiskers and lower whiskers show median , third quartile , first quartile , maxima , and minima , respectively . P - values were calculated by the Student ' s t - test . ( * * * P - value < 0 . 001 ) . ( D , E ) The genomic regions harboring the vasa ( D ) and aub ( E ) genes . The RNA - seq reads in y w , L ( 3 ) mbtts1 , and Lint - OKO brains are shown . The shading in gray corresponds to exons . The y - axis shows the number of RPM . RNA - seq samples were biological triplicates .

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Figure 6(A) Western blotting shows that Vasa, Piwi, and Aub were ectopically expressed in Lint-OKO and L(3)mbtts1 larval brains at 29ºC. Anti-Lint-O, anti-L(3)mbt, anti-Vasa, andi-Piwi, anti-Aub, and anti-Tubulin antibodies were used.(B) Confocal images of y w, Lint-OKO, and L(3)mbtts1 immunostained for Vasa (magenta), MIRA (green), and ELAV (green). Scale bar: 100 μm.(C) Quantification of brain lobe volume for the following genotypes: Lint-OKO -/+ (n = 42 biological replicates), Lint-OKO -/- (n = 36 biological replicates), and L(3)mbtts1 -/- (n = 34 biological replicates). Boxplot central bands, upper edges of boxes, lower edges of boxes, upper whiskers and lower whiskers show median, third quartile, first quartile, maxima, and minima, respectively. P-values were calculated by the Student's t-test. (***P-value < 0.001).(D, E) The genomic regions harboring the vasa (D) and aub (E) genes. The RNA-seq reads in y w, L(3)mbtts1, and Lint-OKO brains are shown. The shading in gray corresponds to exons. The y-axis shows the number of RPM. RNA-seq samples were biological triplicates.


Figure 1 ( B ) Representative spinning disc confocal microscopy images of iDCs cultured in 2D suspension ( left ) or 3D collagen ( middle ) 8h post seeding . Scale bars = 20µm . Cells were stained with phalloidin - Atto390 to visualize F - actin and cell morphology . The dotted lines indicate which area are shown in higher magnification in the panel on the right . Scale bars = 10µm . Comparison of the volume ( G ) of iDCs derived from four donors ( n = 1559 to n = 4675 each , mean + / - SD ) cultured either in 2D suspension or 3D collagen and measured with RT - DC at a flow rate of 0 . 12 µl / s . A significant increase in deformability of iDCs when cultured in collagen was found , whereas no significant difference in cell volume ( G ) For statistical significance testing , linear mixed model analysis was performed to calculate ANOVA p - values ; * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 . ( I - J ) Two days post culturing in 2D suspension or 3D collagen , RNA was extracted and the transcriptome was determined by Clariom ™ S Pico assay . The left panels show the heatmap related to the comparison of the expression between 2D suspension and 3D collagen profile of the untreated ( mock , top ) and LPS treated cells ( bottom ) ( adjusted p - value < 0 . 05 ) . Inside each cluster , two subclusters identify the 2D suspension and the 3D collagen cells . The right panels show the set of statistically enriched pathways identified with the genes in the heatmap using metascape .

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

Figure 1(B) Representative spinning disc confocal microscopy images of iDCs cultured in 2D suspension (left) or 3D collagen (middle) 8h post seeding. Scale bars = 20µm. Cells were stained with phalloidin-Atto390 to visualize F-actin and cell morphology. The dotted lines indicate which area are shown in higher magnification in the panel on the right. Scale bars = 10µm.Comparison of the volume (G) of iDCs derived from four donors (n = 1559 to n = 4675 each, mean +/-SD) cultured either in 2D suspension or 3D collagen and measured with RT-DC at a flow rate of 0.12 µl/s. A significant increase in deformability of iDCs when cultured in collagen was found , whereas no significant difference in cell volume (G) For statistical significance testing, linear mixed model analysis was performed to calculate ANOVA p-values; *p < 0.05, **p < 0.01, ***p < 0.001.(I-J) Two days post culturing in 2D suspension or 3D collagen, RNA was extracted and the transcriptome was determined by Clariom™ S Pico assay. The left panels show the heatmap related to the comparison of the expression between 2D suspension and 3D collagen profile of the untreated (mock, top) and LPS treated cells (bottom) (adjusted p-value < 0.05). Inside each cluster, two subclusters identify the 2D suspension and the 3D collagen cells. The right panels show the set of statistically enriched pathways identified with the genes in the heatmap using metascape.


Figure 2 ( C ) Percentage of p24 + cells at 2 d . p . i . ( left ) and 4 d . p . i . ( right ) . Graphs depict mean values + / - SD for cells from five donors . * * p < 0 . 01 calculated with 2way ANOVA test .

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Figure 2(C) Percentage of p24+ cells at 2 d.p.i. (left) and 4 d.p.i. (right). Graphs depict mean values +/- SD for cells from five donors. ** p<0.01 calculated with 2way ANOVA test.


Figure 3 ( B ) Representative spinning disc confocal microscopy images of iDC cultured in 2D suspension ( left ) or 3D collagen ( right ) at 4 h . p . i . Cells were stained with Phalloidin - Atto390 to visualize F - actin . Vpr - mRuby2 particles are shown in red . Scale bar = 20µm . Arrow heads highlight virus particles in 3D collagen . The dotted lines indicate which area are shown in higher magnification . Scale bars = 2µm ( C ) Vpr signal per cell . Graphs depict means values + / - SD for cells from four donors * * * p < 0 . 001 calculated with 2way ANOVA test . Cells were infected with HIV - 1 R5 Vpr - Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11 ° C to prevent particle fusion during the staining process . For the indicated sample , T20 was added at the moment of infection . ( E ) Percentage of positive cells for the presence of the CFF2 product at 4h p . i . * * p < 0 . 01calculated with 2way ANOVA test . Cells were infected with HIV - 1 R5 Vpr - Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11 ° C to prevent particle fusion during the staining process . For the indicated sample , T20 was added at the moment of infection . ( F - G ) Percentage of positive cells for the presence of the CFF2 product at 4h p . i . ( left ) and 48h p . i . ( right ) * p < 0 . 05 calculated with 2way ANOVA test . The graphs show mean values + / - SD for cells from five and three donors , respectively .

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Figure 3(B) Representative spinning disc confocal microscopy images of iDC cultured in 2D suspension (left) or 3D collagen (right) at 4 h.p.i. Cells were stained with Phalloidin-Atto390 to visualize F-actin. Vpr-mRuby2 particles are shown in red. Scale bar = 20µm. Arrow heads highlight virus particles in 3D collagen. The dotted lines indicate which area are shown in higher magnification. Scale bars = 2µm (C) Vpr signal per cell. Graphs depict means values +/- SD for cells from four donors *** p< 0.001 calculated with 2way ANOVA test.Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (E) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. ** p<0.01calculated with 2way ANOVA test.Cells were infected with HIV-1 R5 Vpr-Blam for 4h or 48h and then incubated with CCF2 for 5 h at 11°C to prevent particle fusion during the staining process. For the indicated sample, T20 was added at the moment of infection. (F-G) Percentage of positive cells for the presence of the CFF2 product at 4h p.i. (left) and 48h p.i. (right) *p<0.05 calculated with 2way ANOVA test. The graphs show mean values +/- SD for cells from five and three donors, respectively.


Figure 4 ( B ) Representative dot plots of FACS analysis to quantify the number of productively infected cells . Shown is the CD3 - PerCp - Cy5 - 5 fluorescence intensity against intracellular p24 - FITC after gating on live cells . The gate denotes the productive infected p24 + cell population with the percentage of p24 + CD4 T cells indicated . ( C ) Percentage of p24 + cells at 2 d . p . i . ( top ) and 4 d . p . i . ( bottom ) . Graphs depict mean values + / - SD for cells from four donors . ( F ) Manual quantification of percentage of iDCs interacting with CD4 T cells over the 2 hour time course . Bars indicates mean value for cells from two donors .

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Figure 4(B) Representative dot plots of FACS analysis to quantify the number of productively infected cells. Shown is the CD3-PerCp-Cy5-5 fluorescence intensity against intracellular p24-FITC after gating on live cells. The gate denotes the productive infected p24+ cell population with the percentage of p24+ CD4 T cells indicated. (C) Percentage of p24+ cells at 2 d.p.i. (top) and 4 d.p.i. (bottom). Graphs depict mean values +/- SD for cells from four donors.(F) Manual quantification of percentage of iDCs interacting with CD4 T cells over the 2 hour time course. Bars indicates mean value for cells from two donors.


Figure 5 ( C ) Representative dot plots of FACS analysis to quantify the number of productively infected cells that upregulated CD86 . Shown is CD86 - PerCp - 770 fluorescence intensity against intracellular p24 - FITC . The gates indicate the percentage of p24 + and p24 - cells together with the percentage of CD86 + and CD86 - cell . ( D ) Ratio between the percentage of CD86 positive and CD86 negative cells for HIV - 1 R5 infected iDC in the presence or absence of Vpx and for LPS treaded iDCs , as positive control . The graph shows mean values + / - SD for cells for five donors . n . s calculated with 2way ANOVA and Sidak ' s multiple comparison test .

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Figure 5(C) Representative dot plots of FACS analysis to quantify the number of productively infected cells that upregulated CD86. Shown is CD86-PerCp-770 fluorescence intensity against intracellular p24-FITC. The gates indicate the percentage of p24+ and p24- cells together with the percentage of CD86+ and CD86- cell. (D) Ratio between the percentage of CD86 positive and CD86 negative cells for HIV-1 R5 infected iDC in the presence or absence of Vpx and for LPS treaded iDCs, as positive control. The graph shows mean values +/- SD for cells for five donors. n.s calculated with 2way ANOVA and Sidak's multiple comparison test.


Figure 6 ( C - E ) Graphs show the fold change increase , over uninfected control , for infection with HIV - 1 R5 in the presence of Vpx ( C ) , HIV - 1 R5 ( D ) or HIV - 1 R5 in the presence of Vpx and Raltegravir control ( E ) of the 16 selected genes with antiviral properties for cells from three donors . Bars represent the mean + / - SD * p < 0 . 05 calculated with 2way ANOVA with Sidak test .

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Figure 6(C-E) Graphs show the fold change increase, over uninfected control, for infection with HIV-1 R5 in the presence of Vpx (C), HIV-1 R5 (D) or HIV-1 R5 in the presence of Vpx and Raltegravir control (E) of the 16 selected genes with antiviral properties for cells from three donors. Bars represent the mean +/- SD * p< 0.05 calculated with 2way ANOVA with Sidak test.


Figure 7 ( A - E ) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen or 2D suspension for two days and the surface expression of CD4 ( A ) , CCR5 ( B ) , DC - SIGN ( C ) , SIGLEC - 1 ( D ) , and MMR ( E ) was analyzed by FACS . Representative histograms of FACS analysis showing fluorescence intensity for each receptor analyzed ( Top ) . The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample for cells from three to six donors ( bottom ) Bars represent mean + / - SD . * * * p < 0 . 001 ; * * p < 0 . 01 calculated with paired t - test . ( F ) Representative images showing sum slice z - projections . Images from the DC - SIGN channel of cells kept in 2D suspension or 3D collagen were identically processed and are shown with identical brightness / contrast settings . The actin signal was adjusted according to its brightness in each image individually to identify the cells . Scale bar = 20µm ( G ) The graph shows the fluorescence intensity mean of the DC - SIGN channel from cells grown in 2D suspension or 3D collagen . Bars define the mean + / - SD and dots represent individual cell analyzed . N of cells in 2D suspension = 34 ; N of cells in 3D collagen = 54 . * * * p < 0 . 001 calculated with Mann - Whitney test . iDCs were spin - transduced with VLPs carrying Vpxmac239 one day prior to infection . Cells were incubated with anti - DC - SIGN and anti - MMR blocking antibody or isotype control for 30 min , infected with HIV - 1 R5 Vpr - Blam for 4 hours to asses fusion or for 48 hours to asses productive infection . ( I ) Percentage of positive cells for the presence of the CFF2 product at 4 hours p . i . ( J ) Percentage of p24 + cells at 48 hours p . i . The graphs show mean values + / - SD for cells from three donors . * p < 0 . 05 calculated with 2way ANOVA test .

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Figure 7(A-E) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen or 2D suspension for two days and the surface expression of CD4 (A), CCR5 (B), DC-SIGN (C), SIGLEC-1 (D), and MMR (E) was analyzed by FACS. Representative histograms of FACS analysis showing fluorescence intensity for each receptor analyzed (Top). The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample for cells from three to six donors (bottom) Bars represent mean +/- SD. *** p< 0.001; ** p<0.01 calculated with paired t-test.(F) Representative images showing sum slice z-projections. Images from the DC-SIGN channel of cells kept in 2D suspension or 3D collagen were identically processed and are shown with identical brightness/contrast settings. The actin signal was adjusted according to its brightness in each image individually to identify the cells. Scale bar = 20µm (G) The graph shows the fluorescence intensity mean of the DC-SIGN channel from cells grown in 2D suspension or 3D collagen. Bars define the mean +/- SD and dots represent individual cell analyzed. N of cells in 2D suspension = 34; N of cells in 3D collagen = 54. *** p< 0.001 calculated with Mann-Whitney test.iDCs were spin-transduced with VLPs carrying Vpxmac239 one day prior to infection. Cells were incubated with anti-DC-SIGN and anti-MMR blocking antibody or isotype control for 30 min, infected with HIV-1 R5 Vpr-Blam for 4 hours to asses fusion or for 48 hours to asses productive infection. (I) Percentage of positive cells for the presence of the CFF2 product at 4 hours p.i. (J) Percentage of p24+ cells at 48 hours p.i. The graphs show mean values +/- SD for cells from three donors. *p<0.05 calculated with 2way ANOVA test.


Figure 8 ( G ) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen 2D collagen or 2D suspension for two days and the surface expression of DC - SIGN was analyzed by FACS . The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample indicated as fold change . Graphs represent the mean for cells from two donors . ( H ) iDC differentiated from peripheral blood monocytes for five days were spin - transduced with VLPs carrying Vpxmac239 . One day later , iDCs were infected with HIV - 1 R5 and cultured in 2D suspension , 2D collagen or 3D collagen . Productive infection was analyzed by p24 intracellular staining and FACS at 4 ( d . p . i ) . The graph depicts mean values for cells from two donors .

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Figure 8(G) iDC differentiated from peripheral blood monocytes for 6 days were cultured in 3D collagen 2D collagen or 2D suspension for two days and the surface expression of DC-SIGN was analyzed by FACS. The graph shows the ratio between the median fluorescence intensity of the stained sample and the unstained sample indicated as fold change. Graphs represent the mean for cells from two donors.(H) iDC differentiated from peripheral blood monocytes for five days were spin-transduced with VLPs carrying Vpxmac239. One day later, iDCs were infected with HIV-1 R5 and cultured in 2D suspension, 2D collagen or 3D collagen. Productive infection was analyzed by p24 intracellular staining and FACS at 4 (d.p.i). The graph depicts mean values for cells from two donors.


Figure 1A - B . Correlation of MCU and MCUb expression levels with breast cancer clinical stages . Median - centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset ( http : / / tcga - data . nci . nih . gov / docs / publications / brca _ 2012 / ) . Data were plotted and analysed against tumor size ( T1 - T4 ) ( A ) and regional lymph node infiltration ( N0 - N3 ) ( B ) , according to the AJCC Cancer Staging Manual ( 7th edition ) . Linear regression analysis with different stages was implemented . Parameters of linear regression are shown . Number of samples for each stage are shown in parentheses . C - E . MCU silencing reduces Ca2 + mit uptake in TNBC cells . Cells were transfected with siMCU or siControl . After 48h , [ Ca2 + ] mit uptake upon ATP ( C , E ) or Histamine ( D ) stimulation was measured ( n = 10 ) . P - values : * * * p = 0 . 0008 , * * * p < 0 . 0001 , * * * p = 0 . 0001 , respectively . F - H . MCU silencing impairs TNBC cell migration . Cells were transfected with siMCU or siControl . The day after transfection , a linear scratch was obtained on the cell monolayer through a vertically held P200 tip ( time - point 0h ) . Cell migration into the scratched area was monitored 48 hours later . The covered area was measured and expressed as a percentage relative to 0 - hour time - point ( n = 12 ) . P - value : * * * p < 0 . 0001 . I - K . Cell proliferation is mainly unaffected by MCU depletion . Cells were transfected with siMCU or siControl . Cell number was counted every 24 hours for 3 days ( the 72 - hour time point corresponds to the 48 - hour time - point of wound healing assay ) . Results are expressed as ratio R / R0 where R0 is the number of cells at the time of transfection ( 0 hour time - point ) ( n = 6 ) . P - value : * p = 0 . 05 .

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Figure 1A-B. Correlation of MCU and MCUb expression levels with breast cancer clinical stages. Median-centered log2 mRNA expression levels of MCU and MCUb were collected from the TCGA breast cancer dataset (http://tcga-data.nci.nih.gov/docs/publications/brca_2012/). Data were plotted and analysed against tumor size (T1-T4) (A) and regional lymph node infiltration (N0-N3) (B), according to the AJCC Cancer Staging Manual (7th edition). Linear regression analysis with different stages was implemented. Parameters of linear regression are shown. Number of samples for each stage are shown in parentheses.C-E. MCU silencing reduces Ca2+mit uptake in TNBC cells. Cells were transfected with siMCU or siControl. After 48h, [Ca2+]mit uptake upon ATP (C, E) or Histamine (D) stimulation was measured (n=10). P-values: ***p= 0.0008, ***p<0.0001, ***p= 0.0001, respectively.F-H. MCU silencing impairs TNBC cell migration. Cells were transfected with siMCU or siControl. The day after transfection, a linear scratch was obtained on the cell monolayer through a vertically held P200 tip (time- point 0h). Cell migration into the scratched area was monitored 48 hours later. The covered area was measured and expressed as a percentage relative to 0-hour time-point (n=12). P-value: ***p<0.0001.I-K. Cell proliferation is mainly unaffected by MCU depletion. Cells were transfected with siMCU or siControl. Cell number was counted every 24 hours for 3 days (the 72-hour time point corresponds to the 48-hour time-point of wound healing assay). Results are expressed as ratio R/R0 where R0 is the number of cells at the time of transfection (0 hour time-point) (n=6). P-value: *p=0.05.


Figure 2A . Re - expression of mMCU rescues cell motility of MCU - silenced cells . Cells were transfected with siMCU or siControl . Ad - mMCU was used to re - express MCU ( Ad - GFP was used as control ) . MCU protein expression was verified by western blot . The day after transduction a linear scratch was made ( time - point 0h ) . Cell migration into the wounded area was monitored at 48 hour time - point and the covered area was measured ( n = 12 ) . P - value : * * * p < 0 . 0001 . B . MCU silencing blunts cell invasiveness . Stable shMCU and shControl - expressing spheroids were plated and let grow into collagen - I ( 0 hour time - point ) . Spheroid area was measured at 0 - hour and 48 - hour ( n = 8 ) . Scale bar 300 μm . P - value : * * * p = 0 . 0003 . C . MCU silencing reduces the clonogenic potential of MDA - MB - 231 cells . Stable shMCU and shControl - expressing cells were plated at low confluence ( 2x103 / well of a 6 - well plate ) . After 7 days the number of colonies was counted ( minimum 30 cells / colony , n = 8 ) . P - value : * * p = 0 . 0027 . D . MCU depletion does not induce cell death . Cells were transfected with siMCU or siControl . 72 hours later , cell apoptosis and necrosis were measured by FITC - Annexin V and Propidium Iodide ( PI ) detection ( Q1 : PI positive , Q2 : PI and FITC - Annexin V positive , Q3 : PI and FITC - Annexin V negative , Q4 : FITC - Annexin V positive . n = 6 ) . E . MCU depletion does not alter cell cycle . Cells were transfected with siMCU or siControl . 72 hours later , cell cycle distribution was monitored by Propidium Iodide ( PI ) detection ( n = 6 ) .

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Figure 2A. Re-expression of mMCU rescues cell motility of MCU-silenced cells. Cells were transfected with siMCU or siControl. Ad-mMCU was used to re-express MCU (Ad-GFP was used as control). MCU protein expression was verified by western blot. The day after transduction a linear scratch was made (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.B. MCU silencing blunts cell invasiveness. Stable shMCU and shControl-expressing spheroids were plated and let grow into collagen-I (0 hour time-point). Spheroid area was measured at 0-hour and 48-hour (n=8). Scale bar 300 μm. P-value: ***p=0.0003.C. MCU silencing reduces the clonogenic potential of MDA-MB-231 cells. Stable shMCU and shControl-expressing cells were plated at low confluence (2x103/well of a 6-well plate). After 7 days the number of colonies was counted (minimum 30 cells/colony, n=8). P-value: **p=0.0027.D. MCU depletion does not induce cell death. Cells were transfected with siMCU or siControl. 72 hours later, cell apoptosis and necrosis were measured by FITC-Annexin V and Propidium Iodide (PI) detection (Q1: PI positive, Q2: PI and FITC-Annexin V positive, Q3: PI and FITC-Annexin V negative, Q4: FITC-Annexin V positive. n=6).E. MCU depletion does not alter cell cycle. Cells were transfected with siMCU or siControl. 72 hours later, cell cycle distribution was monitored by Propidium Iodide (PI) detection (n=6).


Figure 3A . Tumor mass volume was measured at specific time - points until the day of sacrifice ( day 39 post - injection for control , day 46 and 56 p . i . for MCU - / - cl . 1 and cl . 2 , respectively ) . P - values : * * * p = 0 . 0001 , * * * p < 0 . 0001 . B . left : in vivo metastasis at the homolateral axillary area of three representative mice per group at the time of sacrifice . Right : total flux analysis . P - values : * * p = 0 . 01 , * p = 0 . 02 . C . Lymph nodes weight at the time of sacrifice . P - values : * * * p = 0 . 0010 , * * p = 0 . 0014 . D . Human cytokeratin 7 ( CK7 ) IHC staining of three representative lymph nodes per group . Scale bar = 500 µm . E . Left : images of three representative lungs per group collected ex vivo at the time of sacrifice . Right : total flux analysis . P - values : * * p = 0 . 0031 , * * * p = 0 . 0004 .

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Figure 3A. Tumor mass volume was measured at specific time-points until the day of sacrifice (day 39 post-injection for control, day 46 and 56 p.i. for MCU-/- cl.1 and cl.2, respectively). P-values: ***p=0.0001, ***p<0.0001.B. left: in vivo metastasis at the homolateral axillary area of three representative mice per group at the time of sacrifice. Right: total flux analysis. P-values: **p=0.01, *p=0.02.C. Lymph nodes weight at the time of sacrifice. P-values: ***p=0.0010, **p=0.0014.D. Human cytokeratin 7 (CK7) IHC staining of three representative lymph nodes per group. Scale bar= 500 µm.E. Left: images of three representative lungs per group collected ex vivo at the time of sacrifice. Right: total flux analysis. P-values: **p=0.0031, ***p= 0.0004.


Figure 4A - C . FLIM analysis of cellular NADH / NADPH levels . Fluorescence lifetimes of NAD ( P ) H autofluorescence in stable shControl and shMCU expressing cells were imaged . Representative images of the distribution of τbound on an intensity weighted pseudocolored scale ( 2 . 2 - 2 . 5 ns ) are shown . Scale bars = 20 μm ( A ) . Mean + / - SE of τbound ( B ) and relative NADH and NADPH intensities ( C ) calculated from equation in ( Blacker et al . , 2014 ) are shown ( n = 3 ) . P - values : * * p = 0 . 01 , * * p = 0 . 002 , respectively . D - E . Measurement of the redox state of the NADH / NAD + couple . Representative measurements of NADH intensity at steady state and at minimal and maximal reduced state ( D ) . Percentage of the steady state redox state ( E ) ( n = 3 ) . F . MCU depletion impairs the mitochondrial rate of ATP production . Cells were transfected with siMCU or siControl . 48 hours later , cells were treated with 5 . 5 mM 2 - deoxy - D - glucose for 1 hour and cellular ATP levels were quantified ( n = 6 ) . P - value : * * * p = 0 . 0009 .

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

Figure 4A-C. FLIM analysis of cellular NADH/NADPH levels. Fluorescence lifetimes of NAD(P)H autofluorescence in stable shControl and shMCU expressing cells were imaged. Representative images of the distribution of τbound on an intensity weighted pseudocolored scale (2.2-2.5 ns) are shown. Scale bars = 20 μm (A). Mean +/- SE of τbound (B) and relative NADH and NADPH intensities (C) calculated from equation in (Blacker et al., 2014) are shown (n=3). P-values: **p=0.01, **p=0.002, respectively.D-E. Measurement of the redox state of the NADH/NAD+ couple. Representative measurements of NADH intensity at steady state and at minimal and maximal reduced state (D). Percentage of the steady state redox state (E) (n=3).F. MCU depletion impairs the mitochondrial rate of ATP production. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 5.5 mM 2-deoxy-D-glucose for 1 hour and cellular ATP levels were quantified (n=6). P-value: ***p=0.0009.


Figure 5A . Antioxidant treatments decrease cell migration . A linear scratch was obtained on cell monolayer through a vertically held P200 tip ( time - point 0h ) . Cells were treated for 48 hours with N - Acetylcysteine ( NAC ) or Dithioerythritol ( DTE ) . Cell migration into the wounded area was monitored at 48 - hour time - point and the covered area was measured ( n = 12 ) . P - values : * * p = 0 . 008 , * * * p < 0 . 0001 , * * p = 0 . 005 . B . Scavenging of mitochondrial ROS decreases cell migration . A linear scratch was obtained on a cell monolayer through a vertically held P200 tip ( time - point 0h ) . Cells were treated for 48 hours with 50 µM MitoTEMPO . Cell migration into the wounded area was monitored at 48 hour time - point and the covered area was measured ( n = 12 ) . P - value : * * * p < 0 . 0001 . C . MCU silencing does not affect matrix pH . Cell were transfected with siMCU or siControl and SypHer2 probe . 48 hours later , matrix pH was measured ( n = 22 ) . D - E . Mitochondrial H2O2 levels are critically blunted after MCU depletion . Cells were transfected with shMCU or shControl , together with the ratiometric YFP - based biosensor pHyper - dMito ( D ) or the mitochondrial H2O2 - sensitive HyPerRed probe ( E ) . 48 hours later , H2O2 production was measured ( n = 35 ) . P - values : * p = 0 . 02 , * p = 0 . 05 , respectively . F . Mitochondrial superoxide levels are critically blunted after MCU silencing . Cells were transfected with siMCU or siControl . 48 hours later , cells were loaded with the red dye MitoSOXTM and superoxide anion levels were measured ( n = 25 ) . P - value : * p = 0 . 04 . G . Mitochondrial GSSG / GSH ratio is critically reduced after MCU silencing . Cells were transfected with shMCU or shControl , together with the mitochondrial targeted mitGrx1 - roGFP2 probe . 96 hours later , the glutathione redox potential ( EGSH ) was measured ( n = 46 ) . P - value : * * * p < 0 . 0001 .

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

Figure 5A. Antioxidant treatments decrease cell migration. A linear scratch was obtained on cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with N-Acetylcysteine (NAC) or Dithioerythritol (DTE). Cell migration into the wounded area was monitored at 48-hour time-point and the covered area was measured (n=12). P-values: **p=0.008, ***p<0.0001, **p=0.005.B. Scavenging of mitochondrial ROS decreases cell migration. A linear scratch was obtained on a cell monolayer through a vertically held P200 tip (time-point 0h). Cells were treated for 48 hours with 50 µM MitoTEMPO. Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-value: ***p<0.0001.C. MCU silencing does not affect matrix pH. Cell were transfected with siMCU or siControl and SypHer2 probe. 48 hours later, matrix pH was measured (n=22).D-E. Mitochondrial H2O2 levels are critically blunted after MCU depletion. Cells were transfected with shMCU or shControl, together with the ratiometric YFP-based biosensor pHyper-dMito (D) or the mitochondrial H2O2-sensitive HyPerRed probe (E). 48 hours later, H2O2 production was measured (n=35). P-values: *p=0.02, *p=0.05, respectively.F. Mitochondrial superoxide levels are critically blunted after MCU silencing. Cells were transfected with siMCU or siControl. 48 hours later, cells were loaded with the red dye MitoSOXTM and superoxide anion levels were measured (n=25). P-value: *p=0.04.G. Mitochondrial GSSG/GSH ratio is critically reduced after MCU silencing. Cells were transfected with shMCU or shControl, together with the mitochondrial targeted mitGrx1-roGFP2 probe. 96 hours later, the glutathione redox potential (EGSH) was measured (n=46). P-value: ***p<0.0001.


Figure 6A . MCU silencing reduces HIF - 1α protein levels . Cells were transfected with siMCU or siControl . HIF - 1α protein levels were detected 48 hours later . B . MCU silencing reduces MG132 - mediated HIF - 1α and hydroxylated HIF - 1α protein accumulation . Cells were transfected with siMCU or siControl . 48 hours later , cells were treated with 10 μM of the proteasome inhibitor MG132 . Left : Protein levels were revealed by western blot . Right : quantification by densitometry ( n = 5 ) . C . ROS increase HIF - 1α transcription . Cells were treated O / N with 100 µM paraquat to induce ROS production . HIF - 1α mRNA levels were measured by RealTime - PCR ( n = 3 ) . P - value : * * p = 0 . 002 . D - J . MCU silencing reduces mRNA levels of HIF - 1α , HIF - 2α , and HIF - 1α target genes . Cells were transfected with siMCU or siControl . mRNA expression was measured by RealTime - PCR ( n = 3 ) . P - values : ( HIF - 1α ) * * p = 0 . 0031 , * * p = 0 . 009 , ( HIF - 2α ) * * p = 0 . 01 , ( LOX ) * * * p = 0 . 001 , * * * p = 0 . 0005 , ( PDK1 ) * p = 0 . 02 , * * p = 0 . 009 , ( G6PI ) * p = 0 . 02 , * * p = 0 . 005 , ( CA IX ) * * p = 0 . 0026 , * * p = 0 . 0022 , ( HK2 ) * * p = 0 . 0024 , * p = 0 . 03 . K . HIF - 1α overexpression rescues siMCU - mediated migration impairment . Cells were transfected with siMCU or siControl . Wild type ( wt ) and constitutively active ( ca ) HIF - 1α were expressed by retroviral infection ( pBABE was used as control ) . The day after transduction , cells were scratched ( time - point 0h ) . Cell migration into the wounded area was monitored at 48 hour time - point and the covered area was measured ( n = 12 ) . P - values : * * * p < 0 . 0001 , * p = 0 . 04 . L - M . MCU expression levels correlate with HIF1α ( L ) and HIF1α - regulated genes ( M ) . A linear model ( lm ) to test the power of MCU expression levels predicting the expression of HIF1α and HIF1α - regulated genes was calculated and plotted for each of the 532 samples of the TCGA database ( see Figure 1 ) . Equation and R2 values of the linear regression and significance indicating deviation from 0 are shown . The area of 95 % prediction limit is shaded below and above the linear regression line . The HIF1α - regulated gene set was compiled from Broad Institute GSEA database ( http : / / www . broadinstitute . org / gsea / msigdb / cards / V $ HIF1 _ Q5 . html , merged sets of V $ HIF1 _ Q3 and V $ HIF1 _ Q5 ) .

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

Figure 6A. MCU silencing reduces HIF-1α protein levels. Cells were transfected with siMCU or siControl. HIF-1α protein levels were detected 48 hours later.B. MCU silencing reduces MG132-mediated HIF-1α and hydroxylated HIF-1α protein accumulation. Cells were transfected with siMCU or siControl. 48 hours later, cells were treated with 10 μM of the proteasome inhibitor MG132. Left: Protein levels were revealed by western blot. Right: quantification by densitometry (n=5).C. ROS increase HIF-1α transcription. Cells were treated O/N with 100 µM paraquat to induce ROS production. HIF-1α mRNA levels were measured by RealTime-PCR (n=3). P-value: **p=0.002.D-J. MCU silencing reduces mRNA levels of HIF-1α, HIF-2α, and HIF-1α target genes. Cells were transfected with siMCU or siControl. mRNA expression was measured by RealTime-PCR (n=3). P-values: (HIF-1α) **p=0.0031, **p=0.009, (HIF-2α)**p=0.01, (LOX)***p=0.001, ***p=0.0005, (PDK1)*p=0.02, **p=0.009, (G6PI)*p=0.02, **p=0.005, (CA IX)**p=0.0026, **p=0.0022, (HK2) **p=0.0024, *p=0.03.K. HIF-1α overexpression rescues siMCU-mediated migration impairment. Cells were transfected with siMCU or siControl. Wild type (wt) and constitutively active (ca) HIF-1α were expressed by retroviral infection (pBABE was used as control). The day after transduction, cells were scratched (time-point 0h). Cell migration into the wounded area was monitored at 48 hour time-point and the covered area was measured (n=12). P-values: ***p<0.0001, *p=0.04.L-M. MCU expression levels correlate with HIF1α (L) and HIF1α-regulated genes (M). A linear model (lm) to test the power of MCU expression levels predicting the expression of HIF1α and HIF1α-regulated genes was calculated and plotted for each of the 532 samples of the TCGA database (see Figure 1). Equation and R2 values of the linear regression and significance indicating deviation from 0 are shown. The area of 95% prediction limit is shaded below and above the linear regression line. The HIF1α-regulated gene set was compiled from Broad Institute GSEA database (http://www.broadinstitute.org/gsea/msigdb/cards/V$HIF1_Q5.html, merged sets of V$HIF1_Q3 and V$HIF1_Q5).


Figure 1A . SF - 1 , FATE1 and β - tubulin protein levels shown in basal condition and after Dox treatment in H295R / TR , H295R / TRSF - 1 , H295R / TRFATE1 and H295R / TR N - FlagFATE1cells . B . FATE1 mRNA expression is increased in H295R / TRSF - 1 cells by Dox treatment . The efficiency of FATE1 knockdown by specific ( siFATE1 ) vs . control ( siC ) siRNA nucleofection is shown ( mean ± SEM ; n = 4 with 3 replicates / experiment ) . C . Immunofluorescence microscopy showing endogenous FATE1 protein ( red ) induction of expression by Dox - treatment of H295R / TRSF - 1 cells . SF - 1 ( green ) , DAPI ( blue ) . Scale bars , 10 μm . D . Subcellular localization of endogenous FATE1 ( red ) and transfected fluorescent markers for Golgi , ER and mitochondria , respectively ( green ) in Dox - treated H295R / TRSF - 1 cells . DAPI ( blue ) . Scale bars , 10 μm . E . Subcellular localization of the endogenous FATE1 protein ( green ) in Dox - treated H295R / TRSF - 1 cells costained with an antibody against the mitochondrial marker HSP60 ( red ) . SF - 1 ( blue ) . Scale bar , 10 μm . F . Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox - treated H295R / TR N - FlagFATE1 cells . Scale bar , 500 nm . G . Dox - treated H295R / TR N - FlagFATE1 cells were fractioned into nuclear ( N ) , heavy membranes ( HM ) , light membranes ( LM ) and cytosolic ( C ) fractions and localization of SF - 1 , FATE1 , ribosomal protein RPL7 , VDAC1 and GAPDH was revealed by immunoblot . H . Effect of increasing concentrations ( 0 , 1 , 10 and 100 μg / ml ) of proteinase K and 0 . 1 % Triton X - 100 ( TX - 100 ) treatment of the mitochondrial fraction from Dox - treated H295R / TR N - FlagFATE1 cells on VDAC1 , FATE1 , TOM20 and cytochrome c . I . FATE1 and VDAC1 are associated with the pellet ( membrane ; P ) fraction after high - speed centrifugation of the alkaline - extracted mitochondrial fraction of Dox - treated H295R / TR SF - 1 cells , while cytochrome c is found in the supernatant ( S ) .

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

Figure 1A. SF-1, FATE1 and β-tubulin protein levels shown in basal condition and after Dox treatment in H295R/TR, H295R/TRSF-1, H295R/TRFATE1 and H295R/TR N-FlagFATE1cells.B. FATE1 mRNA expression is increased in H295R/TRSF-1 cells by Dox treatment. The efficiency of FATE1 knockdown by specific (siFATE1) vs. control (siC) siRNA nucleofection is shown (mean ± SEM; n=4 with 3 replicates/experiment).C. Immunofluorescence microscopy showing endogenous FATE1 protein (red) induction of expression by Dox-treatment of H295R/TRSF-1 cells. SF-1 (green), DAPI (blue). Scale bars, 10 μm.D. Subcellular localization of endogenous FATE1 (red) and transfected fluorescent markers for Golgi, ER and mitochondria, respectively (green) in Dox-treated H295R/TRSF-1 cells. DAPI (blue). Scale bars, 10 μm.E. Subcellular localization of the endogenous FATE1 protein (green) in Dox-treated H295R/TRSF-1 cells costained with an antibody against the mitochondrial marker HSP60 (red). SF-1 (blue). Scale bar, 10 μm.F. Immunogold electron microscopy showing association of FATE1 with the mitochondrial outer surface in Dox-treated H295R/TR N-FlagFATE1 cells. Scale bar, 500 nm.G. Dox-treated H295R/TR N-FlagFATE1 cells were fractioned into nuclear (N), heavy membranes (HM), light membranes (LM) and cytosolic (C) fractions and localization of SF-1, FATE1, ribosomal protein RPL7, VDAC1 and GAPDH was revealed by immunoblot.H. Effect of increasing concentrations (0, 1, 10 and 100 μg/ml) of proteinase K and 0.1% Triton X-100 (TX-100) treatment of the mitochondrial fraction from Dox-treated H295R/TR N-FlagFATE1 cells on VDAC1, FATE1, TOM20 and cytochrome c.I. FATE1 and VDAC1 are associated with the pellet (membrane; P) fraction after high-speed centrifugation of the alkaline-extracted mitochondrial fraction of Dox-treated H295R/TR SF-1 cells, while cytochrome c is found in the supernatant (S).


Figure 2B . Subcellular localization of EGFP - fusions of full - length ( FL ) FATE1 and its mutants : N - terminal ( aa . 1 - 124 ) , C - terminal ( aa . 125 - 183 ) , 1 - 162 , transmembrane domain ( aa . 155 - 183 ) and L151D ( green ) transfected in H295R / TRSF - 1 cells . Mitochondria were stained by anti - TOM20 antibody ( red ) and DNA by DAPI ( blue ) . Scale bars , 10 μm . C . EMD and Flag - tagged FATE1 , but not the other OMM protein TOM20 , were co - immunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 cells . Control immunoprecipitations were performed with anti - myc ( α - myc ) and non - immune rabbit IgG ( IgG - R ) antibodies . D . Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD , as revealed by immunofluorescence microscopy ( red ) , with ER , stained by ER - GFP BacMam ( green ) . Scale bars , 10 μm . E . Mic60 / mitofilin and Flag - tagged FATE1 were coimmunoprecipitated from Dox - treated H295R / TR N - FlagFATE1 cells . Control immunoprecipitations were performed with anti - myc ( α - myc ) antibody . F . Mitochondrial localization of Mic60 / mitofilin ( green ) and FATE1 ( red ) in Dox - treated H295R / TRSF - 1 cells . Scale bar , 10 μm . G . Left , triple immunofluorescence microscopy labelling of endogenous FATE1 ( green ) , ER labelled by calreticulin ( red ) and mitochondria labelled by HSP60 staining ( blue ) in Dox - treated H295R / TRSF - 1 cells . One area showing close apposition of red , green and blue staining ( white signals ) is shown at higher magnification . Scale bar , 10 μm . Right , graph showing quantification of FATE1 signal in total mitochondria ( white histogram ) vs . ER - mitochondria contact sites ( red histogram ) ( mean ± SEM ; n = 13 ) . * * p < 0 . 01 . H . H295R / TRSF - 1 cells were fractioned into crude mitochondria ( CM ) , endoplasmic reticulum ( ER ) , purified mitochondria ( PM ) and mitochondria - associated membranes ( MAM ) fractions . H , total homogenate . The localization of SERCA2 , SOAT1 , VDAC1 , GRP75 , S1R , EMD , Mic60 / mitofilin , FATE1 and TOM20 proteins in the different fractions was revealed by immunoblot .

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

Figure 2B. Subcellular localization of EGFP-fusions of full-length (FL) FATE1 and its mutants: N-terminal (aa. 1-124), C-terminal (aa.125-183), 1-162, transmembrane domain (aa. 155-183) and L151D (green) transfected in H295R/TRSF-1 cells. Mitochondria were stained by anti-TOM20 antibody (red) and DNA by DAPI (blue). Scale bars, 10 μm.C. EMD and Flag-tagged FATE1, but not the other OMM protein TOM20, were co-immunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) and non-immune rabbit IgG (IgG-R) antibodies.D. Confocal fluorescence microscopy showed colocalization of cytoplasmic EMD, as revealed by immunofluorescence microscopy (red), with ER, stained by ER-GFP BacMam (green). Scale bars, 10 μm.E. Mic60/mitofilin and Flag-tagged FATE1 were coimmunoprecipitated from Dox-treated H295R/TR N-FlagFATE1 cells. Control immunoprecipitations were performed with anti-myc (α-myc) antibody.F. Mitochondrial localization of Mic60/mitofilin (green) and FATE1 (red) in Dox-treated H295R/TRSF-1 cells. Scale bar, 10 μm.G. Left, triple immunofluorescence microscopy labelling of endogenous FATE1 (green), ER labelled by calreticulin (red) and mitochondria labelled by HSP60 staining (blue) in Dox-treated H295R/TRSF-1 cells. One area showing close apposition of red, green and blue staining (white signals) is shown at higher magnification. Scale bar, 10 μm. Right, graph showing quantification of FATE1 signal in total mitochondria (white histogram) vs. ER-mitochondria contact sites (red histogram) (mean ± SEM; n=13). **p<0.01.H. H295R/TRSF-1 cells were fractioned into crude mitochondria (CM), endoplasmic reticulum (ER), purified mitochondria (PM) and mitochondria-associated membranes (MAM) fractions. H, total homogenate. The localization of SERCA2, SOAT1, VDAC1, GRP75, S1R, EMD, Mic60/mitofilin, FATE1 and TOM20 proteins in the different fractions was revealed by immunoblot.


Figure 3A . H295R / TR N - FlagFATE1 cells were transfected with D1 ER marker for ER ( green ) and mtRFP marker for mitochondria ( red ) . Manders ' coefficient for green - red signals colocalization in basal ( white histograms ) and Dox - treated ( black histograms ) cells is shown on the right ( mean ± SEM ; n = 12 ) . * * * p < 0 . 001 . Scale bars , 10 μm . B . Effect of FATE1 expression on ER - mitochondria distance in H295R / TR N - FlagFATE1 cells measured using a split - GFP probe [ 29 ] . Quantification is shown in the graph . White histogram , number of fluorescent objects / cell in basal conditions ; black histogram , number of fluorescent objects / cell in Dox - treated cells ( mean ± SEM ; n = 6 with 90 cells analyzed in total ) . * * p < 0 . 01 . Scale bars , 5 μm . C . Transmission electron microscopy images of H295R / TR N - FlagFATE1 cells cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by white arrowheads . Scale bars , 200 nm . Right , quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions ( white histograms ) and after Dox treatment ( black histograms ) of cells ( mean ± SEM ; n = 38 for basal and 47 for Dox - treated cells ) . * * * p < 0 . 001 . D . Transmission electron microscopy images of H295R / TRSF - 1 cells cultured in basal conditions or treated with Dox . ER - mitochondria contacts are indicated by white arrowheads . Scale bars , 200 nm . Right , quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions ( white histograms ) and after Dox treatment ( black histograms ) of cells ( mean ± SEM ; n = 41 for basal and 42 for Dox - treated cells ) . * * * p < 0 . 001 . E . Mitochondrial shape ( BacMam Mitochondria - RFP ; in red ) in H295R / TR N - FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment . FATE1 ( green ) , DAPI ( blue ) . A higher magnification of merged green and red signal is shown in the insets . Scale bars , 10 μm . F . Mitochondrial fragmentation index in H295R / TR N - FlagFATE1 cells after Dox treatment ( red histogram ) compared to cells cultured in basal conditions ( white histogram ) ( mean ± SEM ; n = 111 for basal and 101 for Dox - treated cells ) . * * * p < 0 . 001 . G . Mitochondrial fragmentation index in H295R / TRSF - 1 cells after Dox treatment ( green histogram ) compared to cells cultured in basal conditions ( white histogram ) . ( mean ± SEM ; n = 37 for basal and 49 for Dox - treated cells ) . * * * p < 0 . 001 . H . Mitochondrial membrane potential ( ∆ Ψ ) measured by TMRM fluorescence after Dox treatment of H295R / TR N - FlagFATE1 cells ( red histogram ) compared to cells cultured in basal conditions ( white histogram ) . Mean ± SEM is shown . ( mean ± SEM ; n = 3 with 12 replicates / experiment ) . ns , not significant .

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

Figure 3A. H295R/TR N-FlagFATE1 cells were transfected with D1 ER marker for ER (green) and mtRFP marker for mitochondria (red). Manders' coefficient for green-red signals colocalization in basal (white histograms) and Dox-treated (black histograms) cells is shown on the right (mean ± SEM; n=12). ***p<0.001. Scale bars, 10 μm.B. Effect of FATE1 expression on ER-mitochondria distance in H295R/TR N-FlagFATE1 cells measured using a split-GFP probe [29]. Quantification is shown in the graph. White histogram, number of fluorescent objects/cell in basal conditions; black histogram, number of fluorescent objects/cell in Dox-treated cells (mean ± SEM; n=6 with 90 cells analyzed in total). **p<0.01. Scale bars, 5 μm.C. Transmission electron microscopy images of H295R/TR N-FlagFATE1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=38 for basal and 47 for Dox-treated cells). ***p<0.001.D. Transmission electron microscopy images of H295R/TRSF-1 cells cultured in basal conditions or treated with Dox. ER-mitochondria contacts are indicated by white arrowheads. Scale bars, 200 nm. Right, quantification of the number of contacts normalized by the number of mitochondria and the percentage of mitochondria with ER contact sites is shown in basal conditions (white histograms) and after Dox treatment (black histograms) of cells (mean ± SEM; n=41 for basal and 42 for Dox-treated cells). ***p<0.001.E. Mitochondrial shape (BacMam Mitochondria-RFP; in red) in H295R/TR N-FlagFATE1 cells shown by fluorescence confocal microscopy in basal conditions and after Dox treatment. FATE1 (green), DAPI (blue). A higher magnification of merged green and red signal is shown in the insets. Scale bars, 10 μm.F. Mitochondrial fragmentation index in H295R/TR N-FlagFATE1 cells after Dox treatment (red histogram) compared to cells cultured in basal conditions (white histogram) (mean ± SEM; n=111 for basal and 101 for Dox-treated cells). ***p<0.001.G. Mitochondrial fragmentation index in H295R/TRSF-1 cells after Dox treatment (green histogram) compared to cells cultured in basal conditions (white histogram). (mean ± SEM; n=37 for basal and 49 for Dox-treated cells). ***p<0.001.H. Mitochondrial membrane potential (∆Ψ) measured by TMRM fluorescence after Dox treatment of H295R/TR N-FlagFATE1 cells (red histogram) compared to cells cultured in basal conditions (white histogram). Mean ± SEM is shown. (mean ± SEM; n=3 with 12 replicates/experiment). ns, not significant.


Figure 4A . MitochondrialCa2 + uptake . H295R / TR parental clone ( white / grey histograms ) H295R / TRSF - 1 ( white / green histograms ) , H295R / TR N - FlagFATE1 ( white / red histograms ) and H295R / TRFATE1 cells ( white / violet histograms ) ( mean ± SEM ; n = 3 - 5 with 12 replicates / condition ) . * * p < 0 . 01 ; * * * p < 0 . 001 ; ns , not significant . B . CytosolicCa2 + concentration . Histograms as in A ( mean ± SEM ; n = 3 - 5 with 12 replicates / condition ) . ns , not significant . C . Mitochondrial ( left ) and cytosolic ( right ) Ca2 + concentrations in Dox - treated H295R / TRSF - 1 cells transfected with control ( siC ; white histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs ( mean ± SEM ; n = 3 - 5 with 12 replicates / condition ) . * * * p < 0 . 001 ; ns , not significant . D . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cells cultured in basal conditions ( white histograms ) or in the presence of Dox ( red histograms ) treated with H2O2 ( 500 μM ) C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) . ( mean ± SEM ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; * * p < 0 . 01 ; ns , not significant . E . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 cells transfected with control ( siC ; white histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs and treated with H2O2 or STS ( mean ± SEM ; n = 5 - 8 with 3 replicates / condition ) . * p < 0 . 05 ; ns , not significant . F . Flow cytometry analysis of TUNEL staining in H295R / TR N - Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle ( control ) H2O2 ( 500 μM ) , C2 - ceramide ( 50 μM ) or staurosporine ( STS ) ( 1 μM ) . G . Caspase 3 / 7 activity in H295R / TR N - FlagFATE1 cells cultured in basal conditions ( white histograms ) or in the presence of Dox ( red histograms ) and treated with mitotane ( 50 μM ) ( mean ± SEM ; n = 5 - 8 with 3 replicates / condition ) . * * p < 0 . 01 . H . Caspase 3 / 7 activity in Dox - treated H295R / TRSF - 1 cells transfected with control ( siC ; white histograms ) or FATE1 - specific ( siFATE1 ; green histograms ) siRNAs and treated with mitotane ( mean ± SEM ; n = 5 - 8 with 3 replicates / condition ) . * p < 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 O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. MitochondrialCa2+ uptake. H295R/TR parental clone (white/grey histograms) H295R/TRSF-1 (white/green histograms), H295R/TR N-FlagFATE1 (white/red histograms) and H295R/TRFATE1 cells (white/violet histograms) (mean ± SEM; n=3-5 with 12 replicates/condition). **p<0.01; ***p<0.001; ns, not significant.B. CytosolicCa2+ concentration. Histograms as in A (mean ± SEM; n=3-5 with 12 replicates/condition). ns, not significant.C. Mitochondrial (left) and cytosolic (right) Ca2+ concentrations in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs (mean ± SEM; n=3-5 with 12 replicates/condition). ***p<0.001; ns, not significant.D. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) treated with H2O2 (500 μM) C2-ceramide (50 μM) or staurosporine (STS) (1 μM). (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; **p<0.01; ns, not significant.E. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with H2O2 or STS (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05; ns, not significant.F. Flow cytometry analysis of TUNEL staining in H295R/TR N-Flag FATE1 cells cultured in basal conditions or in the presence of Dox treated with vehicle (control) H2O2 (500 μM), C2-ceramide (50 μM) or staurosporine (STS) (1 μM).G. Caspase 3/7 activity in H295R/TR N-FlagFATE1 cells cultured in basal conditions (white histograms) or in the presence of Dox (red histograms) and treated with mitotane (50 μM) (mean ± SEM; n=5-8 with 3 replicates/condition). **p<0.01.H. Caspase 3/7 activity in Dox-treated H295R/TRSF-1 cells transfected with control (siC; white histograms) or FATE1-specific (siFATE1; green histograms) siRNAs and treated with mitotane (mean ± SEM; n=5-8 with 3 replicates/condition). *p<0.05.


Figure 5A . Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia , adenoma and carcinoma ( ACC ) . Testis tissue was used as positive control . Scale bars , 200 µm ( low magnification ) ; 50 μ m ( high magnification ) . B . Table summarizing the relative intensity of FATE1IHC staining ( H - score ) in groups of normal , benign and malignant tissues . The highest FATE1 expression levels were found in ACC . n , number of samples studied for each category of tissues . NBST , normal and benign steroidogenic tissues . C . Prognostic parameters in ACC patients . Results are presented as hazard ratios ( HR ) including 95 % confidence interval ( CI ) . The significance level was set at α = 5 % for all comparisons . The results for SF - 1 , FATE1 and combined SF - 1 / FATE1 expression levels are shown both in univariate and mutivariate ( adjusted for age , sex and ENSAT tumour stage ) analysis . D . Overall survival of ACC patients according to FATE1 expression in their tumours , as measured by quantitative immunohistochemistry ( low , green curve ; high , red curve ) in mutivariate analysis . p = 0 . 008 .

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

Figure 5A. Immunohistochemistry analysis of FATE1 expression in normal adrenal cortex and adrenal hyperplasia, adenoma and carcinoma (ACC). Testis tissue was used as positive control. Scale bars, 200 µm (low magnification); 50 μ m (high magnification).B. Table summarizing the relative intensity of FATE1IHC staining (H-score) in groups of normal, benign and malignant tissues. The highest FATE1 expression levels were found in ACC. n, number of samples studied for each category of tissues. NBST, normal and benign steroidogenic tissues.C. Prognostic parameters in ACC patients. Results are presented as hazard ratios (HR) including 95% confidence interval (CI). The significance level was set at α=5% for all comparisons. The results for SF-1, FATE1 and combined SF-1/FATE1 expression levels are shown both in univariate and mutivariate (adjusted for age, sex and ENSAT tumour stage) analysis.D. Overall survival of ACC patients according to FATE1 expression in their tumours, as measured by quantitative immunohistochemistry (low, green curve; high, red curve) in mutivariate analysis. p=0.008.


Figure 1C CBB staining of purified p62 ( 268 - 440 aa ) , p62 ( 320 - 440 aa ) , SNAP - Atg1 , and SNAP - ULK1 . F In vitro kinase assay . Purified recombinant p62 ( 268 - 440 aa ) , p62 ( 320 - 440 aa ) , or mCherry - p62 was incubated for 20 min at 30 ° C with purified SNAP - Atg1 or SNAP - ULK1 in the presence or absence of ATP . Reactions were then terminated by adding LDS sample buffer containing reducing agent , followed by immunoblot analysis with the indicated antibodies . As positive and negative controls , Huh - 1 cell lysates treated with or without lambda protein phosphatase ( λPP ) were used . Data were obtained from three independent experiments . Asterisks show possible dimeric structures of p62 ( 268 - 440 aa ) , p62 ( 320 - 440 aa ) and mCherry - p62 .

O B-PANEL_START O O O O O 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

Figure 1C CBB staining of purified p62 (268-440 aa), p62 (320-440 aa), SNAP-Atg1, and SNAP-ULK1.F In vitro kinase assay. Purified recombinant p62 (268-440 aa), p62 (320-440 aa), or mCherry-p62 was incubated for 20 min at 30 °C with purified SNAP-Atg1 or SNAP-ULK1 in the presence or absence of ATP. Reactions were then terminated by adding LDS sample buffer containing reducing agent, followed by immunoblot analysis with the indicated antibodies. As positive and negative controls, Huh-1 cell lysates treated with or without lambda protein phosphatase (λPP) were used. Data were obtained from three independent experiments. Asterisks show possible dimeric structures of p62 (268-440 aa), p62 (320-440 aa) and mCherry-p62.


Figure 2A In vitro formation of p62 - 8xUb condensates . 10 μM SNAP - 8xUb labelled with Alexa Fluor 649 was mixed with 10 μM mCherry - p62 wild - type , mCherry - p62S403E S407E , or mCherry - p62S349E and observed by fluorescence microscopy . Scale bars : 20 μm . The graph indicates the quantified area of p62 condensates formed by wild - type or mutant p62 . Data are means ± s . d of wild - type p62 , p62S403E , S407E and p62S349E condensates ( n = 4 ) . Statistical analysis was performed by Šidák ' s test after one - way ANOVA . C Immunofluorescence microscopy . Wild - type , FIP200 - or ULK1 - knockout Huh - 1 cells were immunostained with indicated antibodies . The mean fluorescence intensities of ULK1 on p62 bodies per cell were quantified in each genotype ( n = 249 cells ) . Horizontal bars indicate medians , boxes the interquartile range ( 25th - 75th percentiles ) and whiskers 1 . 5 × the interquartile range ; outliers are plotted individually . Statistical analysis was performed by Šidák ' s test after one - way ANOVA . Scale bars , 10 μm ( main panels ) , 1 μm ( inset panels ) . D Immunofluorescence microscopy . Wild - type and FIP200 - knockout Huh - 1 cells were transfected with GFP - ULK1 or GFP - ULK2 and immunostained with anti - p62 antibody . The mean fluorescence intensities of ULK1 on p62 bodies in each cell were quantified for each genotype ( n = 79 cells ) . Horizontal bars indicate medians , boxes indicate interquartile range ( 25th - 75th percentiles ) , and whiskers indicate 1 . 5 × interquartile range ; outliers are plotted individually . Statistical analysis was performed by Welch ' s t - test . Scale bars , 10 μm ( main panels ) , 1 μm ( inset panels ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 In vitro formation of p62-8xUb condensates. 10 μM SNAP-8xUb labelled with Alexa Fluor 649 was mixed with 10 μM mCherry-p62 wild-type, mCherry-p62S403E S407E, or mCherry-p62S349E and observed by fluorescence microscopy. Scale bars: 20 μm. The graph indicates the quantified area of p62 condensates formed by wild-type or mutant p62. Data are means ± s.d of wild-type p62, p62S403E, S407E and p62S349E condensates (n = 4). Statistical analysis was performed by Šidák's test after one-way ANOVA.C Immunofluorescence microscopy. Wild-type, FIP200- or ULK1-knockout Huh-1 cells were immunostained with indicated antibodies. The mean fluorescence intensities of ULK1 on p62 bodies per cell were quantified in each genotype (n = 249 cells). Horizontal bars indicate medians, boxes the interquartile range (25th-75th percentiles) and whiskers 1.5× the interquartile range; outliers are plotted individually. Statistical analysis was performed by Šidák's test after one-way ANOVA. Scale bars, 10 μm (main panels), 1 μm (inset panels).D Immunofluorescence microscopy. Wild-type and FIP200-knockout Huh-1 cells were transfected with GFP-ULK1 or GFP-ULK2 and immunostained with anti-p62 antibody. The mean fluorescence intensities of ULK1 on p62 bodies in each cell were quantified for each genotype (n = 79 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).


Figure 3A Immunoblot analysis . Huh - 1 cells were treated with or without 2 . 5 µM MRT68921 for 6 h , and the cell lysates were subjected to immunoblot analysis with indicated antibodies . The asterisk indicates non - specific bands . Data shown are representative of three separate experiments . Bar graphs show the results of quantitative densitometric analysis of Ser349 - or Ser403 - phosphorylated p62 forms relative to total p62 ( n = 3 ) , and of Ser318 - phosphorylated ATG13 relative to total ATG13 ( n = 3 ) . Data are means ± s . e . Statistical analysis was performed by Welch ' s t - test . B Immunofluorescence microscopy . Huh - 1 cells were treated with or without 2 . 5 µM MRT68921 for 6 h and immunostained with the indicated antibodies . The ratio of p62 ( p - S403 ) to p62 on p62 bodies and the size and number of p62 bodies in each cell were quantified ( n = 500 cells ) . Horizontal bars indicate medians , boxes indicate interquartile range ( 25th - 75th percentiles ) , and whiskers indicate 1 . 5 × interquartile range ; outliers are plotted individually . Statistical analysis was performed by Welch ' s t - test . Scale bars , 10 μm ( main panels ) , 1 μm ( inset panels ) . C Immunofluorescence microscopy . Huh - 1 cells were treated with or without 2 . 5 µM MRT68921 for 6 h and immunostained with the indicated antibodies . The ratio of p62 ( p - S349 ) to p62 and the signal intensity of KEAP1 on p62 bodies in each cell were quantified ( n = 500 cells ) . Horizontal bars indicate medians , boxes indicate interquartile range ( 25th - 75th percentiles ) , and whiskers indicate 1 . 5 × interquartile range ; outliers are plotted individually . Statistical analysis was performed by Welch ' s t - test . Scale bars , 10 μm ( main panels ) , 1 μm ( inset panels ) . D Gene expression of NRF2 targets . Total RNAs were prepared from Huh - 1 cells treated with or without 2 . 5 µM MRT68921 for 6 h . Values were normalized against the amount of mRNA in non - treated Huh - 1 cells . qRT - PCR analyses were performed as technical replicates on each biological sample . Data are means ± s . e . Statistical analysis was performed by two - sided Welch ' s t - test .

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

Figure 3A Immunoblot analysis. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h, and the cell lysates were subjected to immunoblot analysis with indicated antibodies. The asterisk indicates non-specific bands. Data shown are representative of three separate experiments. Bar graphs show the results of quantitative densitometric analysis of Ser349- or Ser403-phosphorylated p62 forms relative to total p62 (n = 3), and of Ser318-phosphorylated ATG13 relative to total ATG13 (n = 3). Data are means ± s.e. Statistical analysis was performed by Welch's t-test.B Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S403) to p62 on p62 bodies and the size and number of p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).C Immunofluorescence microscopy. Huh-1 cells were treated with or without 2.5 µM MRT68921 for 6 h and immunostained with the indicated antibodies. The ratio of p62 (p-S349) to p62 and the signal intensity of KEAP1 on p62 bodies in each cell were quantified (n = 500 cells). Horizontal bars indicate medians, boxes indicate interquartile range (25th-75th percentiles), and whiskers indicate 1.5× interquartile range; outliers are plotted individually. Statistical analysis was performed by Welch's t-test. Scale bars, 10 μm (main panels), 1 μm (inset panels).D Gene expression of NRF2 targets. Total RNAs were prepared from Huh-1 cells treated with or without 2.5 µM MRT68921 for 6 h. Values were normalized against the amount of mRNA in non-treated Huh-1 cells. qRT-PCR analyses were performed as technical replicates on each biological sample. Data are means ± s.e. Statistical analysis was performed by two-sided Welch's t-test.


Figure 4A Fluorescence microscopy . GFP - p62 , GFP - p62S349E , GFP - p62S349A , or GFP - p62T350A were co - transfected with mCherry or mCherry - KEAP1 into Huh1 p62 KEAP1 double - knockout cells . Twenty - four hours after transfection , the fluorescence images were observed . E FRAP analyses of mCherry - KEAP1 localized in p62 bodies comprised of GFP - p62S349E or GFP - p62S349A . The half - time of recovery ( t50 ) and mobile fraction ( MF ) of mCherry - KEAP1 was measured by FRAP of whole p62 bodies ( n = 7 ) . Data are means ± s . d . Statistical analysis was performed by two - sided Welch ' s t - test . F FLIP analyses of mCherry - KEAP1 localized in p62 bodies comprised of GFP - p62S349E or GFP - p62S349A . The fluorescence loss of mCherry - KEAP1 in GFP - p62 bodies ( n = 14 ) was measured 30 min after photobleaching over a large area of cells . G FRAP analyses of mCherry - KEAP1 localized in p62 bodies comprised of GFP - p62S349E or GFP - p62S349A . t50 and MF of mCherry - KEAP1 were measured by FRAP of the central portions of p62 bodies ( n = 10 ) . Data are means ± s . d . Statistical analysis was performed by two - sided Welch ' s t - test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 Fluorescence microscopy. GFP-p62, GFP-p62S349E, GFP-p62S349A, or GFP-p62T350A were co-transfected with mCherry or mCherry-KEAP1 into Huh1 p62 KEAP1 double-knockout cells. Twenty-four hours after transfection, the fluorescence images were observed.E FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. The half-time of recovery (t50) and mobile fraction (MF) of mCherry-KEAP1 was measured by FRAP of whole p62 bodies (n = 7). Data are means ± s.d. Statistical analysis was performed by two-sided Welch's t-test.F FLIP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. The fluorescence loss of mCherry-KEAP1 in GFP-p62 bodies (n = 14) was measured 30 min after photobleaching over a large area of cells.G FRAP analyses of mCherry-KEAP1 localized in p62 bodies comprised of GFP-p62S349E or GFP-p62S349A. t50 and MF of mCherry-KEAP1 were measured by FRAP of the central portions of p62 bodies (n = 10). Data are means ± s.d. Statistical analysis was performed by two-sided Welch's t-test.


Figure 5D RNA - seq analysis of livers of p62 + / + and p62S351E / + mice at P19 ( n = 3 ) . Volcano plots of differentially expressed genes in p62 + / + versus p62S351E / + mice ( red points : FDR < 0 . 05 and log2FC > 1 , blue points : FDR < 0 . 05 and log2FC < - 1 ) . Green points are the targets of transcription factor NRF2 . F Immunoblot analysis of p62 + / + ( n = 7 ) and p62S351E / + ( n = 3 ) mice at P19 . Liver homogenates were subjected to immunoblot analysis with the indicated antibodies . Bar graphs show the results of quantitative densitometric analysis . Data are means ± s . e . Statistical analysis was performed by Welch ' s t - test . Note that both faster and slower migrating bands in the KEAP1 blot represent translational products from the Keap1 gene . G , H Hematoxylin and eosin ( HE ) staining ( G ) and immunohistochemical analysis of NQO1 ( H ) of livers from p62 + / + and p62S351E / + mice at P19 . Scale bars , 100 μm ( low magnification panels ) , and 10 μm ( high magnification panels ) . i Serum levels of aspartate aminotransferase ( AST ) , alanine aminotransferase ( ALT ) , glucose , total cholesterol , blood urea nitrogen ( BUN ) , and creatinine from p62 + / + ( n = 4 at P12 , n = 10 at P15 ) and p62S351E / + ( n = 4 ) mice at P12 and P15 were measured . IU / l , international units / liter . Data are means ± s . e . Statistical analysis was performed by Tukey ' s test after one - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5D RNA-seq analysis of livers of p62+/+ and p62S351E/+ mice at P19 (n = 3). Volcano plots of differentially expressed genes in p62+/+ versus p62S351E/+ mice (red points: FDR < 0.05 and log2FC > 1, blue points: FDR < 0.05 and log2FC < -1). Green points are the targets of transcription factor NRF2.F Immunoblot analysis of p62+/+ (n = 7) and p62S351E/+ (n = 3) mice at P19. Liver homogenates were subjected to immunoblot analysis with the indicated antibodies. Bar graphs show the results of quantitative densitometric analysis. Data are means ± s.e. Statistical analysis was performed by Welch's t-test. Note that both faster and slower migrating bands in the KEAP1 blot represent translational products from the Keap1 gene.G, H Hematoxylin and eosin (HE) staining (G) and immunohistochemical analysis of NQO1 (H) of livers from p62+/+ and p62S351E/+ mice at P19. Scale bars, 100 μm (low magnification panels), and 10 μm (high magnification panels).i Serum levels of aspartate aminotransferase (AST), alanine aminotransferase (ALT), glucose, total cholesterol, blood urea nitrogen (BUN), and creatinine from p62+/+ (n = 4 at P12, n = 10 at P15) and p62S351E/+ (n = 4) mice at P12 and P15 were measured. IU/l, international units/liter. Data are means ± s.e. Statistical analysis was performed by Tukey's test after one-way ANOVA.


Figure 1 ( A and B ) qPCR analysis ( n = 3 ) of BRCA1 mRNA ( A ) and pre - mRNA levels ( B ) in different cell cycle phases of HeLa cells . ( C ) ChIP analysis ( n = 3 ) of Pol II binding to BRCA1 promoter during G1 and S phases in HeLa cells . ( D ) ChIP analysis ( n = 3 ) of Pol II - pSer2 binding to BRCA1 3 ' UTR during G1 and S phases in HeLa cells . Pol II - pSer2 , phosphorylation of RNA Pol II CTD Ser2 . ( G and H ) HeLa cells were synchronized to G1 or S phase , then the degradation rates of endogenous ( G ) and exogenous ( H ) BRCA1 were analyzed by western blot . The quantification of BRCA1 relative levels according to GAPDH level are labeled below the BRCA1 band . CHX , Cycloheximide that inhibits nascent protein production . SFB - BRCA1 , exogenous BRCA1 with flag tag in N - terminal . Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1 ( n = 3 ) ( O ) or BRCA1 exon5 alternative splicing ( n = 3 ) ( AS ) during the indicated cell cycle phases ( P ) by semi - quantitative RT - PCR ( sqRT - PCR ) . Left , bar plot showing the concentration of BRCA1 isoforms in exon5 AS ( O ) or the ratio of BRCA1 WT isoform in exon5 / 11 AS ( P the above is the representative DNA gel . Right , signal figure showing quantitative analysis of PCR products . RFU , relative fluorescence units . ( P ) HeLa cells were transfected with UPF1 siRNA , then BRCA1 WT and 5q isoform mRNA levels during the indicated cell cycle phases were analyzed by qPCR ( n = 3 ) . siNC , transfected with control siRNA . siUPF1 , transfected with UPF1 siRNA . Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1 ( n = 3 ) during the indicated cell cycle phases by semi - quantitative RT - PCR ( sqRT - PCR ) . siNC , transfected with control siRNA . siUPF1 , transfected with UPF1 siRNA .

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

Figure 1(A and B) qPCR analysis (n = 3) of BRCA1 mRNA (A) and pre-mRNA levels (B) in different cell cycle phases of HeLa cells.(C) ChIP analysis (n = 3) of Pol II binding to BRCA1 promoter during G1 and S phases in HeLa cells. (D) ChIP analysis (n = 3) of Pol II-pSer2 binding to BRCA1 3'UTR during G1 and S phases in HeLa cells. Pol II-pSer2, phosphorylation of RNA Pol II CTD Ser2.(G and H) HeLa cells were synchronized to G1 or S phase, then the degradation rates of endogenous (G) and exogenous (H) BRCA1 were analyzed by western blot. The quantification of BRCA1 relative levels according to GAPDH level are labeled below the BRCA1 band. CHX, Cycloheximide that inhibits nascent protein production. SFB-BRCA1, exogenous BRCA1 with flag tag in N-terminal.Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1(n = 3) (O) or BRCA1 exon5 alternative splicing (n = 3) (AS) during the indicated cell cycle phases (P) by semi-quantitative RT-PCR (sqRT-PCR). Left, bar plot showing the concentration of BRCA1 isoforms in exon5 AS (O) or the ratio of BRCA1 WT isoform in exon5/11 AS (P the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. (P) HeLa cells were transfected with UPF1 siRNA, then BRCA1 WT and 5q isoform mRNA levels during the indicated cell cycle phases were analyzed by qPCR (n = 3). siNC, transfected with control siRNA. siUPF1, transfected with UPF1 siRNA.Analysis of BRCA1 WT or 5q isoform RNA levels after the depletion of UPF1(n = 3) during the indicated cell cycle phases by semi-quantitative RT-PCR (sqRT-PCR). siNC, transfected with control siRNA. siUPF1, transfected with UPF1 siRNA.


Figure 2 ( C and D ) RNA pull - down assay shows RBM10 binding to BRCA1 exon5 ( C ) and exon11 ( D ) RNA in vitro . ( G and H ) The BRCA1 protein ( G ) and mRNA levels ( n = 3 ) ( H ) in 293T and RBM10 - KD cells . ( N and O ) qPCR analysis ( n = 3 ) of BRCA1 WT isoform mRNA level in exon11 AS during the indicated cell phases after depleting RBM10 in HeLa ( N ) and MCF - 7 ( O ) cells . ( P ) Results of western blot analyzing BRCA1 levels in HeLa and RBM10 - KD cells in the indicated cell cycle phases .

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

Figure 2(C and D) RNA pull-down assay shows RBM10 binding to BRCA1 exon5 (C) and exon11 (D) RNA in vitro.(G and H) The BRCA1 protein (G) and mRNA levels (n = 3) (H) in 293T and RBM10-KD cells.(N and O) qPCR analysis (n = 3) of BRCA1 WT isoform mRNA level in exon11 AS during the indicated cell phases after depleting RBM10 in HeLa (N) and MCF-7 (O) cells.(P) Results of western blot analyzing BRCA1 levels in HeLa and RBM10-KD cells in the indicated cell cycle phases.


Figure 3 ( A to D ) Analysis of BRCA1 exon5 ( A ) and exon11 ( C ) AS by by semi - quantitative RT - PCR ( sqRT - PCR ) ( n = 3 ) , and analysis of BRCA1 exon5 ( n = 3 ) ( B ) or exon11 ( n = 3 ) ( D ) AS by minigene splicing reporter system after the depletion of RBM10 . Bar plot shows the ratio of BRCA1 WT isoform in exon5 / 11 AS , the figure above is the representative DNA gel , the right is signal figure showing quantitative analysis of PCR products . RFU , relative fluorescence units . ( F ) qPCR ( n = 3 ) analyzing the WT and D11q isoform levels ( exon11 AS ) after RBM10 overexpression in 293T cells .

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

Figure 3(A to D) Analysis of BRCA1 exon5 (A) and exon11 (C) AS by by semi-quantitative RT-PCR (sqRT-PCR) (n = 3), and analysis of BRCA1 exon5 (n = 3) (B) or exon11 (n = 3) (D) AS by minigene splicing reporter system after the depletion of RBM10. Bar plot shows the ratio of BRCA1 WT isoform in exon5/11 AS, the figure above is the representative DNA gel, the right is signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units.(F) qPCR (n = 3) analyzing the WT and D11q isoform levels (exon11 AS) after RBM10 overexpression in 293T cells.


Figure 4 ( A and B ) Western blot analysis of RBM10 phosphorylation in the indicated cell phases ( A ) or G1 / S phase ( B ) . In ( B ) , cells were treated with the indicated CDK inhibitors ( 10 μM ) for 6 h . CDK1i , Cucurbitacin E . CDK2i , SB1317 . CDK4 / 6i , Palbociclib . ( F ) Analysis of BRCA1 exon5 AS by by sqRT - PCR ( n = 3 ) in in G1 / S phase of 293T cells treated with CDK2i . In ( F ) bar plot showing the ratio of BRCA1 WT isoform in exon5 AS , the above is the representative DNA gel . Right , signal figure showing quantitative analysis of PCR products . RFU , relative fluorescence units . Data are presented as mean ± s . d . in all qPCR . ( I ) Mass spectrometry analysis showing RBM10 phosphorylation at S89 site . ( N ) Analysis of BRCA1 exon5 AS by sqRT - PCR ( n = 3 ) , 293T cell was transfected with RBM10 S89D variant in G1 phase . In ( N ) , bar plot showing the ratio of BRCA1 WT isoform in exon5 AS , the above is the representative DNA gel . Right , signal figure showing quantitative analysis of PCR products . RFU , relative fluorescence units . Data are presented as mean ± s . d . in all qPCR . ( O to Q ) Phosphorylation at S89 of the indicated RBM10 variants ( O ) , WT RBM10 in the indicated cell cycle phases ( P ) , and WT / S89A mutant RBM10 after CDK2i treatment in G1 / S phase ( Q ) were analyzed in 293T cells .

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

Figure 4(A and B) Western blot analysis of RBM10 phosphorylation in the indicated cell phases (A) or G1/S phase (B). In (B), cells were treated with the indicated CDK inhibitors (10 μM) for 6 h. CDK1i, Cucurbitacin E. CDK2i, SB1317. CDK4/6i, Palbociclib.(F) Analysis of BRCA1 exon5 AS by by sqRT-PCR (n = 3) in in G1/S phase of 293T cells treated with CDK2i. In (F) bar plot showing the ratio of BRCA1 WT isoform in exon5 AS, the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. Data are presented as mean ± s.d. in all qPCR.(I) Mass spectrometry analysis showing RBM10 phosphorylation at S89 site.(N) Analysis of BRCA1 exon5 AS by sqRT-PCR (n = 3), 293T cell was transfected with RBM10 S89D variant in G1 phase. In (N), bar plot showing the ratio of BRCA1 WT isoform in exon5 AS, the above is the representative DNA gel. Right, signal figure showing quantitative analysis of PCR products. RFU, relative fluorescence units. Data are presented as mean ± s.d. in all qPCR.(O to Q) Phosphorylation at S89 of the indicated RBM10 variants (O), WT RBM10 in the indicated cell cycle phases (P), and WT/S89A mutant RBM10 after CDK2i treatment in G1/S phase (Q) were analyzed in 293T cells.