B . HeLa cells stably expressing CHMP4B - eGFP were treated as in ( A ) and stained for p62 . CHMP4B is recruited before p62 on the damaged lysosomes . The number of LAMP1 - positive foci appears stable independently of LLOMe treatment Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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B. HeLa cells stably expressing CHMP4B-eGFP were treated as in (A) and stained for p62. CHMP4B is recruited before p62 on the damaged lysosomes. The number of LAMP1-positive foci appears stable independently of LLOMe treatment Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


C . Experimental setup as in ( A and B ) where HeLa cells stably expressing CHMP4B - eGFP and mCherry - Galectin3 were stained for LC3 . As shown , CHMP4B is recruited prior to LC3 Data information : Quantification graphs are normalized to the cell number where error bars represent 95 % confidence intervals . Data were obtained from > 150 cells per condition from three independent

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C. Experimental setup as in (A and B) where HeLa cells stably expressing CHMP4B-eGFP and mCherry-Galectin3 were stained for LC3. As shown, CHMP4B is recruited prior to LC3 Data information: Quantification graphs are normalized to the cell number where error bars represent 95% confidence intervals. Data were obtained from >150 cells per condition from three independent experiments


A . HeLa cells were transfected with different siRNAs as indicated . 48 h post - transfection cells were treated with 250 μM LLOMe for 3 , 6 , 10 hours harvested and stained for Annexin V . Cells were immediately processed for flow cytometry . Already after 3 h of treatment with LLOMe , cells co - transfected with TSG101 + ALIX siRNA showed an increase in apoptotic cell death , whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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A. HeLa cells were transfected with different siRNAs as indicated. 48 h post-transfection cells were treated with 250 μM LLOMe for 3, 6, 10 hours harvested and stained for Annexin V. Cells were immediately processed for flow cytometry. Already after 3 h of treatment with LLOMe, cells co-transfected with TSG101+ALIX siRNA showed an increase in apoptotic cell death, whereas TSG101 siRNA transfected cells showed elevated Annexin V staining after 6 h of treatment Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


B . Experimental set up as in ( A ) with additional propidium iodide ( PI ) staining of transfected HeLa cells Data information : Graphs show average data from a set of three independent experiments where > 10 , 000 cells per condition were analyzed . Error bars represent SEM . Statistical significance was determined using one - way ANOVA test . p - values are presented where significant . * p - value ≤ 0 . 05 , * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 .

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B. Experimental set up as in (A) with additional propidium iodide (PI) staining of transfected HeLa cells Data information: Graphs show average data from a set of three independent experiments where >10,000 cells per condition were analyzed. Error bars represent SEM. Statistical significance was determined using one-way ANOVA test. p-values are presented where significant. *p-value≤ 0.05, **p-value≤ 0.01 and ***p-value≤ 0.001


A . HeLa cells stably expressing CHMP4B - eGFP were transfected with the mCherry - Galectin3 plasmid . 24 h later , cells were infected with WT Coxiella burnetti . Live - cell imaging started 24 h post - infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella - containing vacuole ( DRAQ5 labeling ) becoming positive for CHMP4B - eGFP and mCherry - Galectin3 . Data information : Scale bars : 50

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A. HeLa cells stably expressing CHMP4B-eGFP were transfected with the mCherry-Galectin3 plasmid. 24 h later, cells were infected with WT Coxiella burnetti. Live-cell imaging started 24 h post-infection and several time points are indicated during the next 24 h of observatio Arrows indicate Coxiella-containing vacuole (DRAQ5 labeling) becoming positive for CHMP4B-eGFP and mCherry-Galectin3. Data information: Scale bars: 50 μm


B . Bacterial viability and replication assay upon TSG101 KD . HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h . Then cells were infected with mCherry - Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells . 72 h later , infected cells were fixed , DAPI stained and processed for quantitative image analysis . Between 30 and 40 fields representing more than 9 , 000 cells per condition of three independent experiments were analyzed . Error bars represent SD . Statistical significance was determined using one - way ANOVA test . * * p - value ≤ 0 . 01 and * * * p - value ≤ 0 . 001 .

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B. Bacterial viability and replication assay upon TSG101 KD. HeLa cells were treated with either siCtrl or with two different siTSG101 for 48 h. Then cells were infected with mCherry-Coxiella burnetii for 48 h before lysis and serial dilutions were made to infect Vero cells. 72 h later, infected cells were fixed, DAPI stained and processed for quantitative image analysis. Between 30 and 40 fields representing more than 9,000 cells per condition of three independent experiments were analyzed. Error bars represent SD. Statistical significance was determined using one-way ANOVA test. **p-value≤ 0.01 and ***p-value≤ 0.001.


b - d ) Comparative characterization of CHO cell bulk transfection cultures , selected for stable expression of the UnaG - or EGFP - encoding reporter constructs . b ) Flow cytometry revealed efficient induction of green fluorescence , after treatment of both UnaG and EGFP - expressing CHO bulk cultures with CoCl2 , while growth under hypoxia ( 1 % oxygen ) for 12 hours , selectively induced green fluorescence only in dUnaG expressing cells . Under normoxia ( 21 % oxygen ) only background fluorescence was observed . c ) Assessment of the mean fluorescence intensity ( MFI ) to determine the activity of the 5xHRE - CMV promoter after 12 and 24hrs . Fluorescence intensity increased between 12 and 24hrs .

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b - d) Comparative characterization of CHO cell bulk transfection cultures, selected for stable expression of the UnaG- or EGFP-encoding reporter constructs.b) Flow cytometry revealed efficient induction of green fluorescence, after treatment of both UnaG and EGFP-expressing CHO bulk cultures with CoCl2, while growth under hypoxia (1% oxygen) for 12 hours, selectively induced green fluorescence only in dUnaG expressing cells. Under normoxia (21% oxygen) only background fluorescence was observed.c) Assessment of the mean fluorescence intensity (MFI) to determine the activity of the 5xHRE-CMV promoter after 12 and 24hrs. Fluorescence intensity increased between 12 and 24hrs.


d ) Representative images of dUnaG - or dEGFP - expressing CHO cells grown either under normoxia ( 21 % oxygen ) , CoCl2 - treatment or hypoxia ( 1 % oxygen ) for 12 hours . Scale bars =

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d) Representative images of dUnaG- or dEGFP-expressing CHO cells grown either under normoxia (21% oxygen), CoCl2-treatment or hypoxia (1% oxygen) for 12 hours. Scale bars= 50µm


e ) Fluorescence intensity in dUnaG - or dEGFP - expressing CHO cell bulks grown under increasingly hypoxic conditions of 10 % , 5 % and 1 % oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5 % .

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e) Fluorescence intensity in dUnaG- or dEGFP-expressing CHO cell bulks grown under increasingly hypoxic conditions of 10%, 5% and 1% oxygen for 24 hrs indicated efficient induction of the dUnaG sensor at oxygen concentrations below 5%.


f ) Maximal fluorescence of the UnaG - based hypoxia reporter in CHO cells grown at 1 % oxygen for 24 hrs hours was only observed at serum concentrations of 5 % FCS or higher , reflecting the dependence of UnaG on its serum cofactor bilirubin .

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f) Maximal fluorescence of the UnaG-based hypoxia reporter in CHO cells grown at 1% oxygen for 24 hrs hours was only observed at serum concentrations of 5% FCS or higher, reflecting the dependence of UnaG on its serum cofactor bilirubin.


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP . Expression under hypoxia was induced by incubation in 1 % oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia . Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly - destabilized UnaG - variants , while the doubly destabilized doUnaG displayed the most rapid switching behavior , but the least brightness . To reduce phototoxicity , illumination intensity was kept minimal . Data points represent one viewfield of 425 µm2 ± SEM .

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b) Microscopic assessment of the averaged fluorescence intensity (AFI) of CHO cell bulk cultures stably expressing the indicated UnaG variants or destabilized EGFP. Expression under hypoxia was induced by incubation in 1% oxygen for 16 hrs and then culture was continued for 24 hrs under normoxia. Fluorescence hysteresis after switching to normoxia was markedly reduced in the singly-destabilized UnaG-variants, while the doubly destabilized doUnaG displayed the most rapid switching behavior, but the least brightness. To reduce phototoxicity, illumination intensity was kept minimal. Data points represent one viewfield of 425 µm2 ± SEM.


c ) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG . Open bars represent control cultures grown under normoxia . Solid bars indicate culture for 4 , 8 , 12 , 16 , 20 or 24 hrs under hypoxia ( 1 % oxygen ) . To assess deactivation , cultures previously kept under hypoxia ( 1 % oxygen ) for 24hrs were shifted to normoxia ( 21 % oxygen ) for the indicated times . Error bars represent standard errors .

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c) Flow cytometry analysis of the induction and decay kinetics of the fluorescence intensity in CHO cells stably expressing either dUnaG or oUnaG. Open bars represent control cultures grown under normoxia. Solid bars indicate culture for 4, 8, 12, 16, 20 or 24 hrs under hypoxia (1% oxygen). To assess deactivation, cultures previously kept under hypoxia (1% oxygen) for 24hrs were shifted to normoxia (21% oxygen) for the indicated times. Error bars represent standard errors.


d ) Representative maximum intensity projections ( MIPs ) from the life cell cultures depicted in b . Cells were grown for the indicated times at hypoxic ( 1 % oxygen ) conditions and subsequently switched for the indicated times to normoxia ( 21 % oxygen ) ( red arrows in b ) to illustrate the dynamics of the different UnaG - based hypoxia sensors . Increasing destabilization is associated with reduced brightness but , also reduced background and improved switching behavior . Scale bars = 50 µm .

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d) Representative maximum intensity projections (MIPs) from the life cell cultures depicted in b. Cells were grown for the indicated times at hypoxic (1% oxygen) conditions and subsequently switched for the indicated times to normoxia (21% oxygen)(red arrows in b) to illustrate the dynamics of the different UnaG-based hypoxia sensors. Increasing destabilization is associated with reduced brightness but, also reduced background and improved switching behavior. Scale bars = 50 µm.


a ) UnaG - based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model . 500 Gli36 glioblastoma cells , constitutively expressing mCherry and stably transfected with the HRE - dUnaG sensor construct , were stereotactically transplanted into the cortex of a SCIDmouse . Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation . Tumor cells are distinguished from the surrounding cortex by mCherry expression . Blood vessels were contrasted by immunostaining against PECAM - 1 . Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density ( outlined by white line in the composite panel ( bottom right ) . b ) Representative area from the tumor shown in a ) , which is located outside the viewfield in a ) positioned more closely to the tumor border . Also at the tumor border UnaG - expressing cells are preferentially observed at a distance to PECAM - 1 + vessels .

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a) UnaG-based hypoxia sensing is applicable in vivo as shown here using the human Gli36 glioblastoma model. 500 Gli36 glioblastoma cells, constitutively expressing mCherry and stably transfected with the HRE-dUnaG sensor construct, were stereotactically transplanted into the cortex of a SCIDmouse. Shown is a 30 µm cryosection of a growing tumor 10 days after transplantation. Tumor cells are distinguished from the surrounding cortex by mCherry expression. Blood vessels were contrasted by immunostaining against PECAM-1. Expression of dUnaG was visualized by its green fluorescence and predominates in areas with reduced vascular density (outlined by white line in the composite panel (bottom right).b) Representative area from the tumor shown in a), which is located outside the viewfield in a) positioned more closely to the tumor border. Also at the tumor border UnaG-expressing cells are preferentially observed at a distance to PECAM-1+ vessels.


c ) Areas of dUnaG expression correlate well with HIF - 1α stabilization . Immunostaining for HIF - 1α ( cyan ) revealed the predominantly nuclear localization of stabilized HIF - 1α in cells that were also dUnaG - positive ( green ) . d ) Analysis of dUnaG expressing cells in c ) for mCherry fluorescence and HIF - 1α stabilization . dUnaG - positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF - 1α The threshold was set in either case to channel 70 of 256 intensity channels . Using this classification > 60 % of UnaG positive cells displayed only background level mCherry fluorescence . On the other hand , > 98 % of the UnaG expressing cells also expressed HIF - 1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo .

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c) Areas of dUnaG expression correlate well with HIF-1α stabilization. Immunostaining for HIF-1α (cyan) revealed the predominantly nuclear localization of stabilized HIF-1α in cells that were also dUnaG-positive (green).d) Analysis of dUnaG expressing cells in c) for mCherry fluorescence and HIF-1α stabilization. dUnaG-positive cells were classified according to their average fluorescence intensity into background level and above background level expression for mCherry and HIF-1α The threshold was set in either case to channel 70 of 256 intensity channels. Using this classification >60% of UnaG positive cells displayed only background level mCherry fluorescence. On the other hand, >98% of the UnaG expressing cells also expressed HIF-1α , which together suggests that here UnaG acts preferentially as a hypoxia sensor in vivo.


e ) The dUnaG - based senor was efficiently applied in intravital microscopy . 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG - sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy . We noted two distinct areas of intense UnaG fluorescence ( red lines ) . Shown is a MIP of a 60 µm z stack . The vasculature was contrasted by intravenous injection of non - functionalized Qdots . f ) Depth color coding of the MIP shown in e ) revealed the spatial relation between the vasculature , ( contrasted by Qdots ) , areas of increased dUnaG expression ( bordered by green lines ) and tumor cells expressing residual levels of mCherry . The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner ( red asterisk ) and the smaller vessels with a more axial orientation ( white arrows ) . Scale bars =

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e) The dUnaG-based senor was efficiently applied in intravital microscopy. 500 Gli36 glioblastoma cells stably expressing mCherry and harboring the dUnaG-sensor construct were positioned at 200µm depth subcortically in a SCIDmouse and observed 4 days later through a cranial window using multiphoton microscopy. We noted two distinct areas of intense UnaG fluorescence (red lines). Shown is a MIP of a 60 µm z stack. The vasculature was contrasted by intravenous injection of non-functionalized Qdots.f) Depth color coding of the MIP shown in e) revealed the spatial relation between the vasculature, (contrasted by Qdots), areas of increased dUnaG expression (bordered by green lines) and tumor cells expressing residual levels of mCherry. The hypoxic areas were located towards the top of the imaged tissue cube and therefore at different levels compared to the large vessel at the lower left corner (red asterisk) and the smaller vessels with a more axial orientation (white arrows). Scale bars = 100µm


b ) Microscopic assessment of the averaged fluorescence intensity ( AFI ) in CHO cells stably transfected with the dUnOHR sensor . Hypoxia was induced by incubation in 1 % oxygen for 18 hrs , then the culture was switched to normoxia for 24 hrs , followed by another 14 hrs hypoxia and finally normoxia again . As expected UnaG fluorescence is efficiently induced under hypoxia , while mOrange fluorescence only appears after the switch to normoxia only . The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE - mCMV promoter activity . This behavior is repeated in subsequent hypoxia / normoxia cycles . The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period . Plotted is the average of the mean ± SEM . c ) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b . ) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange . Scale bars = 100 µm .

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b) Microscopic assessment of the averaged fluorescence intensity (AFI) in CHO cells stably transfected with the dUnOHR sensor. Hypoxia was induced by incubation in 1% oxygen for 18 hrs, then the culture was switched to normoxia for 24 hrs, followed by another 14 hrs hypoxia and finally normoxia again. As expected UnaG fluorescence is efficiently induced under hypoxia, while mOrange fluorescence only appears after the switch to normoxia only. The increase in both green and orange fluorescence is limited under normoxia by the subsiding HRE-mCMV promoter activity. This behavior is repeated in subsequent hypoxia / normoxia cycles. The increase in absolute fluorescence intensity is due to proliferation during the 72hrs culture period. Plotted is the average of the mean ± SEM.c) Visualization of the fluorescence of the dUnOHR reporter during alternating hypoxia / normoxia cycles as described in b.) MIPs of life cell cultures stably transfected with the dUnOHR sensor construct illustrate the temporally asynchronous fluorescence of UnaG and mOrange. Scale bars = 100 µm.


a ) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct . The perimeter of the developing tumor ( white line in the merged picture , lower right panel ) was delineated by Hoechst 33342 staining . As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density . Vascular endothelium was identified by PECAM - 1 staining . 30 µm cryosection . Scale bar = 500µm . b ) Details of the tumor shown in a ) . Sparse , distinct cells are characterized by intense green and orange fluorescence ( shown as orange ) and are located preferentially to the edge of the hypoxic phalanx ( white arrows ) . A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence ( asterisks ) , indicative of previously hypoxic environment and subsequent reoxygenation . Scale bar = 50 µm .

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a) Overview of a tumor induced by deep cortical deposition of approximately 500 Gli36 cells stably transfected with the dUnOHR sensor construct. The perimeter of the developing tumor (white line in the merged picture, lower right panel) was delineated by Hoechst 33342 staining. As observed for dUnaG the reoxygenation sensor was expressed in compact areas with low vascular density. Vascular endothelium was identified by PECAM-1 staining. 30 µm cryosection. Scale bar = 500µm.b) Details of the tumor shown in a). Sparse, distinct cells are characterized by intense green and orange fluorescence (shown as orange) and are located preferentially to the edge of the hypoxic phalanx (white arrows). A number of cells in close proximity of the nearest blood vessel display weak green and orange fluorescence (asterisks), indicative of previously hypoxic environment and subsequent reoxygenation. Scale bar = 50 µm.


c ) Recently reoxygenated cells ( arrows ) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber ( double arrows ) . Scale bar = 100 µm .

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c) Recently reoxygenated cells (arrows) cluster at the edge of hypoxic areas and are often detected in the proximity of vessels with a homogeneous caliber (double arrows). Scale bar = 100 µm.


d ) Previously hypoxic cells , frequently undergo apoptosis after reoxygenation . We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas . Scale bar = 50µm .

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d) Previously hypoxic cells, frequently undergo apoptosis after reoxygenation. We noted an accumulation of intensely orange fluorescing spots at the edges of compact UnaG positive areas. Scale bar = 50µm.


e ) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis . Scale bar = 50µm .

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e) TUNEL staining of tumor sections revealed that the tissue areas rich in intense mOrange aggregates colocalized with areas of increased apoptosis. Scale bar = 50µm.


A - 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 .

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A-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 .

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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 .

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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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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.


A . 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 .

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A. 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 .

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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 .

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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 ) .

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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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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).


A . 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 .

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A. 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 .

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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 .

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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 .

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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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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.


A - 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 .

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A-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 ) .

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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 .

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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.


A . 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 .

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A. 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 .

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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 ) .

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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 .

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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 .

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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 .

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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.


A . MCU silencing reduces HIF - 1α protein levels . Cells were transfected with siMCU or siControl . HIF - 1α protein levels were detected 48 hours later .

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A. 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 ) .

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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 .

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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 .

O O O O B-CONTROLLED_VAR O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O 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-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O

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 .

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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 ) .

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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).


( C ) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts ( n = 4 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . n . s means no significance .

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(C) Decreased levels of free carnitine and modified carnitine species in PKM2 KO cell lines as compared with their parental counterparts (n=4). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.


( E ) Increased acetyl - CoA in PKM2 KO MCF7 and MDA - MB 231cells as compared with their parental counterparts ( n = 4 ) . ( F ) Decreased total cholesterol in PKM2 KO MCF7 and MDA - MB 231cells as compared with their parental counterparts ( n = 3 ) . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 . n . s means no significance .

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(E) Increased acetyl-CoA in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=4). (F) Decreased total cholesterol in PKM2 KO MCF7 and MDA-MB 231cells as compared with their parental counterparts (n=3). ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001. n.s means no significance.


( G ) Western blot analysis of TMEM33 , ABCA1 , SREBP1 , HMGCR , SCAP , FASN , LDLR , FDFT1 , I DI1 , RNF5 , INSIG 1 and 2 , and Calnexin in PKM2 KO MCF7 and MDA - MB - 231 cells , and their respective parental cells . Arrow indicates nuclear SREBP1 . P : precursor , N : nuclear .

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(G) Western blot analysis of TMEM33, ABCA1, SREBP1, HMGCR, SCAP, FASN, LDLR, FDFT1, I DI1, RNF5, INSIG 1 and 2, and Calnexin in PKM2 KO MCF7 and MDA-MB-231 cells, and their respective parental cells. Arrow indicates nuclear SREBP1. P: precursor, N: nuclear.


( A ) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging . Scale bar , 5μm . ( B ) Quantitation of nuclear SREBP1 intensity in A . n > = 25 cells as in ( A ) from three different fields . ( Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 .

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(A) Immunofluorescence of SREBP1 in MCF7 parental and PKM2 KO cells by confocal imaging. Scale bar, 5μm. (B) Quantitation of nuclear SREBP1 intensity in A. n>=25 cells as in (A) from three different fields. ( Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001.


( G ) Western blot analysis of TMEM33 , RNF5 , SCAP , SREBP1 , HMGCR , LDLR , FDFT1 , IDI1 , and PKM2 in wild type , PKM2 KO , TMEM33 OE ( pool ) , and two TMEM33OE clones ( clones # 1 and # 8 ) MDA - MB - 231 cells . Arrow indicates nuclear SREBP1 . N : nucleus and P : precursor .

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(G) Western blot analysis of TMEM33, RNF5, SCAP, SREBP1, HMGCR, LDLR, FDFT1, IDI1, and PKM2 in wild type, PKM2 KO, TMEM33 OE (pool), and two TMEM33OE clones (clones #1 and #8) MDA-MB-231 cells. Arrow indicates nuclear SREBP1. N: nucleus and P: precursor.


( B ) Western blot analysis of PKM2 , PKM1 , NRF1 , TMEM33 , RNF5 and SCAP levels in MEF ( PKM2fl / fl , Cre - ERT2 ) cells treated with 4 - OHT in a time course experiment .

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(B) Western blot analysis of PKM2, PKM1, NRF1, TMEM33, RNF5 and SCAP levels in MEF (PKM2fl/fl, Cre-ERT2) cells treated with 4-OHT in a time course experiment.


( C ) Co - immunoprecipitation of endogenous SCAP and RNF5 in Flag - tagged TMEM33 stably expressed MCF7 cells . SEC61β is an ER protein , which is found to interact with TMEM33 .

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(C) Co-immunoprecipitation of endogenous SCAP and RNF5 in Flag-tagged TMEM33 stably expressed MCF7 cells. SEC61β is an ER protein, which is found to interact with TMEM33.


( H ) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells . 293T cells were stably transfected with shCtrl or shRNF5 constructs , then the indicated plasmids were co - transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG - 132 treatment for 8 hours . FLAG M2 beads were used for pull - down .

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(H) RNF5 is involved in TMEM33 mediated SCAP polyubiquitination in 293T cells. 293T cells were stably transfected with shCtrl or shRNF5 constructs, then the indicated plasmids were co-transfected into shCtrl or shRNF5 expressing 293T cells for 18 hours followed by MG-132 treatment for 8 hours. FLAG M2 beads were used for pull-down.


( D ) qRT - PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA - MB - 231 cells ( n = 3 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

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(D) qRT-PCR analyses of selected SREBPs downstream genes in shCtrl and shRNF5 PKM2 KO MDA-MB-231 cells (n=3). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( G ) Cell proliferation of Ctrl and HA - RNF5 OE MCF7 cells measured by MTT assays ( n = 8 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

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(G) Cell proliferation of Ctrl and HA-RNF5 OE MCF7 cells measured by MTT assays (n=8). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( E ) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones . ( F ) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells . (

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(E) Western blot analyses of TMEM33 and RNF5 in NRF1 overexpressed MCF7 clones. (F) Western blot analyses of TMEM33 and RNF5 in shCtrl and shNRF1 PKM2 KO MCF7 cells. (


( G ) Co - immunoprecipitation of VCP with FLAG - tagged PKM1 / 2 in PKM2 KO 293T cells . FLAG - tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours , followed by pull - down using FLAG - M2 beads . PKM1 , PKM2 and VCP antibodies were used for immunoblotting .

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(G) Co-immunoprecipitation of VCP with FLAG-tagged PKM1/2 in PKM2 KO 293T cells. FLAG-tagged PKM1 or PKM2 construct was transiently transfected into PKM2 KO or parental 293T cells for 24 hours, followed by pull-down using FLAG-M2 beads. PKM1, PKM2 and VCP antibodies were used for immunoblotting.


( E ) Representative H & E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers . Scale bars , 100µm .

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(E) Representative H&E and IHC staining images of TMEM33 and SCAP in WT and TMEM33 KO mouse livers. Scale bars, 100µm.


( F , G ) Significant decrease of total plasma cholesterol ( F ) and LDL - C ( G ) levels in TMEM33 - / - and + / - mice relative to WT counterparts ( n > = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using two - tailed Student ' s t - test , ∗ p < 0 . 05 and ∗ ∗ ∗ p < 0 . 001 . n . s denotes no significance .

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(F, G) Significant decrease of total plasma cholesterol (F) and LDL-C (G) levels in TMEM33-/- and +/- mice relative to WT counterparts (n>=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using two-tailed Student's t-test, ∗p < 0.05 and ∗∗∗p < 0.001. n.s denotes no significance.


( B ) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice ( n = 5 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

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(B) Tumor growth curves of PKM2 WT or KO py8119 cell line allografts in systemic PKM2 KO or WT mice (n=5). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( E ) The growth curves of py8119 allografts treated with vehicle and TEPP - 46 ( 50mg / kg ) for 17 days in mice ( n = 7 ) . Data information : Data are displayed as means ± SD . Statistical significance was assessed using a two - tailed Student ' s t - test , ∗ p < 0 . 05 , ∗ ∗ p < 0 . 01 and ∗ ∗ ∗ p < 0 . 001 .

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(E) The growth curves of py8119 allografts treated with vehicle and TEPP-46 (50mg/kg) for 17 days in mice (n=7). Data information: Data are displayed as means ± SD. Statistical significance was assessed using a two-tailed Student's t-test, ∗p < 0.05, ∗∗p < 0.01 and ∗∗∗p < 0.001.


( A ) Electrophoretic analysis ( under non - denaturing conditions ) of the association between the indicated IκBα species association and reconstituted nucleosome core particles ( NCP ) .

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(A) Electrophoretic analysis (under non-denaturing conditions) of the association between the indicated IκBα species association and reconstituted nucleosome core particles (NCP).


( B ) Quantification of the IκBα 60 kDa band ( upper panels ) and the 37 kDa band ( lower panels ) is shown as average and s . d . of three independent experiments .

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(B) Quantification of the IκBα 60 kDa band (upper panels) and the 37 kDa band (lower panels) is shown as average and s.d. of three independent experiments.


( D ) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2 - month - old WT mice ( each # represents a mouse ) .

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(D) Western Blot analysis of chromatin extracts from isolated intestinal villus and crypt cells in several 2-month-old WT mice (each # represents a mouse).


( G ) Pulldown assay using GST - SUMO IκBα protein and chromatin lysates from 2 - month - old WT mice villi .

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(G) Pulldown assay using GST-SUMO IκBα protein and chromatin lysates from 2-month-old WT mice villi.


( A ) Western Blot analysis of the crypt - derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times .

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(A) Western Blot analysis of the crypt-derived chromatin fractions incubated with soluble lysates from villi or crypts for the indicated times.


( F ) Western Blot analysis of a cleavage experiment incubating modified H4 peptides ( P1 - P9 ) with soluble lysates from villi or crypts in the presence ( + ) or absence ( - ) of the commercial protease inhibitors cocktail . Lanes indicated as P correspond to the control peptide without lysate incubation . Notice the electrophoretic shift of peptides incubated with villus lysates ( compared with control peptides ) suggestive of post - translational modifications or binding to proteins absent from crypt lysates .

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(F) Western Blot analysis of a cleavage experiment incubating modified H4 peptides (P1-P9) with soluble lysates from villi or crypts in the presence (+) or absence (-) of the commercial protease inhibitors cocktail. Lanes indicated as P correspond to the control peptide without lysate incubation. Notice the electrophoretic shift of peptides incubated with villus lysates (compared with control peptides) suggestive of post-translational modifications or binding to proteins absent from crypt lysates.


WB analysis ( C ) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre - confluence or 7 days post - confluence . Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed .

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WB analysis (C) of the indicated genes or proteins in parental or IκBα KO HT29 cells obtained at pre-confluence or 7 days post-confluence. Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed.


WB analysis ( E ) of MUC5AC in HT29 cells treated with the indicated protease inhibitors . Bars represent mean values ± standard deviation from 3 biological replicates

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WB analysis (E) of MUC5AC in HT29 cells treated with the indicated protease inhibitors. Bars represent mean values ± standard deviation from 3 biological replicates performed


WB analysis ( G ) of MUC5AC in HT29 cells transduced with sh - RNAs against trypsin ( T1 , T2 ) or chymotrypsin ( C1 , C2 ) . Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates

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WB analysis (G) of MUC5AC in HT29 cells transduced with sh-RNAs against trypsin (T1, T2) or chymotrypsin (C1, C2). Bars represent mean values ± standard deviation of 3 technical replicates from 3 biological replicates performed


( B - D ) Kaplan - Meier survival plots comparing the survival probability of all melanoma patients ( B ) , BRAF - mutated patients ( C ) and BRAF wild - type ( WT ) patients ( D ) with high - ( magenta ) and low ( black ) MCUA mRNA expression .

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(B-D) Kaplan-Meier survival plots comparing the survival probability of all melanoma patients (B), BRAF-mutated patients (C) and BRAF wild-type (WT) patients (D) with high- (magenta) and low (black) MCUA mRNA expression.


( E - G ) Kaplan - Meier survival plots comparing the survival probability of patients of indicated cancer types that are divided into either high - ( magenta ) or low ( black ) MCUA mRNA expression . P - values were calculated by using a log - rank test . Abbreviations : KICH = kidney chromophobe ; KIRC = kidney renal clear cell carcinoma ; KIRP = kidney renal papillary cell carcinoma ; LUAD = lung adenocarcinoma ; LUSC = lung squamous cell carcinoma ; PAAD = pancreatic adenocarcinoma .

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(E-G) Kaplan-Meier survival plots comparing the survival probability of patients of indicated cancer types that are divided into either high- (magenta) or low (black) MCUA mRNA expression. P-values were calculated by using a log-rank test. Abbreviations: KICH = kidney chromophobe; KIRC = kidney renal clear cell carcinoma; KIRP = kidney renal papillary cell carcinoma; LUAD = lung adenocarcinoma; LUSC = lung squamous cell carcinoma; PAAD = pancreatic adenocarcinoma.


( C ) Representative images of the migrated stained 1205Lu shCTRL ( black frame ) , shMCU _ 1 ( darker blue frame ) and shMCU _ 2 ( lighter blue frame ) cells on the lower side of the insert . ( D ) Quantification of the transwell migration in 1205Lu stable MCUA _ KD lines , based on the number of stained cells ( n = 4 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

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(C) Representative images of the migrated stained 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) cells on the lower side of the insert. (D) Quantification of the transwell migration in 1205Lu stable MCUA_KD lines, based on the number of stained cells (n=4 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


( E ) Representative images of 1205Lu shCTRL ( black frame ) , shMCU _ 1 ( darker blue frame ) and shMCU _ 2 ( lighter blue frame ) melanoma spheroids after 72 h invasion in collagen . Live cells are shown in green . Scale bar : 100 µm . ( F ) Quantification of 1205Lu stable MCUA _ KD spheroid core size ( n = 6 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

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(E) Representative images of 1205Lu shCTRL (black frame), shMCU_1 (darker blue frame) and shMCU_2 (lighter blue frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (F) Quantification of 1205Lu stable MCUA_KD spheroid core size (n=6 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


( G ) Representative images of WM3734 shCTRL ( black frame ) , shMCU _ 1 ( darker orange frame ) and shMCU _ 2 ( lighter orange frame ) melanoma spheroids . Live cells are shown in green . Scale bar : 100 µm . ( H ) Quantification of WM3734 stable MCUA _ KD spheroid core size ( n = 5 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G) Representative images of WM3734 shCTRL (black frame), shMCU_1 (darker orange frame) and shMCU_2 (lighter orange frame) melanoma spheroids. Live cells are shown in green. Scale bar: 100 µm. (H) Quantification of WM3734 stable MCUA_KD spheroid core size (n=5 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


( L ) Representative images of 451Lu wild - type ( grey frame ) , 451Lu - BR3 ( lighter green frame ) , WM983B wild - type ( grey frame ) and WM983B - BR ( darker green frame ) melanoma spheroids after 72 h invasion in collagen . Live cells are shown in green . Scale bar : 100 µm . ( M - N ) Quantification of 451Lu versus 451Lu - BR3 ( M ) and WM983B versus WM983B - BR ( N ) spheroid core size ( n ≥ 9 biological replicates / condition , shown also by individual data points ) . Data information : Data are presented as mean ± SEM . Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test ( shMCU cells were compared to their respective control , shCTRL ) , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

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

(L) Representative images of 451Lu wild-type (grey frame), 451Lu-BR3 (lighter green frame), WM983B wild-type (grey frame) and WM983B-BR (darker green frame) melanoma spheroids after 72 h invasion in collagen. Live cells are shown in green. Scale bar: 100 µm. (M-N) Quantification of 451Lu versus 451Lu-BR3 (M) and WM983B versus WM983B-BR (N) spheroid core size (n≥9 biological replicates/condition, shown also by individual data points). Data information: Data are presented as mean ± SEM. Statistical significance was determined using unpaired, two‐tailed Student's t‐test (shMCU cells were compared to their respective control, shCTRL), (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


( A - C ) Mitochondrial hydrogen peroxide ( H2O2 ) measurement in 1205Lu and WM3734 with and without stable MCUA _ KD using mito - HyPer . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for all conditions ( A ) . Scale bar : 10 µm . Quantification of mito - HyPer ratio in 1205Lu ( B ) and WM3734 ( C ) under resting state ( 1205Lu ─ shCTRL : n = 217 cells from 6 biological replicates ; shMCU _ 1 : n = 197 cells from 7 biological replicates ; shMCU _ 2 : n = 212 cells from 7 biological replicates ; WM3734 ─ shCTRL : n = 104 cells from 6 biological replicates ; shMCU _ 1 : n = 107 cells from 7 biological replicates ; shMCU _ 2 : n = 114 cells from 7 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(A-C) Mitochondrial hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using mito-HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (A). Scale bar: 10 µm. Quantification of mito-HyPer ratio in 1205Lu (B) and WM3734 (C) under resting state (1205Lu ─ shCTRL: n=217 cells from 6 biological replicates; shMCU_1: n=197 cells from 7 biological replicates; shMCU_2: n=212 cells from 7 biological replicates; WM3734 ─ shCTRL: n=104 cells from 6 biological replicates; shMCU_1: n=107 cells from 7 biological replicates; shMCU_2: n=114 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.


( D - F ) Cytosolic hydrogen peroxide ( H2O2 ) measurement in 1205Lu and WM3734 with and without stable MCUA _ KD using HyPer . Exemplary ratiometric images ( F505 nm / F420 nm ) are shown for all conditions ( D ) . Scale bar : 10 µm . Quantification of HyPer ratio in 1205Lu ( E ) and WM3734 ( F ) under resting state ( 1205Lu ─ shCTRL : n = 232 cells from 6 biological replicates ; shMCU _ 1 : n = 290 cells from 6 biological replicates ; shMCU _ 2 : n = 290 cells from 6 biological replicates ; WM3734 ─ shCTRL : n = 443 cells from 9 biological replicates ; shMCU _ 1 : n = 297 cells from 9 biological replicates ; shMCU _ 2 : n = 233 cells from 9 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control .

O O O O O O B-MEASURED_VAR I-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(D-F) Cytosolic hydrogen peroxide (H2O2) measurement in 1205Lu and WM3734 with and without stable MCUA_KD using HyPer. Exemplary ratiometric images (F505 nm/F420 nm) are shown for all conditions (D). Scale bar: 10 µm. Quantification of HyPer ratio in 1205Lu (E) and WM3734 (F) under resting state (1205Lu ─ shCTRL: n=232 cells from 6 biological replicates; shMCU_1: n=290 cells from 6 biological replicates; shMCU_2: n=290 cells from 6 biological replicates; WM3734 ─ shCTRL: n=443 cells from 9 biological replicates; shMCU_1: n=297 cells from 9 biological replicates; shMCU_2: n=233 cells from 9 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.


( I - K ) Mitochondrial glutathione redox potential , measured with mito - Grx1 - roGFP2 in 1205Lu and WM3734 with and without stable MCUA _ KD . Representative ratiometric images ( F385 nm / F475 nm ) are shown for all conditions ( I ) . Scale bar : 10 µm . Quantification in 1205Lu ( J ) and WM3734 ( K ) under resting state ( 1205Lu ─ shCTRL : n = 231 cells from 10 biological replicates ; shMCU _ 1 : n = 176 cells from 7 biological replicates ; shMCU _ 2 : n = 164 cells from 7 biological replicates ; WM3734 ─ shCTRL : n = 127 cells from 7 biological replicates ; shMCU _ 1 : n = 142 cells from 7 biological replicates ; shMCU _ 2 : n = 116 cells from 7 biological replicates ) . Data information : All data are presented as mean ± SEM . and were measured in Ringer ' s buffer containing 0 . 5 mM Ca2 + statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 . In all statistical analyses , KD cells were compared to their respective control .

O O O O O O B-MEASURED_VAR O O O O O O O B-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(I-K) Mitochondrial glutathione redox potential, measured with mito-Grx1-roGFP2 in 1205Lu and WM3734 with and without stable MCUA_KD. Representative ratiometric images (F385 nm/F475 nm) are shown for all conditions (I). Scale bar: 10 µm. Quantification in 1205Lu (J) and WM3734 (K) under resting state (1205Lu ─ shCTRL: n=231 cells from 10 biological replicates; shMCU_1: n=176 cells from 7 biological replicates; shMCU_2: n=164 cells from 7 biological replicates; WM3734 ─ shCTRL: n=127 cells from 7 biological replicates; shMCU_1: n=142 cells from 7 biological replicates; shMCU_2: n=116 cells from 7 biological replicates). Data information: All data are presented as mean ± SEM. and were measured in Ringer's buffer containing 0.5 mM Ca2+ statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005. In all statistical analyses, KD cells were compared to their respective control.


( G - I ) Mitochondrial ATP , measured using mito - ATEAM in 1205Lu and WM3734 with and without stable MCUA _ KD . Exemplary ratiometric images ( FRET / CFP ) are shown for all conditions ( G ) . Scale bar : 10 µm . Quantification of basal levels in 1205Lu ( H ) and WM3734 ( I ) ( 1205Lu ─ shCTRL : n = 141 cells from 14 biological replicates ; shMCU _ 1 : n = 143 cells from 12 biological replicates ; shMCU _ 2 : n = 143 cells from 13 biological replicates ; WM3734 ─ shCTRL : n = 122 cells from 12 biological replicates ; shMCU _ 1 : n = 103 cells from 12 biological replicates ; shMCU _ 2 : n = 99 cells from 13 biological replicates ) . Data information : Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(G-I) Mitochondrial ATP, measured using mito-ATEAM in 1205Lu and WM3734 with and without stable MCUA_KD. Exemplary ratiometric images (FRET/CFP) are shown for all conditions (G). Scale bar: 10 µm. Quantification of basal levels in 1205Lu (H) and WM3734 (I) (1205Lu ─ shCTRL: n=141 cells from 14 biological replicates; shMCU_1: n=143 cells from 12 biological replicates; shMCU_2: n=143 cells from 13 biological replicates; WM3734 ─ shCTRL: n=122 cells from 12 biological replicates; shMCU_1: n=103 cells from 12 biological replicates; shMCU_2: n=99 cells from 13 biological replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


( J ) Exemplary confocal microscope images of the mitochondrial network ( blue : DAPI staining of the nucleus ; green : TOMM20 staining of mitochondria ) ; scale bar : 50 µm . ( K - O ) Electron microscopy of mitochondria of stable MCUA _ KD cell lines . ( K ) Exemplary images of stable MCUA _ KD cells ' mitochondria . Scale bar : 500 nm . ( L - O ) Quantification of mitochondrial diameter ( L and N ) and intercristae distance ( M and O ) of 1205Lu ( L and M ) and WM3734 ( N and O ) cells with and without stable MCUA _ KD , presented as boxplot . The box presents the 25 % - quartile , median and 75 % - quartile , the X represents the mean and the whiskers the minimum and maximum , outliers are represented as dots ( mitochondrial diameter : 1205Lu ─ shCTRL : n = 47 technical replicates ; shMCU _ 1 : n = 44 technical replicates ; shMCU _ 2 : n = 40 technical replicates ; WM3734 ─ shCTRL : n = 35 technical replicates ; shMCU _ 1 : n = 52 technical replicates ; shMCU _ 2 : n = 38 technical replicates ; intercristace distance : 1205Lu ─ shCTRL : n = 330 technical replicates ; shMCU _ 1 : n = 285 technical replicates ; shMCU _ 2 : n = 255 technical replicates ; WM3734 ─ shCTRL : n = 352 technical replicates ; shMCU _ 1 : n = 433 technical replicates ; shMCU _ 2 : n = 357 technical replicates ) . Data information : Statistical significance was determined using unpaired , two ‐ tailed Student ' s t ‐ test , ( * ) p < 0 . 05 ; ( * * ) p < 0 . 01 ; ( * * * ) p < 0 . 005 ; no asterisk means no statistical significance ( p > 0 . 05 ) .

O O O O O O O O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(J) Exemplary confocal microscope images of the mitochondrial network (blue: DAPI staining of the nucleus; green: TOMM20 staining of mitochondria); scale bar: 50 µm. (K-O) Electron microscopy of mitochondria of stable MCUA_KD cell lines. (K) Exemplary images of stable MCUA_KD cells' mitochondria. Scale bar: 500 nm. (L-O) Quantification of mitochondrial diameter (L and N) and intercristae distance (M and O) of 1205Lu (L and M) and WM3734 (N and O) cells with and without stable MCUA_KD, presented as boxplot. The box presents the 25 %- quartile, median and 75 %-quartile, the X represents the mean and the whiskers the minimum and maximum, outliers are represented as dots (mitochondrial diameter: 1205Lu ─ shCTRL: n=47 technical replicates; shMCU_1: n=44 technical replicates; shMCU_2: n=40 technical replicates; WM3734 ─ shCTRL: n=35 technical replicates; shMCU_1: n=52 technical replicates; shMCU_2: n=38 technical replicates; intercristace distance: 1205Lu ─ shCTRL: n=330 technical replicates; shMCU_1: n=285 technical replicates; shMCU_2: n=255 technical replicates; WM3734 ─ shCTRL: n=352 technical replicates; shMCU_1: n=433 technical replicates; shMCU_2: n=357 technical replicates). Data information: Statistical significance was determined using unpaired, two‐tailed Student's t‐test, (*) p < 0.05; (**) p < 0.01; (***) p < 0.005; no asterisk means no statistical significance (p > 0.05).


E . Total RNAs from naïve CD4 + T cells , PHA - stimulated CD4 + T cells and resting memory CD4 + T cells were extracted and proceeded to RT - qPCR to quantitate the relative expression of CBX4 . The expression of CBX4 in PHA - stimulated and resting memory CD4 + T cells was normalized to naïve CD4 + T cells . Data information : Data represented mean ± SEM in biological triplicate . p - Values were calculated by one - way ANOVA with Tukey ' s multiple comparisons test . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 .

O O O O O O O O O O O B-CONTROLLED_VAR O O O 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-MEASURED_VAR O O O O B-MEASURED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

E. Total RNAs from naïve CD4+ T cells, PHA-stimulated CD4+ T cells and resting memory CD4+ T cells were extracted and proceeded to RT-qPCR to quantitate the relative expression of CBX4. The expression of CBX4 in PHA-stimulated and resting memory CD4+ T cells was normalized to naïve CD4+ T cells. Data information: Data represented mean ± SEM in biological triplicate. p-Values were calculated by one-way ANOVA with Tukey's multiple comparisons test. *p < 0.05, **p < 0.01, ***p < 0.001.


B . Super - resolution SIM image of CBX4 in HEK293T cells . DAPI was used to dye DNA which was colored into blue . FITC - tagged antibodies were used to label endogenous CBX4 which was colored into green . Data information : Scale bars in ( B ) 5 μm . All the samples were imaged to obtain at least three sets of images .

O O O O O O O O B-MEASURED_VAR O 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-MEASURED_VAR O O O O O O O O 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. Super-resolution SIM image of CBX4 in HEK293T cells. DAPI was used to dye DNA which was colored into blue. FITC-tagged antibodies were used to label endogenous CBX4 which was colored into green. Data information: Scale bars in (B) 5 μm. All the samples were imaged to obtain at least three sets of images.


C . The distribution of CBX4 bodies diameters . The black line indicated the median value of diameters which was 512 nm . Data were collected from three cells of three independent samples .

O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O

C. The distribution of CBX4 bodies diameters. The black line indicated the median value of diameters which was 512 nm. Data were collected from three cells of three independent samples.


F - L . RFP - tagged EZH2 , RING1B , SUMO4 , SUZ12 , CHAF1A , DNMT1 and HP1α were co - overexpressed with GFP - tagged CBX4 in HEK293T cells , respectively . SIM imaging was performed for each combination . The line scan profiles of SIM images which showed co - localization and non - co - localization between CBX4 and its partners were shown on the right of each panel . The red arrows in merged images indicated the positions where the line scans profiled . M - O . GFP - tagged CBX4 was overexpressed in HEK293T cells , followed by treating with AF568 - tagged antibodies against H3K4me3 , H3K9me3 and H3K27me3 , respectively . CBX4 and each histone modification were imaged with SIM . Line scan profiles were shown on the right of each panel . Red arrows in merged images indicated the positions where line scans profiled . Data information : Scale bars 5 μm . All the samples were imaged to obtain at least three sets of images .

O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O O O O O O B-MEASURED_VAR O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O 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-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O B-MEASURED_VAR O B-MEASURED_VAR O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

F-L. RFP-tagged EZH2, RING1B, SUMO4, SUZ12, CHAF1A, DNMT1 and HP1α were co-overexpressed with GFP-tagged CBX4 in HEK293T cells, respectively. SIM imaging was performed for each combination. The line scan profiles of SIM images which showed co-localization and non-co-localization between CBX4 and its partners were shown on the right of each panel. The red arrows in merged images indicated the positions where the line scans profiled. M-O. GFP-tagged CBX4 was overexpressed in HEK293T cells, followed by treating with AF568-tagged antibodies against H3K4me3, H3K9me3 and H3K27me3, respectively. CBX4 and each histone modification were imaged with SIM. Line scan profiles were shown on the right of each panel. Red arrows in merged images indicated the positions where line scans profiled. Data information: Scale bars 5 μm. All the samples were imaged to obtain at least three sets of images.