F . Time - lapse imaging of CDK2 activity ( DHB - mCherry , gray ) p21 - YPet ( green ) in etoposide - treated . Schematic shows two arrest trajectories observed following etoposide treatment . that successfully complete G2 ( " Mothers " , N = 32 ) but arrest following division ( " Daughters " , N = 45 ) are shown in two leftmost panels . that arrest in G2 ( N = 40 ) are shown in rightmost panel . solid lines represent population medians shaded indicates 95 % confidence interval .

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F. Time-lapse imaging of CDK2 activity (DHB-mCherry, gray) and p21-YPet (green) intensity in etoposide-treated cells. Schematic shows the two arrest trajectories observed following etoposide treatment. Cells that successfully complete G2 ("Mothers", N = 32 cells) but arrest following cell division ("Daughters", N = 45 cells) are shown in the two leftmost panels. Cells that arrest in G2 (N = 40 cells) are shown in the rightmost panel. The solid lines represent population medians and the shaded area indicates the 95% confidence interval.


A . Arrest architecture of replicative stress . 2C 4C arrest trajectories are shown , regions that correspond to mitotic skipping senescent state are annotated arrest trajectory of 8C polyploid is indicated . N = 4315

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A. Arrest architecture of replicative stress. 2C and 4C arrest trajectories are shown, the regions that correspond to mitotic skipping and the senescent state are annotated and the arrest trajectory of 8C polyploid cells is indicated. N = 4315 cells


B . Progression of arrest states over time following etoposide treatment . Etoposide - treated ( upper panel : colored by condition , lower panel : colored by content ) populating 2C , 4C , 8C senescent arrest states at each day of treatment ( 1 - 4 d ) .

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B. Progression of arrest states over time following etoposide treatment. Etoposide-treated cells (upper panel: colored by condition, lower panel: colored by DNA content) populating 2C, 4C, 8C and senescent arrest states at each day of treatment (1-4 d).


C . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in following etoposide treatment ( 1 μM , 0 - 4 d ) .

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C. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide treatment (1 μM, 0-4 d).


D - K . ( D ) GSK3β , ( E ) phospho ( Thr157 ) - p27 , ( F ) p27 , ( G ) CDK4 , ( H ) cyclin D1 , ( I ) cyclin E , ( J ) ( K ) : cytoplasm of etoposide - treated are plotted on map . Median values are shown in D - I . indicated with a dotted represents senescent region of map .

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D-K. (D) GSK3β, (E) phospho(Thr157)-p27, (F) p27, (G) CDK4, (H) cyclin D1, (I) cyclin E, (J) cell area and (K) DNA:cytoplasm ratio of etoposide-treated cells are plotted on the map. Median nuclear values are shown in D-I. Area indicated with a dotted line represents the senescent region of the map.


L - O . ( L ) Cyclin A , ( M ) cyclin B1 , ( N ) CDH1 ( O ) SKP2 of etoposide - treated are plotted on map . Median values are shown . indicated with a dotted shows trajectory of mitotic skipping transition into senescence .

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L-O. (L) Cyclin A, (M) cyclin B1, (N) CDH1 and (O) SKP2 of etoposide-treated cells are plotted on the map. Median nuclear values are shown. Area indicated with a dotted line shows the trajectory of mitotic skipping and transition into senescence.


A . Distribution of senescence - associated β - galactosidase ( SA - βgal ) activity in following etoposide or palbociclib treatment ( 1 μM , 4 / 8 d ) . Control : N = 6050 , etop 4d : N = 409 , etop 8d : N = 732 , palbo 4d : N = 1837 , palbo 8d : N = 1677 . Statistical significance was determined using a one - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 0001 ) .

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A. Distribution of senescence-associated β-galactosidase (SA-βgal) activity in individual cells following etoposide or palbociclib treatment (1 μM, 4/8 d). Control: N = 6050 cells, etop 4d: N = 409 cells, etop 8d: N = 732 cells, palbo 4d: N = 1837 cells, palbo 8d: N = 1677 cells. Statistical significance was determined using a one-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.0001).


B . Unified cycle map of unperturbed ( gray ) , palbociclib - ( palbo , 1 μM , 4 / 8 d , blue ) etoposide - ( etop , 1 μM , 4 / 8 d , green ) treated . Control : N = 10499 , etop : N = 2692 , palbo : N = 4931

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B. Unified cell cycle map of the unperturbed (gray), palbociclib- (palbo, 1 μM, 4/8 d, blue) and etoposide- (etop,1 μM, 4/8 d, green) treated cells. Control: N = 10499 cells, etop: N = 2692 cells, palbo: N = 4931 cells


C - K . ( C ) content , ( D ) phospho / total RB , ( E ) p16 , ( F ) TGFβ1 , ( G ) IL - 6 , ( H ) IL - 8 , ( I ) phospho - p38 , ( J ) phospho - STAT3 ( K ) phospho - STAT5 are plotted on map . Median values are shown .

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C-K. (C) DNA content, (D) phospho/total RB, (E) p16, (F) TGFβ1, (G) IL-6, (H) IL-8, (I) phospho-p38, (J) phospho-STAT3 and (K) phospho-STAT5 are plotted on the map. Median nuclear values are shown.


D . Representative single - trace demonstrating mitotic skipping endoreduplication following etoposide treatment ( 1 μM ) by time - lapse imaging . CDK2 activity ( DHB - mCherry , blue ) , cycle phase ( PCNA - mTq2 , S phase shown as gray shaded ) p21 - YPet ( green ) are plotted versus time of etoposide treatment .

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D. Representative single-cell trace demonstrating mitotic skipping and endoreduplication following etoposide treatment (1 μM) by time-lapse imaging. CDK2 activity (DHB-mCherry, blue), cell cycle phase (PCNA-mTq2, S phase shown as gray shaded area) and p21-YPet intensity (green) are plotted versus time of etoposide treatment.


E . Cyclin D1 abundance ( left ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following siRNA - mediated knockdown of cyclin D in control etoposide - treated . Boxplots show data from four independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents population .

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E. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following siRNA-mediated knockdown of cyclin D in control and etoposide-treated cells. Boxplots show data from four independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


F . Cyclin D1 abundance ( left ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following doxycycline ( dox ) - induced upregulation of cyclin D1 in control etoposide - treated . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents population .

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F. Cyclin D1 abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin D1 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


G . Cyclin A abundance ( left ) proportion of polyploid ( right ) , as measured by immunofluorescence Hoechst , respectively , following doxycycline ( dox ) - induced upregulation of cyclin A2 in control etoposide - treated . Boxplots show data from six independent replicates ( gray circles ) . Data information : Statistical significance in right panels was determined using a two - way of variance ( ANOVA ) with Sidak ' s post hoc ( * * * p < 0 . 001 , * p = 0 . 02 ) . boxes show interquartile range , whiskers indicate full distribution of points central band represents population .

G. Cyclin A abundance (left) and the proportion of polyploid cells (right), as measured by immunofluorescence and Hoechst staining, respectively, following doxycycline (dox)-induced upregulation of cyclin A2 in control and etoposide-treated cells. Boxplots show data from six independent replicates (gray circles). Data information: Statistical significance in right panels was determined using a two-way analysis of variance (ANOVA) with Sidak's post hoc test (*** p < 0.001, * p = 0.02). boxes show the interquartile range, the whiskers indicate the full distribution of points and the central band represents the population.


( E ) Representative of pilot validation screen for negative control ( no siRNA ) for five sets of siRNA - treated with reduced TGN retrieval . Different that give rise to reduced TGN retrieval ratios are discussed in text . Scale bar , 100 μm ( top two rows ) 38 μm ( bottom row ) .

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(E) Representative images of the pilot validation screen for the negative control (no siRNA) and for five sets of siRNA-treated cells with reduced TGN retrieval ratio. Different phenotypes that give rise to reduced TGN retrieval ratios are discussed in the text. Scale bar, 100 μm (top two rows) and 38 μm (bottom row).


( A ) screen anti - CD8 antibody - uptake for control ( top ) SFT2D2 KD ( bottom ) .

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(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and SFT2D2 KD cells (bottom).


( B ) stably expressing SFT2D2 - Myc show localization of SFT2D2 to perinuclear membranes endosomes , including VPS35 - positive endosomes ( arrowheads in inset ) . Treatment with nocodazole disperses endosomes even more clearly shows colocalization of SFT2D2 - Myc VPS35 ( arrowheads in inset ) .

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(B) Cells stably expressing SFT2D2-Myc show localization of SFT2D2 to perinuclear membranes and endosomes, including VPS35-positive endosomes (arrowheads in inset). Treatment with nocodazole disperses the endosomes and even more clearly shows colocalization of SFT2D2-Myc and VPS35 (arrowheads in inset).


( C ) stably expressing SFT2D2 - Myc were costained with antibodies against various post - Golgi SNARE . Colocalization was quantified by Pearson ' s correlation coefficient ( right - graph ) indicates very extensive colocalization with STX6 , STX7 , VAMP8 . image panels illustrate colocalization of SFT2D2 - Myc STX5 at Golgi ( top , arrowhead ) but much more extensive colocalization of SFT2D2 - Myc STX6 ( bottom , arrowheads ) . In ( B ) ( C ) , dashed box delineates magnified in insets .

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(C) Cells stably expressing SFT2D2-Myc were costained with antibodies against various post-Golgi SNARE proteins. Colocalization was quantified by Pearson's correlation coefficient (right-hand graph) and indicates very extensive colocalization with STX6, STX7, and VAMP8. The image panels illustrate colocalization of SFT2D2-Myc and STX5 at the Golgi (top, arrowhead) but much more extensive colocalization of SFT2D2-Myc and STX6 (bottom, arrowheads). In (B) and (C), the white dashed box delineates the area magnified in the insets.


( D ) SNARE was compared for control SFT2D2 KD HeLa quantified . graph shows change in measured for each post - Golgi SNARE investigated . illustrate increased of VAMP3 in SFT2D2 KD .

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(D) SNARE protein staining was compared for control and SFT2D2 KD HeLa cells and quantified. The graph shows the change in cellular intensity measured for each post-Golgi SNARE investigated. Images illustrate the increased cellular intensity of VAMP3 in SFT2D2 KD cells.


( E ) Control SFT2D2 KD HeLa lysates were separated by LDS - PAGE blotted for indicated SNARE or actin .

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(E) Control and SFT2D2 KD HeLa cell lysates were separated by LDS-PAGE and blotted for the indicated SNARE proteins or actin.


( A ) screen anti - CD8 antibody - uptake for control ( top ) ZDHHC5 KD ( bottom ) .

(A) Primary screen anti-CD8 antibody-uptake images for control cells (top) and ZDHHC5 KD cells (bottom).


( B ) stably expressing ZDHHC5 - Myc show localization of ZDHHC5 to plasma to intracellular tubules vesicles . Some colocalization between ZDHHC5 retromer VPS35 is observed ( arrowheads in inset ) . Following nocodazole treatment endosomes are dispersed some are labeled with ZDHHC5 VPS35 ( arrowheads in inset ) .

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(B) Cells stably expressing ZDHHC5-Myc show localization of ZDHHC5 to the plasma membrane and to intracellular tubules and vesicles. Some colocalization between ZDHHC5 and retromer VPS35 is observed (arrowheads in inset). Following nocodazole treatment endosomes are dispersed and some are labeled with ZDHHC5 and VPS35 (arrowheads in inset).


( C ) Control ZDHHC5 siRNA - treated SFT2D2 - Myc were mixed stained for ZDHHC5 , Myc , VPS35 . KD are marked by an asterisk .

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(C) Control and ZDHHC5 siRNA-treated SFT2D2-Myc cells were mixed and stained for ZDHHC5, Myc, and VPS35. KD cells are marked by an asterisk.


( D ) Control ZDHHC5 siRNA - treated HeLa were mixed stained for ZDHHC5 , α5 - integrin , VPS35 . KD are indicated with an asterisk .

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(D) Control and ZDHHC5 siRNA-treated HeLa cells were mixed and stained for ZDHHC5, α5-integrin, and VPS35. KD cells are indicated with an asterisk.


( E ) Control ( top ) ZDHHC5 KD ( bottom ) HeLa were fixed stained for TGN46 β1 - integrin .

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(E) Control (top) and ZDHHC5 KD (bottom) HeLa cells were fixed and stained for TGN46 and β1-integrin.


( A ) screen anti - CD8 antibody uptake for control ( top ) GRINA KD ( bottom ) .

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(A) Primary screen anti-CD8 antibody uptake images for control cells (top) and GRINA KD cells (bottom).


( B ) Control ( top ) GRINA KD ( bottom ) HeLa were stained for Golgi marker GM130 Golgi glycoprotein - 1 ( GLG1 ) .

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(B) Control (top) and GRINA KD (bottom) HeLa cells were stained for Golgi marker GM130 and Golgi glycoprotein-1 (GLG1).


( C ) stably expressing GFP - Rab6 were treated with GRINA siRNA ( bottom ) compared to control ( top ) upon for TGN46 VPS35 .

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(C) Cells stably expressing GFP-Rab6 were treated with GRINA siRNA (bottom) and compared to control cells (top) upon staining for TGN46 and VPS35.


( D ) Quantitation by western blotting of lysates from control GRINA - silenced HeLa GFP - Rab6 .

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(D) Quantitation by western blotting of lysates from control and GRINA-silenced HeLa cells and GFP-Rab6 cells.


( E - G ) HeLa were transiently transfected with GRINA - Myc for 24 hr before fixing . ( E F ) GRINA - Myc colocalizes with both TGN46 VPS35 . In this example , GRINA - Myc expression ( in marked by an asterisk in E ) reduced ' s TGN46 expression compared to surrounding untransfected caused enlargement of VPS35 - positive endosomes . in ( E ) magnified in ( F ) is indicated by a dashed . ( G ) GRINA - Myc expression also perturbs CIMPR localization . In this example , GRINA - Myc transfection ( in marked by an asterisk ) caused CIMPR to localize to round vesicular structures positive for GRINA - Myc , some of which appeared larger than regular endosomes , whereas TGN46 was almost absent in transfected .

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(E-G) HeLa cells were transiently transfected with GRINA-Myc for 24 hr before fixing and staining. (E and F) GRINA-Myc colocalizes with both TGN46 and VPS35. In this example, GRINA-Myc expression (in the cell marked by an asterisk in E) reduced the cell's TGN46 expression compared to surrounding untransfected cells and caused enlargement of VPS35-positive endosomes. The area in (E) magnified in (F) is indicated by a dashed line. (G) GRINA-Myc expression also perturbs CIMPR localization. In this example, GRINA-Myc transfection (in the cell marked by an asterisk) caused CIMPR to localize to round vesicular structures positive for GRINA-Myc, some of which appeared larger than regular endosomes, whereas TGN46 staining was almost absent in the transfected cell.


( A ) Control HeLa or transfected with indicated siRNAs were treated for 3 hr with cycloheximide , lysed , incubated with agarose - bound wheat germ agglutinin to capture glycosylated . Total lysates ( left ) lectin pull - down samples ( right ) were assayed by western blotting . experiment was repeated three times , representative data are shown .

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(A) Control HeLa cells or cells transfected with the indicated siRNAs were treated for 3 hr with cycloheximide, lysed, and incubated with agarose-bound wheat germ agglutinin to capture glycosylated membrane proteins. Total cell lysates (left) and lectin pull-down samples (right) were assayed by western blotting. The experiment was repeated three times, and representative data are shown.


( B - E ) Quantitative of SFT2D2 , ZDHHC5 , GRINA KD immunofluorescence using automated microscopy ( see Experimental Procedures ) . ( B ) Representative showing VPS35 , TGN46 , CIMPR . Scale bar , 50 μm . ( C ) Quantitative of CIMPR at Golgi indicates a significant increase in SFT2D2 , ZDHHC5 , or GRINA KD . ( D ) SFT2D2 , ZDHHC5 , GRINA KD increase Pearson ' s correlation coefficient for colocalization between CIMPR retromer VPS35 or SNX1 while decreasing correlation between CIMPR Golgi matrix GM130 . In some cases , identical correlation coefficients were measured in replicate experiments . ( E ) Quantitation of TGN46 GLG1 in three types of KD .

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(B-E) Quantitative analysis of SFT2D2, ZDHHC5, and GRINA KD cells immunofluorescence using automated microscopy (see Experimental Procedures). (B) Representative images showing VPS35, TGN46, and CIMPR staining. Scale bar, 50 μm. (C) Quantitative analysis of CIMPR intensity at the Golgi indicates a significant increase in SFT2D2, ZDHHC5, or GRINA KD cells. (D) SFT2D2, ZDHHC5, and GRINA KD increase the Pearson's correlation coefficient for colocalization between CIMPR and retromer proteins VPS35 or SNX1 while decreasing the correlation between CIMPR and Golgi matrix protein GM130. In some cases, identical correlation coefficients were measured in the replicate experiments. (E) Quantitation of the TGN46 and GLG1 intensity in the three types of KD cells.


D : Co - immunoprecipitation of TMBIM5 LETM1 in tandem in left 3 panels . input represents mitochondrial crudely isolated from HEK293 was used as input for co - IP , LETM1 was immunoprecipitated ( left panel , IP : LETM1 ) using a LETM1 monoclonal antibody G magnetic beads ( ProtG ) . ProtG beads alone were used as a negative control for binding , immunoprecipitates were immunoblotted ( IB ) for indicated to demonstrate interaction . 10 % of input was used for immunoblotting . Prohibitin ( PHB ) was used as a control to illustrate no nonspecific binding of inner mitochondrial complexes . middle right panel of coIPs illustrates converse experiment , in middle in TMBIM5WT right TMBIMKO , using TMBIM5 as bait ( right panel , IP : TMBIM5 ) . last two right panels show blots from BN - PAGE conducted in TMBIM5WT KO .

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D: Co-immunoprecipitation of TMBIM5 and LETM1 protein in tandem in the left 3 panels. The input represents the mitochondrial crudely isolated from HEK293 cells and was used as input for the co-IP, LETM1 was immunoprecipitated (left panel, IP: LETM1) using a LETM1 monoclonal antibody and Protein G magnetic beads (ProtG). ProtG beads alone were used as a negative control for binding, immunoprecipitates were immunoblotted (IB) for the indicated proteins to demonstrate interaction. 10% of the input was used for immunoblotting. Prohibitin (PHB) was used as a control to illustrate no nonspecific binding of inner mitochondrial membrane proteins complexes. The middle and right panel of the coIPs illustrates the converse experiment, in the middle in TMBIM5WT and right TMBIMKO, using TMBIM5 as bait (right panel, IP: TMBIM5). The last two right panels show blots from BN-PAGE conducted in TMBIM5WT and KO.


A : Western blot of TMBIM5 in control targeted HeLa HEK293 clones .

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A: Western blot analysis of TMBIM5 in control and targeted HeLa and HEK293 clones.


E : Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT TMBIM5KO1 KO2 were analyzed by immunoblotting using indicated antibodies , HSP60 TOM40 served as mitochondrial loading controls . F : Densitometric of bands in ( E ) normalized to loading control , bar graph of three experiments ( biological replicates ) , mean ± SD , One - way ANOVA with Bonferroni ' s multiple comparisons performed against TMBIM5WT * p < 0 . 05 , * * p < 0 . 008 , Two - way ANOVA with Bonferroni ' s multiple comparisons performed for OPA1 statistics against TMBIM5WT , * * * p = 0 . 0009 , * * * * p < 0 . 0001 .

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E: Isolated mitochondria from three independent replicates of HEK293 TMBIM5WT and TMBIM5KO1 and KO2 were analyzed by immunoblotting using the indicated antibodies, HSP60 and TOM40 served as mitochondrial loading controls. F: Densitometric analysis of the bands in (E) normalized to loading control, bar graph of three individual experiments (biological replicates), mean ± SD, One-way ANOVA with Bonferroni's multiple comparisons test performed against TMBIM5WT *p<0.05, **p<0.008, Two-way ANOVA with Bonferroni's multiple comparisons test performed for the OPA1 statistics against TMBIM5WT, ***p=0.0009, ****p<0.0001.


( A ) Western blot of purified reconstituted hTMBIM5 for evaluating incorporation of hTMBIM5 into proteoliposomes prepared as described in materials methods .

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(A) Western blot analysis of purified and reconstituted hTMBIM5 for evaluating the incorporation of hTMBIM5 into proteoliposomes prepared as described in materials and methods.


( C ) Acoustic startle reflex to compare hearing ability between Wt ( N = 7 ) Hpn - / - ( N = 4 ) mice .

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(C) Acoustic startle reflex test to compare hearing ability between Wt (N = 7) and Hpn-/- (N = 4) mice.


( F ) Immunoblot of phospho - Smad2 / 3 ( TGFβ pathway signaling marker ) total Smad2 / 3 in lysates from indicated tissues isolated from Wt , Hpn + / - Hpn - / - mice . GAPDH was used as loading control . histogram depicts of pSmad2 / 3 compared to Wt normalized to total Smad2 / 3 .

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(F) Immunoblot analysis of phospho-Smad2/3 (TGFβ pathway signaling marker) and total Smad2/3 in lysates from indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mice. GAPDH was used as the loading control. The histogram depicts quantification of pSmad2/3 compared to Wt normalized to total Smad2/3.


( B ) Immunoblot of Wap - Myc mammary lysates for indicated TGFβ signaling markers hepsin ( T # denotes tumors ) . GAPDH was used as loading control . Lysates were derived from Wap - Myc mammary tumors from 6 mice with 6 mice without DOX - induced hepsin

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(B) Immunoblot analysis of Wap-Myc mammary tumor lysates for the indicated TGFβ signaling markers and hepsin (T# denotes individual tumors). GAPDH was used as the loading control. Lysates were derived from Wap-Myc mammary tumors from 6 mice with and 6 mice without DOX-induced hepsin overexpression


( D ) Immunoblot of phospho - Smad2 / 3 , total Smad2 / 3 , GAPDH ( loading control ) expression in Wt tumors transplanted into either Wt or Hpn - / - recipients ( n = 10 tumors each ) .

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(D) Immunoblot analysis of phospho-Smad2/3, total Smad2/3, and GAPDH (loading control) expression in Wt tumors transplanted into either Wt or Hpn-/- recipients (n = 10 tumors each).


( E ) Western blot of phospho - Smad2 / 3 , total Smad2 / 3 , hepsin , β - tubulin ( loading control ) in MCF10A - pIND20 - HPN treated with doxycycline ( DOX ; overexpression of hepsin ) TGFβR1 inhibitor Galunisertib ( TGFβR1i , 10µM ) as indicated .

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(E) Western blot analysis of phospho-Smad2/3, total Smad2/3, hepsin, and β-tubulin (loading control) in MCF10A-pIND20-HPN cells treated with doxycycline (DOX; overexpression of hepsin) and the TGFβR1 inhibitor Galunisertib (TGFβR1i, 10µM) as indicated.


( G ) TGFβ ELISA to detect active total TGFβ in conditioned medium from MCF10A - pIND20 - HPN pL6 - TGFβ1 with ( DOX + ) or without ( DOX - ) hepsin overexpressing . TGFβ ELISA detects active TGFβ ( first 2 columns ) , but heat treatment of conditioned medium activates TGFβ , thus effectively measuring total TGFβ ( right two columns ) .

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(G) TGFβ ELISA assay to detect active and total TGFβ levels in conditioned medium from MCF10A-pIND20-HPN pL6-TGFβ1 cells with (DOX+) or without (DOX-) hepsin overexpressing. The TGFβ ELISA assay detects active TGFβ levels (first 2 columns), but heat treatment of the conditioned medium activates all TGFβ, thus effectively measuring total TGFβ (right two columns).


( H ) Silver - stained gel with samples from an in vitro protease activity with recombinant SLC hepsin . SLC was incubated with increasing concentrations of recombinant hepsin ( numbers indicate of hepsin in nM ) .

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(H) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant SLC and hepsin. SLC was incubated with increasing concentrations of recombinant hepsin (numbers indicate the concentration of hepsin in nM).


( A ) Coomassie - stained gels with lysates concentrated culture supernatants of MCF10A - pIND20 - HPN , with ( + DOX ) or without ( - DOX ) hepsin overexpression . M indicates media only control . R NR above gels indicate reducing non - reducing conditions , respectively . Numbered arrowheads indicate areas of gel that were analyzed by mass spectrometry ; corresponding differently expressed in hepsin overexpressing are listed on left . image with two lanes on right is a copy of indicated with supernatant under reducing conditions , highlighting part from which fibronectin was identified . Red boxes indicate lanes that were analyzed by mass

O O O B-SMALL_MOLECULE O B-EXP_ASSAY O O O O O O O B-CELL_LINE O O O B-GENEPROD O O O O B-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O 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-EXP_ASSAY I-EXP_ASSAY O O O O O B-GENEPROD O O O 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-GENEPROD O O O O O O O O O O O B-EXP_ASSAY

(A) Coomassie-stained protein gels with cell lysates and concentrated culture supernatants of MCF10A-pIND20-HPN cells, with (+DOX) or without (-DOX) hepsin overexpression. M indicates the media only control. R and NR above the gels indicate reducing and non-reducing conditions, respectively. Numbered arrowheads indicate areas of the gel that were analyzed by mass spectrometry; corresponding proteins differently expressed in hepsin overexpressing cells are listed on the left. The image with two lanes on the right is a copy of the indicated area with supernatant under reducing conditions, highlighting the part from which fibronectin was identified. Red boxes indicate the lanes that were analyzed by mass specrtometry


( C ) Immunoblot of fibronectin , hepsin , β - tubulin ( loading control ) in lysates from MCF10A - pIND20 - HPN with ( DOX + ) or without ( DOX - ) hepsin overexpression . graph shows of fibronectin .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) or without (DOX-) hepsin overexpression. The graph shows the quantification of fibronectin levels.


( D ) Silver - stained gel with samples from an in vitro protease activity with recombinant hepsin either full purified plasma fibronectin ( upper panel ) or 70 kDa most N - terminal fragment of fibronectin ( lower panel ) . Full - fibronectin ( 1μg ) or 70 kDa fibronectin fragment ( 1μg ) were incubated with increasing concentrations of recombinant hepsin . Arrowheads indicate full - fibronectin , 70kDa fragment , 50 kDa cleavage fragment generated by hepsin , hepsin . schematic figure shows full - fibronectin 70KDa N - terminal fragment . red arrow indicates location of putative hepsin cleavage site . RGD indicated RGD - binding domain in fibronectin .

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(D) Silver-stained protein gel with samples from an in vitro protease activity assay with recombinant hepsin and either full length purified plasma fibronectin (upper panel) or the 70 kDa most N-terminal fragment of fibronectin (lower panel). Full-length fibronectin (1μg) or the 70 kDa fibronectin fragment (1μg) were incubated with increasing concentrations of recombinant hepsin. Arrowheads indicate full-length fibronectin, the 70kDa fragment, the 50 kDa cleavage fragment generated by hepsin, and hepsin. The schematic figure shows the full-length fibronectin protein and the 70KDa N-terminal fragment. The red arrow indicates the location of the putative hepsin cleavage site. RGD indicated the RGD-binding domain in fibronectin.


( A ) Immunoblot of fibronectin in indicated tissues isolated from Wt , Hpn + / - Hpn - / - mouse ( representative of at least three repeats ) . GAPDH blot is used here as loading control .

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(A) Immunoblot analysis of fibronectin in indicated tissues isolated from Wt, Hpn+/- and Hpn-/- mouse (representative of at least three repeats). GAPDH blot is used here as the loading control.


( C ) Immunoblot of fibronectin , hepsin , β - tubulin ( loading control ) in prostate lysates of control mice or mice with prostate - specific hepsin overexpression N = 2 mice each , two prostate lobes were run separately ( a b ) .

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(C) Immunoblot analysis of fibronectin, hepsin, and β-tubulin (loading control) in prostate lysates of control mice or mice with prostate-specific hepsin overexpression N=2 mice each, the two prostate lobes were run separately (a and b).


( A ) Immunoblot of fibronectin , pSmad2 / 3 , total Smad2 / 3 , GAPDH ( loading control ) in lysates from MCF10A - pIND20 - HPN without hepsin overexpression . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 , compared to siCtrl ( N = 3 biological repeats ) . ( B ) Immunoblot of lysates ( pSmad2 / 3 , total Smad2 / 3 vinculin as loading control ) concentrated culture supernatant ( anti - V5 for TGFβ ) from MCF10A - pIND20 - HPN pL6 - TGFβ1 - V5 without hepsin overexpression . Ponceau is shown as a loading control for western blot with concentrated culture supernatant . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 , compared to siCtrl ( N = 3 biological repeats ) .

O O O B-EXP_ASSAY O B-GENEPROD O O O O O O B-GENEPROD O O O B-GENEPROD O O O O O O O B-CELL_LINE O O O B-GENEPROD O B-GENEPROD O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-GENEPROD O O B-GENEPROD O O O O O O O O O O B-GENEPROD O B-GENEPROD O O B-CELL_LINE O O O B-GENEPROD O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O B-EXP_ASSAY O O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O O O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O

(A) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells without hepsin overexpression. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats). (B) Immunoblot analysis of cell lysates (pSmad2/3, total Smad2/3 and vinculin as loading control) and concentrated cell culture supernatant (anti-V5 for TGFβ) from MCF10A-pIND20-HPN pL6-TGFβ1-V5 cells without hepsin overexpression. Ponceau staining is shown as a loading control for the western blot with concentrated culture supernatant. The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels, compared to siCtrl (N = 3 biological repeats).


( E ) Immunoblot of fibronectin , pSmad2 / 3 , total Smad2 / 3 , hepsin , GAPDH ( loading control ) in lysates from MCF10A - pIND20 - HPN with ( DOX + ) without ( DOX - ) hepsin overexpression , with or without fibronectin silencing ( siFN1 ) . graph depicts of pSmad2 / 3 normalized to total Smad2 / 3 ( N = 3 biological repeats ) .

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(E) Immunoblot analysis of fibronectin, pSmad2/3, total Smad2/3, hepsin, and GAPDH (loading control) in cell lysates from MCF10A-pIND20-HPN cells with (DOX+) and without (DOX-) hepsin overexpression, and with or without fibronectin silencing (siFN1). The graph depicts quantification of pSmad2/3 normalized to total Smad2/3 levels (N = 3 biological repeats).


( A - C ) 10X phase contrast of vero monolayers at 3 days post - inoculation . Panels shown are ( A ) mock , ( B ) NP , ( C ) OP .

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(A-C) 10X phase contrast images of vero monolayers at 3 days post-inoculation. Panels shown are (A) mock, (B) NP, and (C) OP.


( D ) Electron microscopic image of viral isolate showing extracellular spherical particles with cross sections through nucleocapsids , seen as black dots .

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(D) Electron microscopic image of the viral isolate showing extracellular spherical particles with cross sections through the nucleocapsids, seen as black dots.


A ) Two SARS - CoV - 2 passage 4 stocks ( black gray ) were quantified utilizing plaque at day two ( closed circles ) day three ( open circles ) post infection of Vero E6 Vero CCL81 .

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A) Two SARS-CoV-2 passage 4 stocks (black and gray) were quantified utilizing plaque assay at day two (closed circles) and day three (open circles) post infection of Vero E6 and Vero CCL81.


B ) Plaque on Vero E6 Vero CCL81 at day 2 day 3 post inoculation .

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B) Plaque morphology on Vero E6 and Vero CCL81 at day 2 and day 3 post inoculation.


C ) monolayers two days post infection in Vero E6 ( top ) Vero CCL81 ( bottom row ) at multiple dilutions .

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C) Cell monolayers two days post infection in Vero E6 (top) and Vero CCL81 (bottom row) at multiple dilutions.


Viral titer for SARS - CoV - 2 was quantitated by plaque on Vero E6 two days post inoculation .

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Viral titer for SARS-CoV-2 was quantitated by plaque assay on Vero E6 cells two days post inoculation.


lysates were probed by western blot with B ) anti - SARS nucleocapsid ( N ) antibody or C ) anti - SARS - CoV spike ( S ) antibody .

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Protein lysates were probed by western blot with B) anti-SARS nucleocapsid (N) antibody or C) anti- SARS-CoV spike (S) antibody.


A . NIH3T3 transfected with KV10 . 1 * EGFP did not show cilia . were transiently transfected , after 24 h serum was removed for additional 24 hours to induce ciliogenesis , finally were stained with anti - acetylated α - tubulin . While most were ciliated , those showing green were devoid of cilia . Scale bar : 10µm .

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A. NIH3T3 cells transfected with KV10.1*EGFP did not show primary cilia. Cells were transiently transfected, after 24 h serum was removed for additional 24 hours to induce ciliogenesis, and finally cells were stained with anti-acetylated α-tubulin. While most cells were ciliated, those showing green fluorescence were devoid of cilia. Scale bar: 10µm.


B . NIH3T3 transfected with KV10 . 1 ( red bars ) showed markedly less cilia than control ( empty vector , bars ) . Subconfluent cultures grown in presence of FCS were serum starved for 24 hours to induce ciliogenesis . Cilia were stained with anti acetylated α - tubulin antibody . To determine ciliary disassembly , were starved for 24 hours then incubated for 4h in FCS to induce cycle re - entry ciliary resorption .

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B. NIH3T3 cells transfected with KV10.1 (red bars) showed markedly less cilia than control cells (empty vector, white bars). Subconfluent cultures grown in the presence of FCS were serum starved for 24 hours to induce ciliogenesis. Cilia were stained with anti acetylated α-tubulin antibody. To determine ciliary disassembly, cells were starved for 24 hours and then incubated for 4h in FCS to induce cell cycle re-entry and ciliary resorption.


C . Similarly , hTERT - RPE1 transfected with KV10 . 1 also showed less cilia . Ciliogenesis ciliary disassembly were induced as in B cilia were stained using anti acetylated α - tubulin quantified as in A . Inset shows equivalent experiment using structurally related potassium channel KV10 . 2 , which did not alter frequency of expression of cilia . D . Examples of fields of view of hTERT - RPE1 transfected with KV10 . 1 , serum starved for 24 hours cilia were revealed with anti Arl13B antibody ( arrows ) . A majority of control - transfected showed cilia , while KV10 . 1 transfected did not . Scale bar : 10µm .

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C. Similarly, hTERT-RPE1 cells transfected with KV10.1 also showed less cilia. Ciliogenesis and ciliary disassembly were induced as in B and cilia were stained using anti acetylated α-tubulin and quantified as in A. The Inset shows the equivalent experiment using the structurally related potassium channel KV10.2, which did not alter the frequency of expression of cilia.D. Examples of fields of view of hTERT-RPE1 cells transfected with KV10.1, serum starved for 24 hours and cilia were revealed with anti Arl13B antibody (arrows). A majority of control-transfected cells showed cilia, while KV10.1 transfected did not. Scale bar: 10µm.


A . Representative showing hTERT - RPE1 grown in presence of serum stained with anti acetylated α - tubulin antibody . There were more ciliated after treatment with siRNA against KV10 . 1 ( KV10 . 1 KD ) , as compared to scrambled siRNA - transfected , which were devoid of cilia ( control ) . Scale bar : 10 µm . B . of effect depicted in A for number of indicated at base of columns .

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A. Representative images showing hTERT-RPE1 grown in the presence of serum and stained with anti acetylated α-tubulin antibody. There were more ciliated cells after treatment with siRNA against KV10.1 (KV10.1 KD), as compared to scrambled siRNA-transfected cells, which were devoid of cilia (control). Scale bar: 10 µm.B. Quantification of the effect depicted in A for the number of images indicated at the base of the columns.


C . Time course of ciliary disassembly . hTERT - RPE1 were serum starved for 24 hours , fixed at times indicated after serum reintroduction , stained with anti acetylated α - tubulin to reveal cilia . While in presence of scrambled siRNA abundance of cilia decreased rapidly ( black ) , it remained virtually unchanged in where KV10 . 1 was knocked down ( red ) . N = 8 - 13 . D . Time course of ciliary shortening after serum reintroduction . In same experiments as in C , of remaining cilia was measured automatically using ImageJ ( FIJI ) . While ciliary decreased with a similar time course in siRNA - treated as in controls , cilia were longer in KV10 . 1 - knockdown . A re - elongation of cilia was evident in both populations after 6 hours . Values are mean ± for a number of cilia between 62 ( control , 4 . 5 h ) 308 ( both groups , 1 . 5 h ) .

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C. Time course of ciliary disassembly. hTERT-RPE1 cells were serum starved for 24 hours, fixed at the times indicated after serum reintroduction, and stained with anti acetylated α-tubulin to reveal cilia. While in the presence of scrambled siRNA the abundance of cilia decreased rapidly (black line), it remained virtually unchanged in cells where KV10.1 was knocked down (red). N=8-13.D. Time course of ciliary shortening after serum reintroduction. In the same experiments as in C, the length of the remaining cilia was measured automatically using ImageJ (FIJI). While ciliary length decreased with a similar time course in the siRNA-treated cells as in controls, cilia were longer in KV10.1-knockdown cells. A re-elongation of cilia was evident in both populations after 6 hours. Values are mean ± SEM for a number of cilia between 62 (control, 4.5 h) and 308 (both groups, 1.5 h).


E . Blockade of permeation through K + channel altered speed of deciliation . After serum starvation , medium containing serum Astemizole ( 10 µM ) was added , were fixed stained at he indicated time points ( N = 12 ) .

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E. Blockade of permeation through the K+ channel altered the speed of deciliation. After serum starvation, medium containing serum and Astemizole (10 µM) was added, and cells were fixed and stained at he indicated time points (N=12).


A . of amount of ciliated in embryonic fibroblasts from KV10 . 1 knockout mice . Actively growing cultures , cultures after 24 h serum starvation , four hours after subsequent serum reintroduction were stained for presence of cilia using anti - acetylated α - tubulin . An increase in frequency of cilia in knockout was evident under conditions , but especially marked in ciliary resorption ( 4h after serum reintroduction ) , indicating an influence of KV10 . 1 expression on deciliation .

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A. Quantification of the amount of ciliated cells in primary embryonic fibroblasts from KV10.1 knockout mice. Actively growing cultures, cultures after 24 h serum starvation, and four hours after subsequent serum reintroduction were stained for the presence of cilia using anti-acetylated α-tubulin. An increase in frequency of cilia in the knockout cells was evident under all conditions, but especially marked in the ciliary resorption test (4h after serum reintroduction), indicating an influence of KV10.1 expression on deciliation.


B . Exogenous expression of human KV10 . 1 reverted phenotype in MEFs . obtained from three were transfected with KV10 . 1 or empty vector ( mock ) , serum starved for 48 h cilia were revealed by immunocytochemistry using anti - acetylated α - tubulin in indicated number of . KV10 . 1 expression decreased frequency of ciliated .

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B. Exogenous expression of human KV10.1 reverted the phenotype in MEFs. Cells obtained from three animals were transfected with KV10.1 or empty vector (mock), serum starved for 48 h and cilia were revealed by immunocytochemistry using anti-acetylated α-tubulin in the indicated number of images. KV10.1 expression decreased the frequency of ciliated cells.


C . MEFs from knockout mice did not show cilia when transfected with KV10 . 1 . were co - transfected with human KV10 . 1 mVenus , serum starved for 48 hours then fixed stained for cilia ( AcTub ) . Venus - positive did not show cilia . Scale bar : 25 µm .

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C. MEFs from knockout mice did not show primary cilia when transfected with KV10.1. Cells were co-transfected with human KV10.1 and mVenus, serum starved for 48 hours and then fixed and stained for cilia (AcTub). Venus-positive cells did not show cilia. Scale bar: 25 µm.


D . In wild type MEFs , transfection with KV10 . 1 reduced presence of cilia . When a gain - of - function mutant ( L352V ) that originates developmental defects in human was transfected , frequency of with cilia was further reduced .

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D. In wild type MEFs, transfection with KV10.1 reduced the presence of primary cilia. When a gain-of-function mutant (L352V) that originates developmental defects in human patients was transfected, the frequency of cells with cilia was further reduced.


E . Sensitivity to SHH signaling was significantly increased in fibroblasts from KV10 . 1 knockout mice , in agreement with higher abundance of cilia . were serum starved SHH was stimulated after 24 hours by addition of culture supernatant of SHH - expressing HEK293 . Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT - PCR .

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E. Sensitivity to SHH signaling was significantly increased in primary fibroblasts from KV10.1 knockout mice, in agreement with the higher abundance of primary cilia. Cells were serum starved and SHH was stimulated after 24 hours by addition of culture supernatant of SHH-expressing HEK293 cells. Expression of mRNA for Gli1 was used as reporter of SHH activation by qRT-PCR.


A . Actively proliferating hTERT - RPE1 were transfected with scrambled or KV10 . 1 siRNA , cycle distribution was determined using acridine orange . Knockdown of KV10 . 1 induces an increase in with / content compatible with G0 ( rectangle ) . However , quantitative increase ( to approximately 10 % ) was insufficient to explain abundance of ciliated ( over 40 % )

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A. Actively proliferating hTERT-RPE1 cells were transfected with scrambled or KV10.1 siRNA, and cell cycle distribution was determined using acridine orange staining. Knockdown of KV10.1 induces an increase in cells with DNA/RNA content compatible with G0 (rectangle). However, the quantitative increase (to approximately 10%) was insufficient to explain the abundance of ciliated cells (over 40%)


B . proliferation marker Ki67 frequently coexists with cilia in KV10 . 1 knockdown . Exponentially growing were stained for acetylated α - tubulin ( red , AvTub ) Ki67 ( green ) . Nuclei were counterstained with Draq5 ( blue ) . Scale bar : 10 µm . C . of effects observed in B . Although positivity for Ki67 ( proliferating ) presence of cilia were not always excluding each other even in transfected with control siRNA , KV10 . 1 knockdown increased frequency of ciliated , while it reduced amount of Ki67 positive , actively proliferating . Of those presenting a cilium , fraction of simultaneously Ki - 67 - positive was much larger upon KV10 . 1 knockdown . Scale bar , 10 µm .

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B. The proliferation marker Ki67 frequently coexists with primary cilia in KV10.1 knockdown cells. Exponentially growing cells were stained for acetylated α-tubulin (red, AvTub) and Ki67 (green). Nuclei were counterstained with Draq5 (blue). Scale bar: 10 µm.C. Quantification of the effects observed in B. Although the positivity for Ki67 (proliferating cells) and the presence of cilia were not always excluding each other even in cells transfected with control siRNA, KV10.1 knockdown increased the frequency of ciliated cells, while it reduced the amount of Ki67 positive, actively proliferating cells. Of those cells presenting a cilium, the fraction of simultaneously Ki-67-positive cells was much larger upon KV10.1 knockdown. Scale bar, 10 µm.


D . cilia were present in actively synthesizing ( measured by EdU incorporation ) . were transfected with siRNA against KV10 . 1 , 24 hours later were incubated in presence of EdU for 4h before fixation . KV10 . 1 knockdown resulted in presence of abundant EdU - positive simultaneously ciliated , suggesting a decoupling between deciliation cycle progression . Scale bar :

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D. Primary cilia were present in cells actively synthesizing DNA (measured by EdU incorporation). Cells were transfected with siRNA against KV10.1, and 24 hours later were incubated in the presence of EdU for 4h before fixation. KV10.1 knockdown resulted in the presence of abundant EdU-positive and simultaneously ciliated cells, suggesting a decoupling between deciliation and cell cycle progression. Scale bar: 10µm


A . An anti - KV10 . 1 antibody directed against an extracellular epitope labels a structure related to centrosome ( pericentrin - positive ) positive for Arl13b with an appearance suggestive of cilium in HFF ( human foreskin fibroblast ) . were serum starved for 24 or 48h , fixed , blocked ( 10 % BSA ) then stained without permeabilization with anti - KV10 . 1 antibody recognizing an extracellular epitope . Immunostaining for pericentrin was performed after subsequent permeabilization additional block . Scale bars : 10 µm ( pericentrin ) , 20 µm ( Arl13b ) .

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A. An anti-KV10.1 antibody directed against an extracellular epitope labels a structure related to the centrosome (pericentrin-positive) and positive for Arl13b with an appearance suggestive of the primary cilium in HFF (human foreskin fibroblast) cells. Cells were serum starved for 24 or 48h, fixed, blocked (10% BSA) and then stained without permeabilization with anti-KV10.1 antibody recognizing an extracellular epitope. Immunostaining for pericentrin was performed after subsequent permeabilization and additional block. Scale bars: 10 µm (pericentrin), 20 µm (Arl13b).


B . Anti - KV10 . 1 antibodies recognize ciliary structures in hTERT - RPE1 . After serum starvation for 48h , serum was reintroduced for 4h to induce KV10 . 1 expression ciliary resorption . were then fixed stained with a mixture of anti - KV10 . 1 mouse monoclonals a rabbit monoclonal anti acetylated α - tubulin . Scale bar : 2 µm .

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B. Anti-KV10.1 antibodies recognize ciliary structures in hTERT-RPE1 cells. After serum starvation for 48h, serum was reintroduced for 4h to induce KV10.1 expression and ciliary resorption. Cells were then fixed and stained with a mixture of anti-KV10.1 mouse monoclonals and a rabbit monoclonal anti acetylated α-tubulin. Scale bar: 2 µm.


C . Specificity of monoclonal anti KV10 . 1 ( mAb 62 ) as revealed by MEFs from wild type ( left ) or knockout ( right ) Scale bar : 50 µm :

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C. Specificity of the monoclonal anti KV10.1 (mAb 62) as revealed by staining MEFs from wild type (left) or knockout animals (right) Scale bar: 50 µm:


D . from cilia of MEFs from KV10 . 1 knockout mice obtained using mAb62 a rabbit polyclonal anti acetylated a - tubulin antibody . No recognizable structures were stained by mAb62 . Scale bar : 1 µm . E . In contrast , mAb62 did stain ciliary - related structures ( acetylated - tubulin - positive ) in wild type mice . Scale bar : 1 µm .

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D. Images from primary cilia of MEFs from KV10.1 knockout mice obtained using mAb62 and a rabbit polyclonal anti acetylated a-tubulin antibody. No recognizable structures were stained by mAb62. Scale bar: 1 µm.E. In contrast, mAb62 did stain ciliary-related structures (acetylated-tubulin-positive) in wild type mice. Scale bar: 1 µm.


A . KV10 . 1 localizes to pericentrin - positive structures during ciliary resorption . After short ( 4h ) reintroduction of FCS in serum starved hTERT RPE1 , signal of anti - KV10 . 1 antibody co - localized with pericentrin . Scale bar : 25µm .

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A. KV10.1 localizes to pericentrin-positive structures during ciliary resorption. After short (4h) reintroduction of FCS in serum starved hTERT RPE1 cells, the signal of anti-KV10.1 antibody co-localized with pericentrin. Scale bar: 25µm.


B . expressing a KV10 . 1 * mVenus fusion under control of endogenous promoter to render a mild overexpression were fixed immunostained for pericentrin . Venus colocalized with pericentrin - positive structures . Scale bar : 5µm .

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B. Cells expressing a KV10.1*mVenus fusion under the control of the endogenous promoter to render a mild overexpression were fixed and immunostained for pericentrin. Venus fluorescence colocalized with pericentrin-positive structures. Scale bar: 5µm.


A . Density centrifugation of hTERT - RPE1 extracts followed by immunoblot revealed presence of KV10 . 1 in same fractions as Aurora A , phospho - aurora A a - tubulin ( centrosomal fractions ) .

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A. Density centrifugation of hTERT-RPE1 cell extracts followed by immunoblot revealed the presence of KV10.1 in the same fractions as Aurora A, phospho-aurora A and a-tubulin (centrosomal fractions).


B . KV10 . 1 is also present in microsomal fractions . fractions obtained as in A were ultracentrifuged to sediment membranes . In those preparations , immunoprecipitation revealed presence of full - KV10 . 1 in both centrosomal microsomal fractions ( obtained by ultracentrifugation ) .

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B. KV10.1 is also present in microsomal fractions. Cytoplasmic fractions obtained as in A were ultracentrifuged to sediment membranes. In those preparations, immunoprecipitation revealed the presence of full-length KV10.1 in both the centrosomal and the microsomal fractions (obtained by ultracentrifugation).


B . Deletion of CLS does not preclude functional expression at plasma . Two electrode - voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10 . 1 channel lacking CLS at C - terminus revealed voltage - gated currents after stimulation using potential depicted in inset . C . Conductance / voltage relationship of deleted channel was similar to that of wild - type ( shown for comparison as a solid ) .

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B. Deletion of the CLS does not preclude functional expression at the plasma membrane. Two electrode-voltage clamp recordings from Xenopus oocytes injected with cRNA for a KV10.1 channel lacking the CLS at the C-terminus revealed voltage-gated currents after stimulation using the potential depicted in the inset.C. The Conductance/voltage relationship of the deleted channel was similar to that of wild-type (shown for comparison as a solid line).


D . In absence of serum , C - terminus of KV10 . 1 reduced number of cilia ( maintained deciliated ) also after deletion of CLS ( data for controls is reproduced from Figure 5A ) CLS is required to induce deciliation . When hTERT - RPE1 3T3 transfected with KV10 . 1 or KV10 . 1ΔCLS were serum starved , serum was reintroduced after 24 hours for 4 h , KV10 . 1ΔCLS failed to reduce fraction of ciliated .

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D. In the absence of serum, the C-terminus of KV10.1 reduced the number of cilia (maintained cells deciliated) also after deletion of the CLS (data for controls is reproduced from Figure 5A)The CLS is required to induce deciliation. When hTERT-RPE1 and 3T3 cells transfected with KV10.1 or KV10.1ΔCLS were serum starved, and serum was reintroduced after 24 hours for 4 h, KV10.1ΔCLS failed to reduce the fraction of ciliated cells.


E . CLS is required for KV10 . 1 - induced tumorigenesis . CHO were transfected with indicated constructs implanted into flank of nude mice . KV10 . 1ΔCLS transfected did not induce larger tumors than control .

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E. The CLS is required for KV10.1-induced tumorigenesis. CHO cells were transfected with the indicated constructs and implanted into the flank of nude mice. KV10.1ΔCLS transfected cells did not induce larger tumors than the control.


A . Overexpression of cortactin compensated for knockdown of KV10 . 1 in ciliary disassembly ( 4 h serum reintroduction after starvation ) .

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A. Overexpression of cortactin compensated for the knockdown of KV10.1 in ciliary disassembly (4 h serum reintroduction after starvation).


B . Deletion of domain of KV10 . 1 responsible for interaction with cortactin abolished effects of KV10 . 1 overexpression on abundance of cilia .

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B. Deletion of the domain of KV10.1 responsible for interaction with cortactin abolished the effects of KV10.1 overexpression on the abundance of cilia.


C . In mouse embryonic fibroblasts , active ( phosphorylated ) cortactin is increased as compared to wild type , possibly compensating lack of KV10 . 1 .

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C. In mouse embryonic fibroblasts, active (phosphorylated) cortactin is increased as compared to wild type, possibly compensating the lack of KV10.1.


( A ) Scheme of actin filaments polymerizing on functionalized glass surfaces from profilin - actin in solution . ( B ) Time - lapse TIRF microscopy of single filament growth from Alexa488phalloidin - stabilized seeds ( green ) in presence of 5μM profilin - actin .

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(A) Scheme of individual actin filaments polymerizing on functionalized glass surfaces from profilin-actin in solution. (B) Time-lapse TIRF microscopy of single filament growth from Alexa488phalloidin-stabilized seeds (green) in the presence of 5μM profilin-actin.


( C ) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF - lipid - coated microspheres with profilin - actin , CP Arp2 / 3 in solution . ( D ) Widefield epifluorescence of indicated network components of bead - attached dendritic networks after kinetic arrest at indicated times .

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(C) Scheme of branched actin filaments polymerizing within dendritic actin networks assembled from NPF- and lipid-coated microspheres with profilin-actin, CP and Arp2/3 in solution. (D) Widefield epifluorescence images of indicated network components of bead-attached dendritic networks after kinetic arrest at indicated times.


Biochemical conditions are matching ( B ) with exception of additional presence of 200nM CP 50nM Arp2 / 3 ( E ) Scheme of dendritic network growth from square WAVE1 - micropatterns on a 2 - dimensional coverslip with profilin - actin , CP Arp2 / 3 in solution . ( F ) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth . Conditions are as in ( D )

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Biochemical conditions are matching (B) with the exception of the additional presence of 200nM CP and 50nM Arp2/3 (E) Scheme of dendritic network growth from square WAVE1-micropatterns on a 2-dimensional coverslip with profilin-actin, CP and Arp2/3 in solution. (F) Reconstructed axial view for indicated network components from confocal imaging of dendritic network growth. Conditions are as in (D)


( B ) TIRF microscopy of actin monomers ( blue , 1uM total , 30 % Cy5 labeled ) in presence of equimolar profilin recruited to a WAVE1 pattern ( magenta ) .

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(B) TIRF microscopy of actin monomers (blue, 1uM total, 30% Cy5 labeled) in the presence of equimolar profilin recruited to a WAVE1 pattern (magenta).


( C ) TIRF microscopy of actin polymerization in - ( blue arrows ) outside ( red arrows ) of WAVE1 - micropatterns ( magenta ) in presence of 1uM profilin - actin visualized by a filament - binding probe ( 5nM Alexa488 - UTRNN ) .

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(C) TIRF microscopy of actin polymerization in- (blue arrows) and outside (red arrows) of WAVE1-micropatterns (magenta) in the presence of 1uM profilin-actin visualized by a filament-binding probe (5nM Alexa488-UTRNN).


( D ) Kymograph of a growing filament barbed end entering a WAVE1 micropattern . Conditions as in ( C )

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(D) Kymograph of a growing filament barbed end entering a WAVE1 micropattern. Conditions as in (C)


( G ) Surface mCherry - NPF corresponding kymographs of barbed end growth for indicated WAVE1 , N - WASP or mCherry control constructs in 1uM profilin - actin .

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(G) Surface mCherry-NPF fluorescence and corresponding kymographs of barbed end growth for indicated WAVE1, N-WASP or dark mCherry control constructs in 1uM profilin-actin.


( B ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) representative kymographs ( lower panel ) from multi - color TIRF imaging of actin polymerization on WAVE1 - coated coverslips . Conditions are 1uM actin , 5nM Alexa488 - UTRNN .

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(B) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of actin polymerization on WAVE1-coated coverslips. Conditions are 1uM actin, 5nM Alexa488-UTRNN.


( E ) TIRF microscopy of actin polymerization from 2uM actin in absence ( upper panel ) or presence of near - saturating amounts of WAVE1 WCA .

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(E) TIRF microscopy of actin polymerization from 2uM actin in the absence (upper panel) or presence of near-saturating amounts of WAVE1 WCA.


( F ) Barbed end polymerization rates as a function of actin WAVE1 - actin ratios . red is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on - rates as indicated ( see Methods ) .

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(F) Barbed end polymerization rates as a function of actin and WAVE1-actin ratios. The red line is a fit to a minimal kinetic model assuming two polymerization competent actin species that associate to filament ends with different on-rates as indicated (see Methods).


( B ) Widefield epifluorescence ( Alexa488 - actin ) of bead - attached dendritic networks following kinetic arrest after 10 min of network growth for spike - in experiments ( i . e . 60 % his10 - darkCherry - PWCA , 40 % his10 - Cherry - W ; molar ) using indicated WAVE1 variants . Conditions are 5μM profilin - actin , 200nM CP 50nM Arp2 / 3 . For conditions , actin network assembly was quenched by adding 5M excess Latrunculin B phalloidin 10 minutes after initiating reaction .

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(B) Widefield epifluorescence images (Alexa488-actin) of bead-attached dendritic networks following kinetic arrest after 10 min of network growth for spike-in experiments (i.e. 60% his10-darkCherry-PWCA, 40% his10-Cherry-W; molar percentage) using indicated WAVE1 variants. Conditions are 5μM profilin-actin, 200nM CP and 50nM Arp2/3. For all conditions, actin network assembly was quenched by adding 5M excess Latrunculin B and phalloidin 10 minutes after initiating the reaction.


( C ) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control ( N = 57 - 73 beads per condition ) . Errors bars represent SD .

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(C) Average network growth rates for indicated WAVE1 variants or mCherry as a negative control (N= 57-73 beads per condition). Errors bars represent SD.


( D ) Scheme of domain architecture of surface - immobilized WAVE1 variants ( top panel ) , time averages ( middle panels , 7 min total ) representative kymographs ( lower panel ) from multi - color TIRF imaging of polymerization from profilin - actin on WAVE1 - coated coverslips . Conditions are 2µM actin , 3µM profilin , 5nM Alexa488 - UTRNN .

O O O O O O O O O O O B-GENEPROD O O O O O O O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O B-EXP_ASSAY O O O O B-GENEPROD O B-GENEPROD O B-GENEPROD O O O O O O O B-GENEPROD O O B-GENEPROD O O B-SMALL_MOLECULE O B-GENEPROD O

(D) Scheme of domain architecture of surface-immobilized WAVE1 variants (top panel), time averages (middle panels, 7 min total) and representative kymographs (lower panel) from multi-color TIRF imaging of polymerization from profilin-actin on WAVE1-coated coverslips. Conditions are 2µM actin, 3µM profilin, 5nM Alexa488-UTRNN.


( E ) Average barbed end polymerization rates for indicated WAVE1 variants . Errors bars represent SD .

O O O O O O B-EXP_ASSAY I-EXP_ASSAY O O B-GENEPROD O O O O O O O

(E) Average barbed end polymerization rates for indicated WAVE1 variants. Errors bars represent SD.


( A ) weighted mass average of WAVE1 PRD ( 15 μM ) determined by sedimentation equilibrium ultracentrifugation with LatB - stabilized monomers ( 15 μM ) various profilin concentrations as indicated . lower dashed indicates expected mass of WAVE1 PRD ; 5 - 7 kDa upshift is due to non - specific actin binding ( WAVE1 PRD [ null ] , black symbols ) . upper dashed mark equilibrium mass observed for 15 μM WAVE1 PRD [ B ] in presence of 90 μM profilin alone .

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(A) The weighted mass average of WAVE1 PRD (15 μM) determined by sedimentation equilibrium ultracentrifugation with LatB-stabilized monomers (15 μM) and various profilin concentrations as indicated. The lower dashed line indicates the expected mass of WAVE1 PRD; the 5-7 kDa upshift is due to non-specific actin binding (WAVE1 PRD[null], black symbols). The upper dashed line mark the equilibrium mass observed for 15 μM WAVE1 PRD[B] in the presence of 90 μM profilin alone.


( C ) FRET efficiencies for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P _ CA = grey ) as a function of LatB - stabilized , quencher - ( Atto540Q - ) labeled actin monomers . Lines are fits to a single site binding curve .

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(C) FRET efficiencies for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P_CA=grey) as a function of LatB-stabilized, quencher- (Atto540Q-) labeled actin monomers concentration. Lines are fits to a single site binding curve.


( D ) WH2 occupancies ( left axis , Methods ) FRET efficiencies ( right axis ) for donor - ( Alexa488 - ) labeled WAVE1 variants ( c = 7 . 5nM , PWCA = blue , WCA = red , P * WCA ( null ) = cyan , P _ CA = grey ) as a function of profilin . of LatB - stabilized , Atto540Q - actin monomers was constant ( 175nM ) at conditions . Lines are fits to a competitive inhibition model ( see Methods ) .

O O O O O O O O O O O B-EXP_ASSAY O O O O O O O O O B-SMALL_MOLECULE O O O B-GENEPROD O O O O O O O O O O O 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-GENEPROD O O B-SMALL_MOLECULE O O O B-SMALL_MOLECULE O B-GENEPROD O O O O O O O O O O O O O O O O O O O O O O

(D) WH2 occupancies (left axis, Methods) and FRET efficiencies (right axis) for donor- (Alexa488-) labeled WAVE1 variants (c=7.5nM, PWCA=blue, WCA=red, P*WCA(null)=cyan, P_CA=grey) as a function of profilin concentration. Concentration of LatB-stabilized, Atto540Q-actin monomers was constant (175nM) at all conditions. Lines are fits to a competitive inhibition model (see Methods).


UMAP plot revealing type annotations of three samples . B ) composition of three healthy kidney samples .

UMAP plot revealing the cell type annotations of three samples. B) The cellular composition of three healthy kidney samples.


Dotplot exhibition of canonical markers used for type classification .

Dotplot exhibition of canonical markers used for cell type classification.


gene expression patterns of ACE2 of detected types in kidney .

The gene expression patterns of ACE2 of all detected cell types in kidney.


type annotations visualized by UMAP . B ) sample specific UMAP visualization of two samples .

cell type annotations visualized by UMAP. B) The sample specific UMAP visualization of two samples.


of canonical markers applied for type assignment .

Heatmap of canonical markers applied for cell type assignment.


Violin plot displaying expression signals of kidney types .

Violin plot displaying the expression signals of kidney cell types.


two - dimensional visualization of bladder atlas by UMAP .

two-dimensional visualization of bladder cell atlas by UMAP.


Expression pattern of canonical markers used for identifying bladder types .

Expression pattern of canonical markers used for identifying bladder cell types.


ACE2 expression across 12 subgroups in bladder dataset .

ACE2 expression across 12 cell subgroups in the bladder dataset.


( a , b ) Changes in HEK293T ( a ) mitochondrial ( b ) [ Ca2 + ] in response to ionomycin ( 2 . 5 μM ) were simultaneously measured by fluo - 4 rhod - 2 imaging , respectively . Each bar represents one target gene silenced with pooled siRNA .

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(a,b) Changes in HEK293T cell cytoplasmic (a) and mitochondrial (b) [Ca2+] in response to ionomycin (2.5 μM) were simultaneously measured by fluo-4 and rhod-2 imaging, respectively. Each bar represents one target gene silenced with pooled siRNA.


( c ) qrtPCR of MCU , MCUR1 MICU1 mRNA from mouse tissues ( n = 3 ; mean ± s . e . m . ) .

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(c) qrtPCR of MCU, MCUR1 and MICU1 mRNA from mouse tissues (n = 3; mean ± s.e.m.).


( d ) qrtPCR of MCUR1 mRNA from HEK293T clones ( n = 3 ; mean ± s . e . m . ) . KD , knockdown . Neg shRNA , negative shRNA .

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(d) qrtPCR of MCUR1 mRNA from HEK293T cell clones (n = 3; mean ± s.e.m.). KD, knockdown. Neg shRNA, negative shRNA.


( e ) qrtPCR of MCUR1 mRNA from HeLa clones of rescued MCUR1 mRNA in shHe2 clone ( n = 3 ; mean ± s . e . m . ) . same lentiviral shRNAs were used to generate shHK4 shHe1 shHK5 shHe2 , respectively .

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(e) qrtPCR of MCUR1 mRNA from HeLa cell clones and of rescued MCUR1 mRNA levels in shHe2 clone cells (n = 3; mean ± s.e.m.). The same lentiviral shRNAs were used to generate shHK4 and shHe1 and shHK5 and shHe2, respectively.


( f ) MCUR1 expression ( top ) densitometric ( bottom left ) ( n = 3 ; ± s . e . m . ) . Bottom right , Flag - tagged MCUR1 expression in clone shHe2 reconstituted with shRNA - resistant MCUR1 cDNA plasmid . WB , western blot .

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(f) MCUR1 protein expression levels (top) and densitometric analysis (bottom left) (n = 3; ± s.e.m.). Bottom right, Flag-tagged MCUR1 protein expression in clone shHe2 cells reconstituted with shRNA-resistant MCUR1 cDNA plasmid. WB, western blot.


( g , h ) Representative from movies of HEK293T negative shRNA or shHK5 showing ( green ) mitochondrial ( red ) [ Ca2 + ] before , during after ionomycin exposure . Scale bar , 20 μm .

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(g,h) Representative images from movies of HEK293T negative shRNA or shHK5 cells showing cytosolic (green) and mitochondrial (red) [Ca2+] before, during and after ionomycin exposure. Scale bar, 20 μm.


( i - p ) ( green ) mitochondrial matrix ( red ) [ Ca2 + ] responses in HEK293T ( i - l ) challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( i ) Wild - type HEK293T . ( j ) expressing negative shRNA . ( k ) Clone shHK5 ( n = 4 ) . ( l ) of peak rhod - 2 . * * P0 . 01 ( mean ± s . e . m . ) .

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(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HEK293T (i-l) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (i) Wild-type HEK293T cells. (j) Cells expressing negative shRNA. (k) Clone shHK5 (n = 4). (l) Quantification of peak rhod-2 fluorescence. **P0.01 (mean±s.e.m.).


( i - p ) ( green ) mitochondrial matrix ( red ) [ Ca2 + ] responses in HeLa ( m - p ) challenged with ionomycin or histamine ( 100 μM ) , respectively . ( n = 3 ) . ( m ) HeLa expressing negative shRNA . ( n ) Clone shHe2 . ( o ) Clone shHe2 re - expressing MCUR1 ( n = 3 ) . ( p ) of peak rhod - 2 . * P0 . 05 , * * P0 . 01 ( mean ± s . e . m . ) .

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(i-p) Cytoplasmic (green) and mitochondrial matrix (red) [Ca2+] responses in HeLa (m-p) cells challenged with ionomycin or histamine (100 μM), respectively. (n = 3). (m) HeLa cells expressing negative shRNA. (n) Clone shHe2. (o) Clone shHe2 re-expressing MCUR1 (n = 3). (p) Quantification of peak rhod-2 fluorescence. *P0.05, **P0.01 (mean±s.e.m.).


( q ) [ Ca2 + ] c [ Ca2 + ] m signals evoked by ATP ( 100 μM ) thapsigargin ( Tg , 2 μM ) were monitored simultaneously using fura - 2 / AM mitopericam ( ipcam490 ) , respectively in control ( left ) MCUR1 - knockdown ( right ) HeLa . [ Ca2 + ] c is calibrated in nanomolar concentrations ( black ) , whereas mitopericam is inversely normalized to baseline ( F0 / F ; red ) .

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(q) [Ca2+]c and [Ca2+]m signals evoked by ATP (100 μM) and thapsigargin (Tg, 2 μM) were monitored simultaneously using fura-2/AM and mitopericam (ipcam490), respectively in control (left) and MCUR1-knockdown (right) HeLa cells. [Ca2+]c is calibrated in nanomolar concentrations (black), whereas mitopericam fluorescence is inversely normalized to baseline (F0/F; red).


( r ) Summary mean [ Ca2 + ] c [ Ca2 + ] m peaks during ATP stimulation ( negative shRNA n = 29 ; MCUR1 knockdown n = 36 ) . * P0 . 05 ( mean ± s . e . m . ) .

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(r) Summary mean [Ca2+]c and [Ca2+]m peaks during ATP stimulation (negative shRNA n = 29; MCUR1 knockdown n = 36 cells). *P0.05 (mean±s.e.m.).


( s ) Increase in bath [ Ca2 + ] ( Rfura2 ) [ Ca2 + ] m ( Ripcam ) signals in response to CaCl2 ( 1 μM ) InsP3 ( IP3 , 7 . 5 μM ) addition in permeabilized . Uncropped of blots / gels are shown in Supplementary Fig . S6 .

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(s) Increase in bath [Ca2+](Rfura2) and [Ca2+]m (Ripcam) signals in response to CaCl2 (1 μM) and InsP3 (IP3, 7.5 μM) addition in permeabilized cells. Uncropped images of blots/gels are shown in Supplementary Fig. S6.


Digitonin - permeabilized HeLa bathed in intracellular - like solution containing thapsigargin ( Tg ) were loaded with ΔΨm indicator JC - 1 Ca2 + indicator Fura2FF , to which pulses of 10 μM Ca2 + were added before addition of mitochondrial uncoupler CCCP ( carbonyl cyanide m - chloro phenyl hydrazone ) . ( a - e ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] ΔΨm in expressing negative shRNA ( Neg shRNA ; a ) , clone shHe1 ( b ) , clone shHe2 ( c ) , clone shHe2 re - expressing MCUR1 ( d ) HeLa stably overexpressing MCUR1 ( e ) . Under similar conditions , 1 μM Ru360 was added before 10 μM Ca2 + pulses until addition of CCCP .

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Digitonin-permeabilized HeLa cells bathed in intracellular-like solution containing thapsigargin (Tg) were loaded with the ΔΨm indicator JC-1 and the Ca2+ indicator Fura2FF, to which pulses of 10 μM Ca2+ were added before the addition of the mitochondrial uncoupler CCCP (carbonyl cyanide m-chloro phenyl hydrazone). (a-e) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in cells expressing negative shRNA (Neg shRNA; a), clone shHe1 (b), clone shHe2 (c), clone shHe2 re-expressing MCUR1 (d) and HeLa cells stably overexpressing MCUR1 (e). Under similar conditions, 1 μM Ru360 was added before 10 μM Ca2+ pulses until the addition of CCCP.


( f - i ) Representative traces from three independent experiments depict simultaneous changes of bath [ Ca2 + ] ΔΨm in negative shRNA ( f ) , MCUR1 - knockdown clone shHe1 ( g ) shHe2 ( h ) , in control overexpressing MCUR1 ( i ) .

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(f-i) Representative traces from three independent experiments depict simultaneous changes of bath [Ca2+] and ΔΨm in negative shRNA cells (f), MCUR1-knockdown clone shHe1 (g) and shHe2 (h), and in control cells overexpressing MCUR1 (i).


( j , k ) Negative shRNA ( j ) MCUR1 - overexpressing ( k ) HEK293T were permeabilized with digitonin in intracellular - like medium containing thapsigargin bath [ Ca2 + ] indicator Fura2FF , then pulsed with 10 μM Ca2 + . After mitochondrial clearance of bath Ca2 + , Ru360 caused an elevation of bath [ Ca2 + ] , indicating that steady - state bath [ Ca2 + ] after was maintained by a balance of MCU - mediated Ca2 + uptake CGP37157 ( 10 μM ) - sensitive Na + - Ca2 + exchanger - mediated extrusion . CCCP was added as indicated . solid is mean ; shaded areas are ± s . e . m . ( n = 3 ) .

O O O O O O O O O O B-GENEPROD O O O O O B-CELL_LINE O O O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-EXP_ASSAY O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O B-SUBCELLULAR O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O B-GENEPROD O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE O O O O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O B-SMALL_MOLECULE O O O 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,k) Negative shRNA (j) and MCUR1-overexpressing (k) HEK293T cells were permeabilized with digitonin in intracellular-like medium containing thapsigargin and bath [Ca2+] indicator Fura2FF, and then pulsed with 10 μM Ca2+. After mitochondrial clearance of bath Ca2+, Ru360 caused an elevation of bath [Ca2+], indicating that steady-state bath [Ca2+] after the pulse was maintained by a balance of MCU-mediated Ca2+ uptake and CGP37157 (10 μM)-sensitive Na+-Ca2+ exchanger-mediated extrusion. CCCP was added as indicated. The solid line is the mean; shaded areas are ±s.e.m. (n = 3).


( l ) [ Ca2 + ] m efflux rate derived from j k during initial 60 s following Ru360 addition ( NS ; not significant ; n = 3 ) .

O O O O B-SMALL_MOLECULE I-SMALL_MOLECULE O O O O O O O O O O O O O B-SMALL_MOLECULE O O O O O O O O O O O O

(l) [Ca2+]m efflux rate derived from j and k during the initial 60 s following Ru360 addition (NS; not significant; n = 3).


O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 , b ) Confocal of HeLa transiently co - transfected for 48 h with GFP - tagged MCUR1 DsRed - Mito plasmids ( a ) or mRFP - tagged MCUR1 EYFP - Mito ( b ) . Scale bar , 20 μm .

O O O O O B-EXP_ASSAY O B-CELL_LINE O O O O O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O B-GENEPROD O O B-GENEPROD I-GENEPROD O B-GENEPROD O O O O O O O O O O O

(a,b) Confocal images of HeLa cells transiently co-transfected for 48 h with GFP-tagged MCUR1 and DsRed-Mito plasmids (a) or mRFP-tagged MCUR1 and EYFP-Mito (b). Scale bar, 20 μm.


( c ) Immunoblot of Flag - tagged MCUR1 in HeLa crude mitochondrial fraction , outer mitochondrial ( OMM ) mitoplasts , using antibodies against VDAC1 ( outer mitochondrial ) , COII ( integral marker ) Flag .

O O O B-EXP_ASSAY O B-GENEPROD O O B-GENEPROD O B-CELL_LINE O B-SUBCELLULAR O O B-SUBCELLULAR I-SUBCELLULAR O B-SUBCELLULAR O B-SUBCELLULAR O O O O B-GENEPROD O B-SUBCELLULAR I-SUBCELLULAR O O B-GENEPROD O O O O B-GENEPROD O

(c) Immunoblot analysis of Flag-tagged MCUR1 in the HeLa cell crude mitochondrial fraction, outer mitochondrial membrane (OMM) and mitoplasts, using antibodies against VDAC1 (outer mitochondrial membrane protein), COII (integral membrane marker) and Flag.