Figure 7 ( A - D ) HeLa cells transfected with GFP ( black ) or ANO8 ( red ) were stimulated with 0 . 5 and then 1 µM ATP to activate the native P2Y2 receptors . The oscillations were analyzed in terms of the % of responding cells ( B ) , oscillation frequency ( C ) , and the amplitude of the Ca2 + signal ( D ) . Data information : All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test . ( E - H ) : HeLa cells were treated with scrambled siRNA ( black ) or siANO8 ( red ) and were stimulated with 1 and then 5 µM ATP . The oscillations were analyzed in terms of the % of responding cells ( F ) , oscillations frequency ( G ) , and the amplitude of the Ca2 + signal ( H ) . Data information : All results are given as mean ± s . e . m of the indicated number of experiments or cells analyzed , and difference were analyzed by unpaired t test .

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

Figure 7(A-D) HeLa cells transfected with GFP (black) or ANO8 (red) were stimulated with 0.5 and then 1 µM ATP to activate the native P2Y2 receptors. The oscillations were analyzed in terms of the % of responding cells (B), oscillation frequency (C), and the amplitude of the Ca2+ signal (D). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.(E-H): HeLa cells were treated with scrambled siRNA (black) or siANO8 (red) and were stimulated with 1 and then 5 µM ATP. The oscillations were analyzed in terms of the % of responding cells (F), oscillations frequency (G), and the amplitude of the Ca2+ signal (H). Data information: All results are given as mean±s.e.m of the indicated number of experiments or cells analyzed, and difference were analyzed by unpaired t test.


Figure 1 ( B ) Dose - dependent inhibition of nirmatrelvir ( left panel ) and GC376 ( right panel ) on SARS - CoV - 2 Mpro . Prior to adding the substrate for the biochemical reaction , the protease was preincubated for 30 minutes at 37 ° C with the indicated concentrations of the compound . ( C ) Dose - dependent antiviral activity of nirmatrelvir in HEK293T - hACE2 cells infected with SARS - CoV - 2 D614G ( purple symbols ) , B . 1 . 617 . 2 ( green symbols ) or B . 1 . 1 . 529 ( blue symbols ) . Antiviral activity was determined as percent inhibition of the virus - induced cytopathic effect . ( F ) Quantification of SARS - CoV - 2 RNA ( left panel ) and viral titers ( right panel ) in the lung 3 days after infection . RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line . Viral titers were determined by median tissue culture infectious dose ( TCID50 ) . ( G ) Quantification of SARS - CoV - 2 RNA in the nasal turbinates 3 days after infection . RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line .

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

Figure 1(B) Dose-dependent inhibition of nirmatrelvir (left panel) and GC376 (right panel) on SARS-CoV-2 Mpro. Prior to adding the substrate for the biochemical reaction, the protease was preincubated for 30 minutes at 37°C with the indicated concentrations of the compound.(C) Dose-dependent antiviral activity of nirmatrelvir in HEK293T-hACE2 cells infected with SARS-CoV-2 D614G (purple symbols), B.1.617.2 (green symbols) or B.1.1.529 (blue symbols). Antiviral activity was determined as percent inhibition of the virus-induced cytopathic effect.(F) Quantification of SARS-CoV-2 RNA (left panel) and viral titers (right panel) in the lung 3 days after infection. RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line. Viral titers were determined by median tissue culture infectious dose (TCID50).(G) Quantification of SARS-CoV-2 RNA in the nasal turbinates 3 days after infection. RNA values are expressed as copy number per ng of total RNA and the limit of detection is indicated as a dotted line.


Figure 2Quantification of anti - S1 RBD IgG levels by ELISA in the plasma of the indicated mice ( C ) 7 , 14 and 21 days p . i . Neutralization dose 50 ( ND50 ) against SARS - CoV - 2 B . 1 . 1 . 529 pseudovirus in the plasma of the indicated mice ( D ) Quantification of anti - S1 RBD IgG levels by ELISA in the plasma of the indicated mice ( E ) 4 days post re - challenge . Neutralization dose 50 ( ND50 ) against SARS - CoV - 2 B . 1 . 1 . 529 pseudovirus in the plasma of the indicated mice ( F ) 4 days post re - challenge . ( I - N ) Representative flow cytometry plots of ( I ) CD8 + T cells or ( L ) CD4 + T cells expressing IFN - γ and TNF - α in the lungs of the indicated mice 4 days post re - challenge . Unstimulated cells are shown in the upper panels , whereas cells re - stimulated with a pool of SARS - CoV - 2 peptides for 4 hours at 37 ° C are shown in the bottom panels . Plots were pre - gated as ( I ) live + / B220 - / CD19 - / CD4 - / CD8 + cells or ( L ) live + / B220 - / CD19 - / CD8 - / CD4 + . Frequency ( J , M ) and absolute number ( K , N ) of IFN - γ − and TNF - α − producing CD8 + T cells ( J , K ) or CD4 + T cells ( M , N ) in the lung of the indicated mice 4 days post re - challenge .

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

Figure 2Quantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice (C) 7, 14 and 21 days p.i. Neutralization dose 50 (ND50) against SARS-CoV-2 B.1.1.529 pseudovirus in the plasma of the indicated mice (D)Quantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice (E) 4 days post re-challenge. Neutralization dose 50 (ND50) against SARS-CoV-2 B.1.1.529 pseudovirus in the plasma of the indicated mice (F) 4 days post re-challenge.(I-N) Representative flow cytometry plots of (I) CD8+ T cells or (L) CD4+ T cells expressing IFN-γ and TNF-α in the lungs of the indicated mice 4 days post re-challenge. Unstimulated cells are shown in the upper panels, whereas cells re-stimulated with a pool of SARS-CoV-2 peptides for 4 hours at 37°C are shown in the bottom panels. Plots were pre-gated as (I) live+/ B220-/CD19-/CD4-/CD8+ cells or (L) live+/B220-/CD19-/CD8-/CD4+. Frequency (J, M) and absolute number (K, N) of IFN-γ− and TNF-α−producing CD8+ T cells (J, K) or CD4+ T cells (M, N) in the lung of the indicated mice 4 days post re-challenge.


Figure 3 ( B , C ) Quantification of anti - S1 RBD IgG levels by ELISA in the plasma of the indicated mice ( B ) 14 , 21 days p . i . and ( C ) 4 days post re - challenge . ( E ) Quantification of anti - S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7 days p . i . . ( F ) Flow cytometry histogram ( left panel ) and geometric mean fluorescent intensity ( gMFI ) quantification ( right panel ) of CD44 expression by CD8 + T cells in the blood of the indicated mice 7 days p . i . ( pre - gated on live + / B220 - / CD19 - / CD4 - / CD8 + cells ) . ( G , H ) Frequency of ( G ) Granzyme - B − and ( H ) IFN - γ − producing CD8 + T cells in the blood of the indicated mice 7 days p . i . . Cells were stimulated in vitro with a pool of SARS - CoV - 2 peptides for 4 hours at 37 ° C . Plots were pre - gated as above . ( J ) Quantification of anti - S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7 , 14 and 21 days p . i . .

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

Figure 3(B, C) Quantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice (B) 14, 21 days p.i. and (C) 4 days post re-challenge.(E) Quantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7 days p.i..(F) Flow cytometry histogram (left panel) and geometric mean fluorescent intensity (gMFI) quantification (right panel) of CD44 expression by CD8+ T cells in the blood of the indicated mice 7 days p.i. (pre-gated on live+/ B220-/ CD19-/ CD4-/ CD8+ cells).(G, H) Frequency of (G) Granzyme-B− and (H) IFN-γ−producing CD8+ T cells in the blood of the indicated mice 7 days p.i.. Cells were stimulated in vitro with a pool of SARS-CoV-2 peptides for 4 hours at 37°C. Plots were pre-gated as above.(J) Quantification of anti-S1 RBD IgG levels by ELISA in the plasma of the indicated mice 7, 14 and 21 days p.i..


Figure 3CAC was induced in control ( n = 7 ) and ColVIcre - Kif3aflx / flx ( n = 7 ) female mice according to the timeline shown in Figure 2A . ( E ) Representative images of hematoxylin and eosin staining of paraffin - embedded colon sections . Areas with high - grade dysplasia are delimited by black lines . CAC was induced in control ( n = 7 ) and ColVIcre - Kif3aflx / flx ( n = 7 ) female mice according to the timeline shown in Figure 2A . ( F , G , H , I ) Box - and - whisker plots depicting area ( F , G ) and number ( H , I ) of low - and high - grade dysplasia per mouse .

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

Figure 3CAC was induced in control (n = 7) and ColVIcre-Kif3aflx/flx (n = 7) female mice according to the timeline shown in Figure 2A. (E) Representative images of hematoxylin and eosin staining of paraffin-embedded colon sections. Areas with high-grade dysplasia are delimited by black lines.CAC was induced in control (n = 7) and ColVIcre-Kif3aflx/flx (n = 7) female mice according to the timeline shown in Figure 2A. (F,G,H,I) Box-and-whisker plots depicting area (F,G) and number (H,I) of low- and high-grade dysplasia per mouse.


Figure 4 ( D , E , F ) At the end of the protocol described in ( A ) mice were sacrificed and colons were analyzed by histology for colitis severity including areas of irregular crypts ( i . e . , non - parallel crypts , variable crypt diameters , bifurcation and branched crypts ; D , E ) and crypt loss ( i . e . mucosa devoid of crypts ; D , F ) . ( H ) Elevated numbers of F4 / 80 + macrophages in colons of DSS - treated ColVIcre - Kif3aflx / flx mice . Representative images for F4 / 80 staining in areas with crypt loss are shown . At least 5 fields in the regions of crypt loss were analyzed from each colon of control ( n = 6 ) and ColVIcre - Kif3aflx / flx ( n = 6 ) mice . Mean cell numbers were scored as low ( < 500 cells / mm2 ) or high ( > 500 cells / mm2 ) . ( N ) Decreased number of PC in inflamed tissue of patients with ulcerative colitis . Shown are the results of matched - pair analysis for PC expression per vimentin + cells as arbitrary unit ( AU , see material and methods ) . The lines link values obtained for regions of ulceration in the ileum with those of normal regions from the same patient , demonstrating the difference in each pair .

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

Figure 4(D,E,F) At the end of the protocol described in (A) mice were sacrificed and colons were analyzed by histology for colitis severity including areas of irregular crypts (i.e., non-parallel crypts, variable crypt diameters, bifurcation and branched crypts; D,E) and crypt loss (i.e. mucosa devoid of crypts; D,F).(H) Elevated numbers of F4/80+ macrophages in colons of DSS-treated ColVIcre-Kif3aflx/flx mice. Representative images for F4/80 staining in areas with crypt loss are shown. At least 5 fields in the regions of crypt loss were analyzed from each colon of control (n=6) and ColVIcre-Kif3aflx/flx (n=6) mice. Mean cell numbers were scored as low (<500 cells/mm2) or high (>500 cells/mm2).(N) Decreased number of PC in inflamed tissue of patients with ulcerative colitis. Shown are the results of matched-pair analysis for PC expression per vimentin+ cells as arbitrary unit (AU, see material and methods). The lines link values obtained for regions of ulceration in the ileum with those of normal regions from the same patient, demonstrating the difference in each pair.


Figure 5Elevated numbers of IL - 6 - expressing F4 / 80 + macrophages in ColVIcre - Kif3aflx / flx mice treated with DSS as described in Figure 4A . At least 5 fields in the regions of crypt loss were analyzed . Scale bar represent 100 µm . * p < 0 , 05 was calculated by chi - squared test . Elevated IL - 6 expression in colonic epithelial cells and fibroblasts of PC - deficient mice upon DSS - treatment as described in Figure 4A . Representative images and corresponding quantification of vimentinpositive fibroblasts ( in green ) and vimentinnegative epithelial cells ( identified by histological criteria ) labeled for IL - 6 ( upper panel ) and p - STAT3 ( lower panel ) in red . Nuclei were stained with DAPI ( in blue ) . White arrow heads indicate IL - 6 ( upper panel ) and nuclear staining for pSTAT3 ( lower panel ) for vimentinpositive fibroblasts , blue arrow heads the respective labeling for epithelial cells . Scale bars represent 100 µm . * p < 0 . 05 , * * * * p < 10 - 4 , n . s . ( not significant ) was calculated by chi - squared test on absolute numbers and is presented as % of cells positive for the indicated marker within vimentinpositive and epithelial cells , respectively .

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

Figure 5Elevated numbers of IL-6-expressing F4/80+ macrophages in ColVIcre-Kif3aflx/flx mice treated with DSS as described in Figure 4A. At least 5 fields in the regions of crypt loss were analyzed. Scale bar represent 100 µm. *p<0,05 was calculated by chi-squared test.Elevated IL-6 expression in colonic epithelial cells and fibroblasts of PC-deficient mice upon DSS-treatment as described in Figure 4A. Representative images and corresponding quantification of vimentinpositive fibroblasts (in green) and vimentinnegative epithelial cells (identified by histological criteria) labeled for IL-6 (upper panel) and p-STAT3 (lower panel) in red. Nuclei were stained with DAPI (in blue). White arrow heads indicate IL-6 (upper panel) and nuclear staining for pSTAT3 (lower panel) for vimentinpositive fibroblasts, blue arrow heads the respective labeling for epithelial cells. Scale bars represent 100 µm. *p<0.05, ****p<10-4, n.s. (not significant) was calculated by chi-squared test on absolute numbers and is presented as % of cells positive for the indicated marker within vimentinpositive and epithelial cells, respectively.


Figure 7 ( A ) Representative image of vimentin + fibroblasts ( in green ) expressing PC ( identified using Arl13b , in red ) in a normal colon . Nuclei were stained with DAPI ( in blue ) . Most PC are detected on vimentin + cells ( arrow ) , and few on vimentin - negative epithelial cells ( arrow head ) . ( B , C ) Quantitative analysis of PC presence on tumoral and peri - tumoral regions of colons from CRC patients ( n = 28 ) at tumor ( T ) stages 1 to 4 . The panels represent quantification of PC expression ( B ) of vimentinpositive cells per mm2 and ( C ) per vimentinpositive cells as arbitrary unit ( AU , see material and methods ) . Images were acquired on an Andor Dragonfly Spinning Disk Confocal microscope and analyzed with the Imaris software . Quantification of PC and Vimentin + fibroblasts was done on at least 5 random fields ( about 1mm2 each ) per sample using ImageJ software .

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

Figure 7(A) Representative image of vimentin+ fibroblasts (in green) expressing PC (identified using Arl13b, in red) in a normal colon. Nuclei were stained with DAPI (in blue). Most PC are detected on vimentin+ cells (arrow), and few on vimentin-negative epithelial cells (arrow head). (B,C) Quantitative analysis of PC presence on tumoral and peri-tumoral regions of colons from CRC patients (n=28) at tumor (T) stages 1 to 4. The panels represent quantification of PC expression (B) of vimentinpositive cells per mm2 and (C) per vimentinpositive cells as arbitrary unit (AU, see material and methods). Images were acquired on an Andor Dragonfly Spinning Disk Confocal microscope and analyzed with the Imaris software. Quantification of PC and Vimentin+ fibroblasts was done on at least 5 random fields (about 1mm2 each) per sample using ImageJ software.


Figure 1HeLa cells were transfected with a WT or mutant Parkin plasmid ( FLAG ) , shown in blue , along with reporters for either PA or DAG shown in green . DMSO or 10 µM CCCP was added for the indicated periods of time . Mitochondria were visualized via anti - Tom20 staining ( red ) . ( A ) PA accumulated on Parkin - positive mitochondria after 1 hour of 10uM CCCP treatment , with a more dramatic increase by 3hr and 18 hr . Scale bar = 25 µM , and zoom is 9x . ( B ) Quantification of imaging experiments shown in A and B . Bars represent mean with SEM from three biological independent experiments ; two - way ANOVA analysis was performed for statistical analysis ( * * * P < 0 . 0001 ) . ( C ) DAG ( diacylglycerol ) accumulated on Parkin - positive mitochondria after 5 . 5 hr of 10uM CCCP treatment . Arrowheads and arrows point to the DAGR concentration at the Golgi and mitochondria , respectively . Scale bar = 25 µM , and zoom is 9x .

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

Figure 1HeLa cells were transfected with a WT or mutant Parkin plasmid (FLAG), shown in blue, along with reporters for either PA or DAG shown in green. DMSO or 10 µM CCCP was added for the indicated periods of time. Mitochondria were visualized via anti-Tom20 staining (red). (A) PA accumulated on Parkin-positive mitochondria after 1 hour of 10uM CCCP treatment, with a more dramatic increase by 3hr and 18 hr. Scale bar = 25 µM, and zoom is 9x. (B) Quantification of imaging experiments shown in A and B. Bars represent mean with SEM from three biological independent experiments; two-way ANOVA analysis was performed for statistical analysis (***P<0.0001). (C) DAG (diacylglycerol) accumulated on Parkin-positive mitochondria after 5.5 hr of 10uM CCCP treatment. Arrowheads and arrows point to the DAGR concentration at the Golgi and mitochondria, respectively. Scale bar = 25 µM, and zoom is 9x.


Figure 2 ( B ) HEK - 293T cells were transfected with Parkin - FLAG and DAGR - YFP followed by CCCP treatment for indicated times . Mitochondrial and cytosolic fractions were purified and analyzed by Western blots by indicated antibodies . Citrate synthetase and α - tubulin were used as a mitochondrial and cytosolic marker , respectively . Note that PLD2 and Parkin levels were elevated in the mitochondrial fraction in response to CCCP treatment . ( C ) Hela cells were co - transfected with mCherry - Parkin and the PA reporter , followed by CCCP treatment alone or with a PLD2 inhibitor VU 0364739 ( 3 µM ) for 5 . 5 hrs . Line scan analysis ( Image J software ) corresponding to the line drawn in the images indicate colocalization between the PA reporter ( green ) and mitochondria ( red ) . Note that VU 0364739 suppressed mitochondrial PA accumulation . Scale bar = 10 µM . ( D ) Quantification of the numbers of cells with the PA reporter positive mitochondria shown in ( C ) . Asterisks indicate statistical significance ( * * * P < 0 . 001 , Student ' s t - test ) from three biological independent experiments . The bars indicate mean ± SEM . ( E ) Hela cells were transfected with a lipin1 siRNA , followed by the DAG reporter and mCherry - Parkin , treated with CCCP ( 10 µM ) for 9 hrs and then subject to image analysis . Scale bar = 25 um , and zoom is 4x . Lipin 1 KD reduced mitochondrial DAGR , but not Golgi - DAGR , accumulation ( arrowheads ) ( F ) The numbers of cells with DAG - reporter positive mitochondria shown in ( E ) and in Lipin - 1 knockdown cells transfected with siRNA - resistant wildtype ( WT ) and catalytically dead ( CD ; D712E ; D714E ) mutant cDNA . Note that Lipin1 KD reduced mitochondrial DAG accumulation , which can be significantly restored by the re - expression of WT , but not CD mutant , Lipin - 1 . The graph shows the means with SEM ( error bars ) from three biological independent experiments . Asterisks indicate statistical significance by one - way ANOVA ( * * P < 0 . 01 , * * * P < 0 . 001 ) .

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

Figure 2(B) HEK-293T cells were transfected with Parkin-FLAG and DAGR-YFP followed by CCCP treatment for indicated times. Mitochondrial and cytosolic fractions were purified and analyzed by Western blots by indicated antibodies. Citrate synthetase and α-tubulin were used as a mitochondrial and cytosolic marker, respectively. Note that PLD2 and Parkin levels were elevated in the mitochondrial fraction in response to CCCP treatment.(C) Hela cells were co-transfected with mCherry-Parkin and the PA reporter, followed by CCCP treatment alone or with a PLD2 inhibitor VU 0364739 (3 µM) for 5.5 hrs. Line scan analysis (Image J software) corresponding to the line drawn in the images indicate colocalization between the PA reporter (green) and mitochondria (red). Note that VU 0364739 suppressed mitochondrial PA accumulation. Scale bar = 10 µM. (D) Quantification of the numbers of cells with the PA reporter positive mitochondria shown in (C). Asterisks indicate statistical significance (***P<0.001, Student's t-test) from three biological independent experiments. The bars indicate mean± SEM.(E) Hela cells were transfected with a lipin1 siRNA, followed by the DAG reporter and mCherry-Parkin, treated with CCCP (10 µM) for 9 hrs and then subject to image analysis. Scale bar = 25 um, and zoom is 4x. Lipin 1 KD reduced mitochondrial DAGR, but not Golgi-DAGR, accumulation (arrowheads) (F) The numbers of cells with DAG-reporter positive mitochondria shown in (E) and in Lipin-1 knockdown cells transfected with siRNA-resistant wildtype (WT) and catalytically dead (CD; D712E;D714E) mutant cDNA. Note that Lipin1 KD reduced mitochondrial DAG accumulation, which can be significantly restored by the re-expression of WT, but not CD mutant, Lipin-1. The graph shows the means with SEM (error bars) from three biological independent experiments. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001).


Figure 3 ( A - B ) Hela cells were transfected with a Lipin1 or control ( cKD ) siRNA followed by a GFP - Parkin expression plasmid . Transfected cells were treated with CCCP at 10 µM for 18 hrs , and subject to immuno - staining . ( B ) Quantification of Parkin - positive cells in ( A ) that have lost a majority of mitochondria ( marked by TOM20 , arrows ) . Note that mitochondrial clearance is reduced in Lipin - 1 knockdown cells ( arrowheads in ( A ) ) . Scale bar = 25 µM . ( C ) . Control or lipin1 knockdown Hela cells stably expressing parkin - mCherry were treated with DMSO or CCCP ( 10 µM for 18 hrs ) and Bafilomycin A1 ( 1μM , lysosomal inhibitor ) treatment , followed by immunoblotting with indicated antibodies and quantified by the Image J . software . Note that lipin1 silencing suppressed CCCP - induced mitochondrial protein degradation . Lipin - 1 - mediated mitochondrial DAG production is required for mitophagosome production . ( D - F ) . Hela cells were transfected with control or a Lipin1 siRNA followed by the expression plasmids of GFP - Parkin and RFP - DAGR . Transfected cells were treated with DMSO or CCCP at 10 µM for 9 hr , with or without further incubation with 1 , 2 - Dipalmitoyl - sn - glycerol ( DPG ) at 100 µM , as indicated . Autophagosome formation was assessed by an LC3 antibody ( pseudocolored in white in single - channel and blue in the overlay images ) . Arrows point to DAGR concentration at the Golgi under basal conditions . The number ( E ) and size ( F , arbitrary unit ) of LC3 vesicles were quantified . n = 4 biological replicates . The bars indicates mean ± SEM . Asterisks indicate statistical significance by one - way ANOVA ( * P < 0 . 05 , * * P < 0 . 01 ) . Scale bar = 25 µM and zoom is 5x . Note that both the number and size of LC3 - vesicles in Lipin - 1 knockdown cells were much smaller than those in WT cells , and these defects were corrected by DPG treatment .

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

Figure 3(A-B) Hela cells were transfected with a Lipin1 or control (cKD) siRNA followed by a GFP-Parkin expression plasmid. Transfected cells were treated with CCCP at 10 µM for 18 hrs, and subject to immuno-staining. (B) Quantification of Parkin-positive cells in (A) that have lost a majority of mitochondria (marked by TOM20, arrows). Note that mitochondrial clearance is reduced in Lipin-1 knockdown cells (arrowheads in (A)). Scale bar = 25 µM.(C). Control or lipin1 knockdown Hela cells stably expressing parkin-mCherry were treated with DMSO or CCCP (10 µM for 18 hrs) and Bafilomycin A1 (1μM, lysosomal inhibitor) treatment, followed by immunoblotting with indicated antibodies and quantified by the Image J. software. Note that lipin1 silencing suppressed CCCP-induced mitochondrial protein degradation. Lipin-1-mediated mitochondrial DAG production is required for mitophagosome production.(D-F). Hela cells were transfected with control or a Lipin1 siRNA followed by the expression plasmids of GFP-Parkin and RFP-DAGR. Transfected cells were treated with DMSO or CCCP at 10 µM for 9 hr, with or without further incubation with 1,2-Dipalmitoyl-sn-glycerol (DPG) at 100 µM, as indicated. Autophagosome formation was assessed by an LC3 antibody (pseudocolored in white in single-channel and blue in the overlay images). Arrows point to DAGR concentration at the Golgi under basal conditions. The number (E) and size (F, arbitrary unit) of LC3 vesicles were quantified. n=4 biological replicates. The bars indicates mean± SEM. Asterisks indicate statistical significance by one-way ANOVA (*P< 0.05, **P<0.01). Scale bar = 25 µM and zoom is 5x. Note that both the number and size of LC3-vesicles in Lipin-1 knockdown cells were much smaller than those in WT cells, and these defects were corrected by DPG treatment.


Figure 4 ( A ) E3 ligase - deficient Parkin mutants ( T240R and T415N ) have no effect on PA accumulation ( green ) on depolarized , Parkin - positive mitochondria . The expression of WT and Parkin mutants were determined by Immunoblotting . Scale bar = 25 µM , and zoom is 3x . ( B ) DAG accumulation depends upon intact Parkin E3 ligase activity . Scale bar = 25µm , and zoom is 3x . ( C ) Quantification of imaging experiments shown in ( A ) and ( B ) . Bars represent mean with SEM ; two - way ANOVA analysis was performed for statistical analysis ( * * * P < 0 . 001 ) . n = 3 biological replicates . ( D ) Hela cells were transfected with control or optineurin - and NDP52 - siRNAs and expression plasmids of FLAG - Parkin and YFP - DAGR . These cells were treated with DMSO or 10 µM CCCP for 5 . 5 hrs , as indicated . Note that OPTN / NDP52 double knockdown prevented mitochondrial YFP - DAGR accumulation . ( Scale bar = 25 µM and zoom is 3x ) ( E ) Hela OPTN / NDP52 knockdown cells were transfected with a siRNA - resistant wildtype or ubiquitin - binding deficient E478G OPTN mutant , as indicated . The percentage of cells with mitochondrial YFP - DAGR following CCCP treatment was quantified . Asterisks indicate statistical significance by one - way ANOVA ( * * P < 0 . 01 , * * * P < 0 . 001 ) from three biological independent experiments . The bars indicate mean ± SEM . Bottom panel : Expression of OPTN , OPTN E478 and NDP52 was determined by immunoblotting . ( F - G ) Hela OPTN / NDP52 knockdown cells were incubated with 1 , 2 - Dipalmitoyl - sn - glycerol ( DPG ) followed by CCCP treatment for 18 hrs . Mitophagy efficiency was assessed by TOM20 staining and quantified in ( G ) ( n = 2 biological replicates ) . Note that DPG significantly restored mitophagy in OPTN / NDP52 knockdown cells . Scale bar = 10 µM .

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

Figure 4(A) E3 ligase-deficient Parkin mutants (T240R and T415N) have no effect on PA accumulation (green) on depolarized, Parkin-positive mitochondria. The expression of WT and Parkin mutants were determined by Immunoblotting. Scale bar = 25 µM, and zoom is 3x. (B) DAG accumulation depends upon intact Parkin E3 ligase activity. Scale bar = 25µm, and zoom is 3x. (C) Quantification of imaging experiments shown in (A) and (B). Bars represent mean with SEM; two-way ANOVA analysis was performed for statistical analysis(***P<0.001). n=3 biological replicates.(D) Hela cells were transfected with control or optineurin- and NDP52- siRNAs and expression plasmids of FLAG-Parkin and YFP-DAGR. These cells were treated with DMSO or 10 µM CCCP for 5.5 hrs, as indicated. Note that OPTN/NDP52 double knockdown prevented mitochondrial YFP-DAGR accumulation. (Scale bar = 25 µM and zoom is 3x) (E) Hela OPTN/NDP52 knockdown cells were transfected with a siRNA-resistant wildtype or ubiquitin-binding deficient E478G OPTN mutant, as indicated. The percentage of cells with mitochondrial YFP-DAGR following CCCP treatment was quantified. Asterisks indicate statistical significance by one-way ANOVA (**P< 0.01, ***P<0.001) from three biological independent experiments. The bars indicate mean± SEM. Bottom panel: Expression of OPTN, OPTN E478 and NDP52 was determined by immunoblotting.(F-G) Hela OPTN/NDP52 knockdown cells were incubated with 1,2-Dipalmitoyl-sn-glycerol (DPG) followed by CCCP treatment for 18 hrs. Mitophagy efficiency was assessed by TOM20 staining and quantified in (G) (n = 2 biological replicates). Note that DPG significantly restored mitophagy in OPTN/NDP52 knockdown cells. Scale bar = 10 µM.


Figure 5 ( B ) Mitochondrial and cytosolic fractions obtained from control and CCCP treated cells , were immunoblotted for EndoB1 and OPTN , as indicated . ( C ) . Cytosolic and mitochondrial fractions purified from control and OPTN / NDP52 knockdown cells treated with CCCP were immunoblotted with indicated antibodies . Note that mitochondrial EndoB1 levels were reduced in OPTN / NDP52 knockdown cells ( D - E ) Hela cells were transfected with an EndoB1 siRNA , followed by the expression plasmids for mCherry - Parkin and YFP - DAGR , and CCCP treatment ( 10 µM ) for 9 hrs . Cells with mitochondrial YFP - DAGR were quantified in ( E ) ( * P < 0 . 05 , Student ' s t - test , n = 3 biological replicates ) . Scale bar = 25 µM and zoom is 5x . Note that knockdown of EndoB1 suppressed mitochondrial DAG production .

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

Figure 5(B) Mitochondrial and cytosolic fractions obtained from control and CCCP treated cells, were immunoblotted for EndoB1 and OPTN, as indicated.(C). Cytosolic and mitochondrial fractions purified from control and OPTN/NDP52 knockdown cells treated with CCCP were immunoblotted with indicated antibodies. Note that mitochondrial EndoB1 levels were reduced in OPTN/NDP52 knockdown cells(D-E) Hela cells were transfected with an EndoB1 siRNA, followed by the expression plasmids for mCherry-Parkin and YFP-DAGR, and CCCP treatment (10 µM) for 9 hrs. Cells with mitochondrial YFP-DAGR were quantified in (E) (*P<0.05, Student's t-test, n=3 biological replicates). Scale bar = 25 µM and zoom is 5x. Note that knockdown of EndoB1 suppressed mitochondrial DAG production.


Figure 1 ( A ) WT and CITK - / - embryonic ( E14 . 5 ) mousecerebral cortex was stained for γ - tubulin ( green ) and DNA ( gray ) . The ventricular plane is marked by a red dashed line , and the spindle axis of apical progenitors is indicated by a white dashed line . The angle between these two lines represents the mitotic angle . Scale bars = 5 μm . ( B ) Quantification of vertical divisions of apical progenitors in WT and CITK - / - mice . n = 3 per each genotype . ( C ) Pregnant CITK + / - females , crossed with CITK + / - males , were injected with BrdU at E13 . 5 ; 24 hours later ( E14 . 5 ) WT and CITK - / - embryonic mouse cerebral cortex were fixed in 4 % PFA , cryosectioned and stained for Ki67 ( red ) , BrdU ( green ) , Tbr2 ( blue ) and DAPI ( gray ) . The different cortical regions are indicated : CP = cortical plate ; IZ = intermediate zone ; SVZ = subventricular zone ; VZ = ventricular zone . Scale bars = 10 μm . White arrows in the inset indicate two Ki67 - cells positive for BrdU and Tbr2 . ( D ) Quantification of cell cycle exit ( ratio of cells BrdU + / Ki67 − ) in the proliferative regions ( VZ / SVZ ) and in the neuronal layers ( IZ and CP ) of sections prepared as in panel C ( n = 4 per each genotype ) . ( E ) Sections prepared as in C were stained for TUBB3 ( TuJ ) to reveal post - mitotic neurons and with DAPI . Red arrowheads indicate apoptotic neurons ( defined by pyknotic nuclei ) . Quantification of neuronal distribution in VZ - SVZ versus total neurons population is showed on the right . Scale bars = 10 μm . ( F ) Quantification of the percentage of Tbr2 - positive cells in the BrdU - positive population of the same sections analyzed for panel D ( n = 4 per each genotype ) . ( G ) Neuroblasts of wild type and dck alleles immunostained for α - tubulin and Miranda . Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis , in dck alleles the spindle show a more oblique orientation respect to Mira crescent . Scale bar = 5 μm . ( H ) Distribution of spindle angle amplitude ( ° ) in neuroblasts of wild type and ck alleles ( n = 58 ) .

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

Figure 1(A) WT and CITK -/- embryonic (E14.5) mousecerebral cortex was stained for γ-tubulin (green) and DNA (gray). The ventricular plane is marked by a red dashed line, and the spindle axis of apical progenitors is indicated by a white dashed line. The angle between these two lines represents the mitotic angle. Scale bars = 5 μm.(B) Quantification of vertical divisions of apical progenitors in WT and CITK -/- mice. n= 3 per each genotype.(C) Pregnant CITK +/- females, crossed with CITK +/- males, were injected with BrdU at E13.5; 24 hours later (E14.5) WT and CITK -/- embryonic mouse cerebral cortex were fixed in 4% PFA, cryosectioned and stained for Ki67 (red), BrdU (green), Tbr2 (blue) and DAPI (gray). The different cortical regions are indicated: CP=cortical plate; IZ= intermediate zone; SVZ= subventricular zone; VZ= ventricular zone. Scale bars = 10 μm. White arrows in the inset indicate two Ki67- cells positive for BrdU and Tbr2.(D) Quantification of cell cycle exit (ratio of cells BrdU+/ Ki67−) in the proliferative regions (VZ/SVZ) and in the neuronal layers (IZ and CP) of sections prepared as in panel C (n=4 per each genotype).(E) Sections prepared as in C were stained for TUBB3 (TuJ) to reveal post-mitotic neurons and with DAPI. Red arrowheads indicate apoptotic neurons (defined by pyknotic nuclei). Quantification of neuronal distribution in VZ-SVZ versus total neurons population is showed on the right. Scale bars = 10 μm.(F) Quantification of the percentage of Tbr2-positive cells in the BrdU-positive population of the same sections analyzed for panel D (n=4 per each genotype).(G) Neuroblasts of wild type and dck alleles immunostained for α-tubulin and Miranda. Note that whereas in wild type cells there is a tight coupling of the mitotic spindle with the polarity axis, in dck alleles the spindle show a more oblique orientation respect to Mira crescent. Scale bar = 5 μm.(H) Distribution of spindle angle amplitude (°) in neuroblasts of wild type and ck alleles (n=58).


Figure 2 ( A ) Selected frames from time - lapse imaging experiments ( see Movies EV1 and EV2 ) showing two dividing cells transfected with either control ( CTRL ) or CITK - specific siRNAs , respectively . Note one of the two daughter cells in the lower panel ( red arrow ) going out of focus as a consequence of oblique division . ( B ) Graphical representation of vertical and oblique divisions . The cleavage plane is indicated by a black dashed line . ( C ) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis ( oblique division ) in CITK siRNA treated HeLa cells compared to control ( n > 50 cells , 3 independent experiments ) . ( D ) Control or CITK - depleted cells were immunostained for γ - tubulin ( red ) and DNA ( blue ) and imaged in z ( 0 . 3 µm - thick sections ) . Upper panel : maximum intensity projections of confocal z - stacks are shown . Lower panel : cross - section ( XZ ) through the two poles of the same cell . ( E ) Distribution of spindle angles ( ° ) in control and in CITK - depleted cells . The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip . ( n ≥ 150 cells , 6 independent experiments ) . ( F ) Quantification of spindle angles average in control cells and in cells depleted of CITK , ASPM or co - depleted of the two proteins ( n ≥ 150 cells , in at least 3 independent experiments ) .

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

Figure 2(A) Selected frames from time-lapse imaging experiments (see Movies EV1 and EV2) showing two dividing cells transfected with either control (CTRL) or CITK-specific siRNAs, respectively. Note one of the two daughter cells in the lower panel (red arrow) going out of focus as a consequence of oblique division.(B) Graphical representation of vertical and oblique divisions. The cleavage plane is indicated by a black dashed line.(C) Quantification of divisions showing uneven timing of daughter cell flattening onto the substrate after mitosis (oblique division) in CITK siRNA treated HeLa cells compared to control (n > 50 cells, 3 independent experiments).(D) Control or CITK-depleted cells were immunostained for γ-tubulin (red) and DNA (blue) and imaged in z (0.3 µm-thick sections). Upper panel: maximum intensity projections of confocal z-stacks are shown. Lower panel: cross-section (XZ) through the two poles of the same cell.(E) Distribution of spindle angles (°) in control and in CITK-depleted cells. The values represent the angles between the axis crossing the two poles of metaphase spindles and the coverslip. (n ≥ 150 cells, 6 independent experiments).(F) Quantification of spindle angles average in control cells and in cells depleted of CITK, ASPM or co-depleted of the two proteins (n ≥ 150 cells, in at least 3 independent experiments).


Figure 3 ( A ) HeLa cells pre - extracted for 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , γ - tubulin ( red ) and DNA ( blue ) . Scale bars , 10μm ( B ) HeLa cells expressing CITK - GFP ( green ) from a BAC transgene treated as in ( A ) and immunostained for γ - tubulin ( red ) and DNA ( blue ) . Scale bars , 5μm ( C ) Western blot of centrosome - containing fractions from HeLa cells , showing that CITK co - purifies with γ ‑ tubulin . ( D ) HeLa cells pre - extracted 1 min with 0 . 5 % Triton X - 100 in PHEM buffer and immunostained for CITK ( green ) , ASPM ( red ) and DNA ( blue ) . ( E ) HeLa cells expressing CITK - GFP treated as in ( A ) and immunostained for GFP ( green ) , ASPM ( red ) and DNA ( blue ) . ( F ) Close physical proximity between CITK and ASPM , revealed by PLA on ASPM - GFP - expressing HeLa cells , incubated with GFP and CITK antibodies and developed with PLA - specific secondary reagents . ( G ) Control or CITK - depleted cells immunostained for ASPM ( green ) , γ - tubulin ( red ) and DNA ( blue ) . ( H ) Control or ASPM - depleted cells treated as in ( A ) and immunostained for CITK ( green ) , γ - tubulin ( red ) and DNA ( blue ) . ( I ) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity , in control and ASPM - depleted cells . ( n ≥ 75 cells , 4 independent experiments ) .

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

Figure 3(A) HeLa cells pre-extracted for 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), γ-tubulin (red) and DNA (blue). Scale bars, 10μm(B) HeLa cells expressing CITK-GFP (green) from a BAC transgene treated as in (A) and immunostained for γ-tubulin (red) and DNA (blue). Scale bars, 5μm(C) Western blot of centrosome-containing fractions from HeLa cells, showing that CITK co-purifies with γ‑tubulin.(D) HeLa cells pre-extracted 1 min with 0.5% Triton X-100 in PHEM buffer and immunostained for CITK (green), ASPM (red) and DNA (blue).(E) HeLa cells expressing CITK-GFP treated as in (A) and immunostained for GFP (green), ASPM (red) and DNA (blue).(F) Close physical proximity between CITK and ASPM, revealed by PLA on ASPM-GFP-expressing HeLa cells, incubated with GFP and CITK antibodies and developed with PLA-specific secondary reagents.(G) Control or CITK-depleted cells immunostained for ASPM (green), γ-tubulin (red) and DNA (blue).(H) Control or ASPM-depleted cells treated as in (A) and immunostained for CITK (green), γ-tubulin (red) and DNA (blue).(I) Quantification of CITK positive centrosomes and of the ratio of CITKspindle pole intensity versus total cell mean intensity, in control and ASPM-depleted cells. (n ≥ 75 cells, 4 independent experiments).


Figure 4 ( A ) Control , CITK or ASPM - depleted HeLa cells immunostained for α - tubulin ( red ) and DNA ( blue ) . Maximum intensity projections of confocal z - stacks are shown . ( B ) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL , CITK or ASPM siRNA ( n > 130 cells in 3 independent experiments ) . ( C ) Quantification of the rescue of astral MT number after CITK overexpression in ASPM - depleted HeLa cells ( n > 50 cells in 3 independent experiments ) . ( D ) Quantification of spindle orientation rescue after overexpression of CITK , CITN ( lacking the kinase domain ) or CKD ( Citron kinase dead ) mutants in ASPM - depleted HeLa cells ( n > 50 cells in 3 independent experiments ) . ( E ) Synchronized HeLa transfected with control , ASPM - or CITK - specific siRNAs treated with DMSO or with 250 pM Paclitaxel ( taxol ) 30 min before immunostaining for α - Tubulin . Spindle angles were measured as above ( n > 100 cells in 6 independent experiments ) .

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Figure 4(A) Control, CITK or ASPM-depleted HeLa cells immunostained for α-tubulin (red) and DNA (blue). Maximum intensity projections of confocal z-stacks are shown.(B) Quantification of astral MT number and maximal length in HeLa cells transfected with CTRL, CITK or ASPM siRNA (n > 130 cells in 3 independent experiments).(C) Quantification of the rescue of astral MT number after CITK overexpression in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).(D) Quantification of spindle orientation rescue after overexpression of CITK, CITN (lacking the kinase domain) or CKD (Citron kinase dead) mutants in ASPM-depleted HeLa cells (n > 50 cells in 3 independent experiments).(E) Synchronized HeLa transfected with control, ASPM- or CITK-specific siRNAs treated with DMSO or with 250 pM Paclitaxel (taxol) 30 min before immunostaining for α-Tubulin. Spindle angles were measured as above (n > 100 cells in 6 independent experiments).


Figure 5 ( A ) Exemplar image of EB3 - tdTomatoHeLa cell used for kymograph analyses . To assess astral MT number and dynamics a line ( blue ) was added to images from the pole to the cortex . ( B ) Kymographs of pole ( bottom ) - to - cortex ( top ) line scans of control and CITK - depleted EB3 - tdTomatoHeLa cells . The maximum projection of a 20 - pixel - wide line ( exemplified above ) was plotted at 500 ms time intervals from left to right . MT growth results in diagonal lines . ( C ) Quantification of astral MT dynamics . MT stability , length , growth speed ( n > 250 MT , 30 cells ) and nucleation frequency were calculated ( n > 30 cells , 3 independent experiments ) . ( D ) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0 , 1 , and 2 min after nocodazole washout . Control and CITK - depleted cells were immunostained for α - tubulin ( red ) and DNA ( blue ) . ( E ) Percentage of cells showing detectable MT nucleation ( aster size > 1 µm ) 0 , 1 , and 2 min after nocodazole washout ( n > 100 cells , 4 independent experiments ) . ( F ) Quantification of aster size 2 min after nocodazole washout ( n > 100 cells , 4 independent experiments ) .

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

Figure 5(A) Exemplar image of EB3-tdTomatoHeLa cell used for kymograph analyses. To assess astral MT number and dynamics a line (blue) was added to images from the pole to the cortex.(B) Kymographs of pole (bottom)-to-cortex (top) line scans of control and CITK-depleted EB3-tdTomatoHeLa cells. The maximum projection of a 20-pixel-wide line (exemplified above) was plotted at 500 ms time intervals from left to right. MT growth results in diagonal lines.(C) Quantification of astral MT dynamics. MT stability, length, growth speed (n > 250 MT, 30 cells) and nucleation frequency were calculated (n > 30 cells, 3 independent experiments).(D) Immunofluorescence microscopy images showing MT growth at mitotic spindle poles 0, 1, and 2 min after nocodazole washout. Control and CITK-depleted cells were immunostained for α-tubulin (red) and DNA (blue).(E) Percentage of cells showing detectable MT nucleation (aster size > 1 µm) 0, 1, and 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).(F) Quantification of aster size 2 min after nocodazole washout (n > 100 cells, 4 independent experiments).


Figure 1Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro - B cells and plasmablasts . Open chromatin was determined by ATAC - seq ( Buenrostro et al . , 2013 ) , and active ( H3K4me2 , H3K4me3 , H3K9ac ) and repressive ( H3K27me3 ) histone marks were mapped by ChIP - seq analysis in ex vivo sorted pro - B cells ( ATAC - seq and H3K27me3 , this study ) , short - term in vitro cultured pro - B cells ( H3K4me2 , H3K4me3 , H3K9ac ) ( Revilla - i - Domingo et al . , 2012 ) and in vitro LPS - induced plasmablasts ( Minnich et al . , 2016 ) ( Table EV4 ) . The indicated upstream regions were previously shown by 3C - analysis to interact with the Prdm1 promoter ( Wöhner et al . , 2016 ) . The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs ( rs658431 and rs548234 ) are indicated . RPM , reads per million mapped sequence readsMapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO - seq or RNA - seq analyses of ex vivo sorted pro - B and FO B cells , respectively ( Table EV4 ) . Strand - specific GRO - seq reads are indicated in blue and red , respectively Quantification of nascent transcript and mRNA levels . The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value ( TPM ) with SEM based on two different GRO - seq or RNA - seq experiments for each B cell type , respectively . TPM , transcripts per million ( Wagner et al . , 2012 )

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Figure 1Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro-B cells and plasmablasts. Open chromatin was determined by ATAC-seq (Buenrostro et al., 2013), and active (H3K4me2, H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks were mapped by ChIP-seq analysis in ex vivo sorted pro-B cells (ATAC-seq and H3K27me3, this study), short-term in vitro cultured pro-B cells (H3K4me2, H3K4me3, H3K9ac) (Revilla-i-Domingo et al., 2012) and in vitro LPS-induced plasmablasts (Minnich et al., 2016) (Table EV4). The indicated upstream regions were previously shown by 3C-analysis to interact with the Prdm1 promoter (Wöhner et al., 2016). The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs (rs658431 and rs548234) are indicated. RPM, reads per million mapped sequence readsMapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO-seq or RNA-seq analyses of ex vivo sorted pro-B and FO B cells, respectively (Table EV4). Strand-specific GRO-seq reads are indicated in blue and red, respectively Quantification of nascent transcript and mRNA levels. The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value (TPM) with SEM based on two different GRO-seq or RNA-seq experiments for each B cell type, respectively. TPM, transcripts per million (Wagner et al., 2012)


Figure 2Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro - B and pre - B cells from the bone marrow of Prdm1ihCd2 / + or wild - type ( WT ) mice . mRNA expression is shown as mean expression value ( TPM ) with SEM based on two independent RNA - seq experiments for each cell type and genotypeRT - qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre - B cells and in in vitro differentiated plasmablasts ( PB ) of the indicated genotypes . Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS . The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild - type pre - B cells ( set as 1 ) . Nascent transcripts were PCR - amplified with primers located in the indicated introns ( Table EV3 ) . Statistical data are shown as mean value with SEM and were analyzed by the Student " s t - test ; * P < 0 . 05 , * * P < 0 . 01 . Each dot corresponds to oneFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types . Bone marrow of Prdm1ihCd2 / + ( red ) or wild - type ( WT , gray ) mice was used to analyz ALPs , BLPs , pro - B , pre - B , immature B cells whereas the spleen was used for flow cytometric analysis o FO B , MZ B and plasma cell of both genotypes The histograms show hCD2 ( top row ) and Blimp1 ( bottom row ) expression for the different cell types Wild - type FO B cells ( dashed line ) were used as negative control for the Blimp1 staining in plasma cells . The difference in mean fluorescence intensity ( ΔMFI ) between the two genotypes is shown for each cell typeFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types . Bone marrow of Prdm1ihCd2 / + ( red ) or wild - type ( WT , gray ) mice was used to analyz NK cells and granulocytes , whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 ( top row ) and Blimp1 ( bottom row ) expression for the different cell types The difference in mean fluorescence intensity ( ΔMFI ) between the two genotypes is shown for each cell typeImmunoblot analysis of Blimp1 and the TATA - binding protein ( TBP ) in nuclear extracts prepared from short - term cultured wild - type or Prdm1ihCd2 / + pro - B cells . Marker proteins of the indicated size ( in kilodaltons ) are shown to the

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

Figure 2Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ or wild-type (WT) mice. mRNA expression is shown as mean expression value (TPM) with SEM based on two independent RNA-seq experiments for each cell type and genotypeRT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student"s t-test; *P < 0.05, **P < 0.01. Each dot corresponds to oneFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz ALPs, BLPs, pro-B, pre-B, immature B cells whereas the spleen was used for flow cytometric analysis o FO B, MZ B and plasma cell of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types Wild-type FO B cells (dashed line) were used as negative control for the Blimp1 staining in plasma cells. The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell typeFlow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyz NK cells and granulocytes, whereas the spleen was used for flow cytometric analysis o naïve CD4 T and naïve CD8 T cells of both genotypes The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell typeImmunoblot analysis of Blimp1 and the TATA-binding protein (TBP) in nuclear extracts prepared from short-term cultured wild-type or Prdm1ihCd2/+ pro-B cells. Marker proteins of the indicated size (in kilodaltons) are shown to the right


Figure 3Flow cytometric analysis of total B cells ( A ) as well as pro - B , pre - B , immature ( imm ) B and recirculating ( recirc ) B cells ( B ) from the bone marrow of wild - type ( WT , gray ) , Prdm1ihCd2 / + ( red ) and Prdm1ihCd2 / ihCd2 ( white ) mice at the age of 6 - 8 weeks Flow cytometric analysis of total B , FO B and MZ B cells from the spleens of wild - type , Prdm1ihCd2 / + and Prdm1ihCd2 / ihCd2 miceDetermination of cell death of ex vivo pro - B and pre - B cells from the bone marrow of Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice by flow cytometric analysis of the loss of plasma membrane asymmetry ( F2N12S ratiometric dye staining ) and the loss of cell membrane integrity ( SYTOX staining ) . Representative flow cytometry plots ( left ) and the quantification of dead and apoptotic ( apop ) cells ( right ) are shownBrdU labeling of immature ( imm ) and FO B cells in the spleen of 3 - month - old Prdm1ihCd2 / + mice ( red dots ) and control wild - type littermates ( gray dots ) . The percentage of BrdU + B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling ( day 10 , white bar ) or after a subsequent 15 - day chase period ( day 25 ; hatched bar ) without BrdU in the drinking wateBrdU labeling of immature ( imm ) and FO B cells in the spleen of 3 - month - old Prdm1ihCd2 / + mice ( red dots ) and control wild - type littermates ( gray dots ) A diagram ( below ) indicates the design of the BrdU labeling and chase experiments . Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in ( F ) The percentage of BrdU + FO B cells is shown above the indicated gates ( F )

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

Figure 3Flow cytometric analysis of total B cells (A) as well as pro-B, pre-B, immature (imm) B and recirculating (recirc) B cells (B) from the bone marrow of wild-type (WT, gray), Prdm1ihCd2/+ (red) and Prdm1ihCd2/ihCd2 (white) mice at the age of 6-8 weeks Flow cytometric analysis of total B, FO B and MZ B cells from the spleens of wild-type, Prdm1ihCd2/+ and Prdm1ihCd2/ihCd2 miceDetermination of cell death of ex vivo pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice by flow cytometric analysis of the loss of plasma membrane asymmetry (F2N12S ratiometric dye staining) and the loss of cell membrane integrity (SYTOX staining). Representative flow cytometry plots (left) and the quantification of dead and apoptotic (apop) cells (right) are shownBrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots). The percentage of BrdU+ B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling (day 10, white bar) or after a subsequent 15-day chase period (day 25; hatched bar) without BrdU in the drinking wateBrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots) A diagram (below) indicates the design of the BrdU labeling and chase experiments. Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in (F) The percentage of BrdU+ FO B cells is shown above the indicated gates (F)


Figure 4Scatter plot of gene expression differences in ex vivo sorted Prdm1ihCd2 / + and wild - type ( WT ) pro - B ( A ) and pre - B ( B ) cells , based on two RNA - seq experiments for each cell type and genotype . The expression data of individual genes ( indicated by dots ) were plotted as normalized ( norm ) rlog values . Genes with an expression difference of > 3 - fold , an adjusted P value of < 0 . 05 and a TPM value of > 5 ( in one of the two pro - B or pre - B cell types , respectively ) are colored in blue or red , corresponding to activation or repression by Blimp1 . ( C ) Expression of commonly activated ( blue ) and commonly repressed ( red ) genes in pro - B and pre - B cells . The log2 - fold expression change observed between Prdm1ihCd2 / + and wild - type pro - B cells ( horizontal axis ) as well as between Prdm1ihCd2 / + and wild - type pre - B cells ( vertical axis ) is plotted for each gene . Lines indicated 2 - fold ( 2x ) and 3 - fold ( 3x ) expression differencesIdentification of 762 Blimp1 peaks in short - term cultured Prdm1ihCd2 / + pro - B cells , as detected by ChIP - seq with an anti - V5 antibody and MACS peak calling with a P value of < 10 - 10 . The 9320 Blimp1 peaks identified in plasmablasts ( PB ; Minnich et al . , 2016 ) are shown for comparison . Common ( black ) and unique ( white ) peaks are shown for both cell types Densities of Blimp1 binding . Average read density profiles aligned at the center of the Blimp1 peak are shown for the common Blimp peaks of both cell typesBlimp1 binding at activated and repressed genes in Prdm1ihCd2 / + pro - B and pre - B cells . Regulated genes , which are bound by Blimp1 in Prdm1ihCd2 / + pro - B cells , are shown in blackBlimp1 binding and regulation of the commonly repressed target genes Sell ( CD62L ) and B3gnt5 . The ChIP - seq data ( left ) were obtained with Prdm1ihCd2 / + pro - B cells ( this study ) and Prdm1Bio / Bio Rosa26BirA / BirA plasmablasts ( PB ; Minnich et al . , 2016 ) . The RNA - seq data ( right ) were determined in pro - B and pre - B cells of the Prdm1ihCd2 / + ( red ) and wild - type ( gray ) genotype and in Blimp1 - deficient ( Prdm1Gfp / ∆ , blue ) and wild - type ( grey ) pre - plasmablasts ( Pre - PB ; Minnich et al . , 2016 ) . Cell types expressing Blimp1 ( + ) are indicatedExpression of selected Blimp1 - activated and Blimp1 - repressed genes coding for transcriptional regulators ( H ) and intracellular signal transducer in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) pre - B cells . Blimp1 - bound genes are underlined . The mRNA expression of the indicated genes is shown as mean expression value ( TPM ) with SEM , based on two different RNA - seq experiments for pre - B cells of each

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

Figure 4Scatter plot of gene expression differences in ex vivo sorted Prdm1ihCd2/+ and wild-type (WT) pro-B (A) and pre-B (B) cells, based on two RNA-seq experiments for each cell type and genotype. The expression data of individual genes (indicated by dots) were plotted as normalized (norm) rlog values. Genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in one of the two pro-B or pre-B cell types, respectively) are colored in blue or red, corresponding to activation or repression by Blimp1. (C) Expression of commonly activated (blue) and commonly repressed (red) genes in pro-B and pre-B cells. The log2-fold expression change observed between Prdm1ihCd2/+ and wild-type pro-B cells (horizontal axis) as well as between Prdm1ihCd2/+ and wild-type pre-B cells (vertical axis) is plotted for each gene. Lines indicated 2-fold (2x) and 3-fold (3x) expression differencesIdentification of 762 Blimp1 peaks in short-term cultured Prdm1ihCd2/+ pro-B cells, as detected by ChIP-seq with an anti-V5 antibody and MACS peak calling with a P value of < 10-10. The 9320 Blimp1 peaks identified in plasmablasts (PB; Minnich et al., 2016) are shown for comparison. Common (black) and unique (white) peaks are shown for both cell types Densities of Blimp1 binding. Average read density profiles aligned at the center of the Blimp1 peak are shown for the common Blimp peaks of both cell typesBlimp1 binding at activated and repressed genes in Prdm1ihCd2/+ pro-B and pre-B cells. Regulated genes, which are bound by Blimp1 in Prdm1ihCd2/+ pro-B cells, are shown in blackBlimp1 binding and regulation of the commonly repressed target genes Sell (CD62L) and B3gnt5. The ChIP-seq data (left) were obtained with Prdm1ihCd2/+ pro-B cells (this study) and Prdm1Bio/Bio Rosa26BirA/BirA plasmablasts (PB; Minnich et al., 2016). The RNA-seq data (right) were determined in pro-B and pre-B cells of the Prdm1ihCd2/+ (red) and wild-type (gray) genotype and in Blimp1-deficient (Prdm1Gfp/∆, blue) and wild-type (grey) pre-plasmablasts (Pre-PB; Minnich et al., 2016). Cell types expressing Blimp1 (+) are indicatedExpression of selected Blimp1-activated and Blimp1-repressed genes coding for transcriptional regulators (H) and intracellular signal transducer in Prdm1ihCd2/+ (red) and wild-type (gray) pre-B cells. Blimp1-bound genes are underlined. The mRNA expression of the indicated genes is shown as mean expression value (TPM) with SEM, based on two different RNA-seq experiments for pre-B cells of each genotype


Figure 5Flow cytometric analysis of plasma cells from the spleen ( A ) and bone marrow ( B ) of Prdm1ihCd2 / + and wild - type ( WT ) mice under steady - state conditions . Representative flow cytometric data are shown for 12 - month - old mice ( left ) , while bar graphs present absolute numbers of plasma cells for Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice at the age of 2 , 4 and 12 months ( right ) T cell - dependent immune responses . Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 months were immunized with NP - KLH ( in alum ) and analyzed at day 14 after immunization by ELISPOT assa The number of anti - NP - IgM antibody - secreting cells ( ASCs ) in the spleen was determined by ELISPOT assay ( C ) using NP24 - BSA - coated plates for detecting cells secreting total anti - NP - IgM antibodies . ELISPOT results are shown as representative pictures ( left ) or absolute ASC numbers ( right ) ( C ) Dot plots display the serum immunoglobulin titers of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice NP - specific IgG1 concentrations ( μg / ml ) were determined relative to a standard NP - binding IgG1 antibodyT cell - dependent immune responses . Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 months were immunized with NP - KLH ( in alum ) and analyzed at day 14 after immunization b ELISA The serum titers of anti - NP - specific IgM , IgG1 and IgG2b antibodies were analyzed by ELISA using NP7 - BSA - or NP24 - BSA - coated plates for detecting high - affinity IgG1 or total IgM , IgG1 and IgG2b antibodies , respectivel Dot plots display the serum immunoglobulin titers of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice . NP - specific IgG1 concentrations ( μg / ml ) were determined relative to a standard NP - binding IgG1 antibody , whereas IgM and IgG2b amounts are indicated as relative units ( RU ) by setting the WT data to 1Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization . Bar graphs display absolute numbers of GC B cells detected in the spleen of both genotype The absence of Bcl6 expression in non - GC B cells ( black ) is shown as referenceFlow cytometric analysis of splenic GC B cells in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2 / + ( red ) and wild - type ( gray ) GC B cell The absence of Bcl6 expression in non - GC B cells ( black ) is shown as referenceFlow cytometric analysi in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Flow cytometric analysis of splenic TFH cell Bar graphs indicate absolute numbers of TFH cell in hCD2lo ( black ) and hCD2hi ( blue ) Prdm1ihCd2 / + as well as wild - type ( gray ) TFH cellsFlow cytometric analysi in Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice ( at the age of 2 months ) at day 14 after NP - KLH immunization Flow cytometric analysis of splenic TFH cell histograms display cell surface expression of hCD2 and intracellular staining of Bcl6 ( H ) in hCD2lo ( black ) and hCD2hi ( blue ) Prdm1ihCd2 / + as well as wild - type ( gray ) TFH cellsEfficient in vitro plasmablast differentiation of immature B cells . Immature B cells sorted from the bone marrow of Prdm1ihCd2 / + ( red ) and wild - type ( gray ) mice were stimulated with CpG oligodeoxynucleotides or LPS for 60 hours followed by flow cytometric analysis of CD138 + CD22lo plasmablasts ( PB ) . Bar graphs ( right ) summarize the plasmablast data of the CpG and LPS stimulation experimentsELISA detection of Igκ - containing antibodies in the supernatants of immature B cells at 60 h of CpG or LPS

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Figure 5Flow cytometric analysis of plasma cells from the spleen (A) and bone marrow (B) of Prdm1ihCd2/+ and wild-type (WT) mice under steady-state conditions. Representative flow cytometric data are shown for 12-month-old mice (left), while bar graphs present absolute numbers of plasma cells for Prdm1ihCd2/+ (red) and wild-type (gray) mice at the age of 2, 4 and 12 months (right)T cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization by ELISPOT assa The number of anti-NP-IgM antibody-secreting cells (ASCs) in the spleen was determined by ELISPOT assay (C) using NP24-BSA-coated plates for detecting cells secreting total anti-NP-IgM antibodies. ELISPOT results are shown as representative pictures (left) or absolute ASC numbers (right) (C) Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibodyT cell-dependent immune responses. Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2 months were immunized with NP-KLH (in alum) and analyzed at day 14 after immunization b ELISA The serum titers of anti-NP-specific IgM, IgG1 and IgG2b antibodies were analyzed by ELISA using NP7-BSA- or NP24-BSA-coated plates for detecting high-affinity IgG1 or total IgM, IgG1 and IgG2b antibodies, respectivel Dot plots display the serum immunoglobulin titers of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice. NP-specific IgG1 concentrations (μg/ml) were determined relative to a standard NP-binding IgG1 antibody, whereas IgM and IgG2b amounts are indicated as relative units (RU) by setting the WT data to 1Flow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization. Bar graphs display absolute numbers of GC B cells detected in the spleen of both genotype The absence of Bcl6 expression in non-GC B cells (black) is shown as referenceFlow cytometric analysis of splenic GC B cells in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Cell surface expression of hCD2 and intracellular staining of Bcl6 are shown for Prdm1ihCd2/+ (red) and wild-type (gray) GC B cell The absence of Bcl6 expression in non-GC B cells (black) is shown as referenceFlow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell Bar graphs indicate absolute numbers of TFH cell in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cellsFlow cytometric analysi in Prdm1ihCd2/+ (red) and wild-type (gray) mice (at the age of 2 months) at day 14 after NP-KLH immunization Flow cytometric analysis of splenic TFH cell histograms display cell surface expression of hCD2 and intracellular staining of Bcl6 (H) in hCD2lo (black) and hCD2hi (blue) Prdm1ihCd2/+ as well as wild-type (gray) TFH cellsEfficient in vitro plasmablast differentiation of immature B cells. Immature B cells sorted from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice were stimulated with CpG oligodeoxynucleotides or LPS for 60 hours followed by flow cytometric analysis of CD138+CD22lo plasmablasts (PB). Bar graphs (right) summarize the plasmablast data of the CpG and LPS stimulation experimentsELISA detection of Igκ-containing antibodies in the supernatants of immature B cells at 60 h of CpG or LPS stimulation


Figure 6Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow of non - immunized Eβ - / - Prdm1ihCd2 / + ( red ) and Eβ - / - Prdm1 + / + ( gray ) chimeric mice . Bar graphs indicate absolute numbers of the different B cell types in these chimeric miceFlow cytometric analysis of total B cells and different B cell subsets in th splee of non - immunized Eβ - / - Prdm1ihCd2 / + ( red ) and Eβ - / - Prdm1 + / + ( gray ) chimeric mice . Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 ( Blimp1 ) in splenic FO B , GC B and naïve CD4 T cells from the chimeric mice of the indicated

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

Figure 6Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric miceFlow cytometric analysis of total B cells and different B cell subsets in th splee of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice Histograms display the expression of hCD2 (Blimp1) in splenic FO B, GC B and naïve CD4 T cells from the chimeric mice of the indicated genotypes


Figure 7Presence of IgG antibodies detecting dsDNA , cardiolipin , SSA ( Ro - 52 ) and SSB ( La ) in the serum of Prdm1ihCd2 / + ( red dots ) and wild - type ( gray dots ) mice at the age of 2 , 4 and 12 months . The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units ( AU ) . The serum of 6 - month - old Faslgld / gld mice was used as positive controlRepresentative images of anti - nuclear antibody ( ANA ) staining obtained with serum from 12 - month - old Prdm1ihCd2 / + or wild - type mice ( right ) , as detected by indirect immunofluorescence assay using HEp - 2 cells and an Alexa488 - conjugated anti - mouse IgG detection antibody . The serum of Faslgld / gld mice was used as positive control . The presence of ANA - IgG antibodies was analyzed for 22 Prdm1ihCd2 / + and 12 wild - type mice , as summarized in the pie - chart ( right ) Periodic acid - Schiff ( PAS ) staining of paraffin - embedded kidney sections of Prdm1ihCd2 / + and wild - type mice at 12 months of age . The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods . Representative pictures of glomeruli of Prdm1ihCd2 / + kidneys with different pathology scores are shown to the left , and dot plots with average scores for male and female mice ( Table EV2 ) are shown to the right . Arrows denote intracapillary hyaline " thrombi " , asterisks indicate mesangial sclerosis with obscured capillary loops , and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture . Statistical data ( A , C ) are shown as mean value with SEM and were analyzed by the Mann - Whitney test ( A ) or the Student " s t - test ( C ) ; * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Each dot

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

Figure 7Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive controlRepresentative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right)Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline "thrombi", asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student"s t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot correspon


Figure 1 ( A ) The intracellular distribution of DOR is modified by starvation . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) , HBSS ( starvation ) , 50 μM chloroquine or 10 mM 3MA . The intracellular localization of DOR was analysed by immunofluorescence and is shown in red . The nuclei are shown in blue ( DAPI staining ) . Scale bars , 10 μm . ( B ) Endogenous DOR leaves the nucleus under starvation . C2C12 myoblasts were incubated for 30 min at 37 ° C with DMEM ( basal ) or HBSS ( starvation ) . DOR is shown in red and nuclei are in blue ( DAPI staining ) . Scale bars , 5 μm . ( C , D ) De ‐ activation of autophagy returns DOR to the nucleus . HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM ( basal ) or HBSS ( starvation ) . Cells were then incubated with normal DMEM for an additional hour ( starvation + GM ) . The intracellular distribution of DOR was determined by immunofluorescence . DOR is shown in red and the nuclei are in blue . Scale bars , 5 μm . Cell lysates were also obtained and western blot assays were performed with specific antibodies . 3MA , 3 ‐ methyladenine ; DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; HBSS , Hank ' s balanced salt solution ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

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Figure 1(A) The intracellular distribution of DOR is modified by starvation. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal), HBSS (starvation), 50 μM chloroquine or 10 mM 3MA. The intracellular localization of DOR was analysed by immunofluorescence and is shown in red. The nuclei are shown in blue (DAPI staining). Scale bars, 10 μm.(B) Endogenous DOR leaves the nucleus under starvation. C2C12 myoblasts were incubated for 30 min at 37°C with DMEM (basal) or HBSS (starvation). DOR is shown in red and nuclei are in blue (DAPI staining). Scale bars, 5 μm.(C,D) De‐activation of autophagy returns DOR to the nucleus. HeLa cells were transiently transfected with DOR and incubated for 1 h with DMEM (basal) or HBSS (starvation). Cells were then incubated with normal DMEM for an additional hour (starvation+GM). The intracellular distribution of DOR was determined by immunofluorescence. DOR is shown in red and the nuclei are in blue. Scale bars, 5 μm. Cell lysates were also obtained and western blot assays were performed with specific antibodies. 3MA, 3‐methyladenine; DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; HBSS, Hank's balanced salt solution; LC3, microtubule‐associated protein 1A/1B‐light chain 3.


Figure 2DOR localizes to autophagosomes when autophagy is activated . ( A ) Confocal images of HeLa cells transiently transfected with DOR and GFP ‐ LC3 , and incubated for 1h with DMEM , HBSS ( starvation ) , 2 μM rapamycin or HBSS containing 50 μM chloroquine . Nuclei were labelled with DAPI . Scale bars , 10 μm . ( B , C ) Confocal images of HeLa cells transiently transfected with DOR or GFP ‐ LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS . Scale bars , 10 μm . ( D ) Confocal images of HeLa cells transiently transfected with DOR and LAMP1 ‐ GFP , or with YFP ‐ LC3 and LAMP1 ‐ GFP . Scale bars , 10 μm . Contrast ‐ corrected merge RGB pictures and the Z ‐ projection of Product of the differences from the mean ( PDM ) images are shown in all panels . Colour scales of PDM images have different maximal values , so the different conditions are not comparable from these projections . Product of the differences from the mean ( PDM ) values closer to 1 show reliable colocalized pixels . DAPI , 4 ′ , 6 ‐ diamidino ‐ 2 ‐ phenylindole ; DMEM , Dulbecco ' s modified Eagle medium ; DOR , diabetes ‐ and obesity ‐ regulated gene ; GFP , green fluorescent protein ; HBSS , Hank ' s balanced salt solution ; LAMP1 , lysosomal ‐ associated membrane protein 1 ; LC3 , microtubule ‐ associated protein 1A / 1B ‐ light chain 3 .

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Figure 2DOR localizes to autophagosomes when autophagy is activated. (A) Confocal images of HeLa cells transiently transfected with DOR and GFP‐LC3, and incubated for 1h with DMEM, HBSS (starvation), 2 μM rapamycin or HBSS containing 50 μM chloroquine. Nuclei were labelled with DAPI. Scale bars, 10 μm.(B,C) Confocal images of HeLa cells transiently transfected with DOR or GFP‐LC3 and incubated with 50 nM Lysotracker in DMEM or HBSS. Scale bars, 10 μm.(D) Confocal images of HeLa cells transiently transfected with DOR and LAMP1‐GFP, or with YFP‐LC3 and LAMP1‐GFP. Scale bars, 10 μm. Contrast‐corrected merge RGB pictures and the Z‐projection of Product of the differences from the mean (PDM) images are shown in all panels. Colour scales of PDM images have different maximal values, so the different conditions are not comparable from these projections. Product of the differences from the mean (PDM) values closer to 1 show reliable colocalized pixels. DAPI, 4′,6‐diamidino‐2‐phenylindole; DMEM, Dulbecco's modified Eagle medium; DOR, diabetes‐ and obesity‐regulated gene; GFP, green fluorescent protein; HBSS, Hank's balanced salt solution; LAMP1, lysosomal‐associated membrane protein 1; LC3, microtubule‐associated protein 1A/1B‐light chain 3.