Figure 4B The time course of the distance between the geometrical centers of domains a and a ′ and the angle among domains b , b ′ and a ′ from the simulation of pS357 - PDI ( red ) and PDI ( black ) ( Yang et al . , 2014 ) . C ( Left ) ANS fluorescence spectra of PDI WT and S357E . Controls with ANS alone was shown . A . U . , arbitrary units . ( Right ) Enhancement factor were quantified . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . D Limited proteolysis of PDI WT ( Left ) and S357E ( Right ) proteins by proteinase K ( Pro K ) for indicated times . Main digested fragments were indicated on the right margin . Band abb ' x * is a digested product with cleavage after Met356 in the x - linker region ( Wang et al . , 2010 ) . E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min , and in some aliquots of cells Tg was removed for 30 min . Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis . F HepG2 cells expressing HA - tagged PDI and FLAG - tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis . Chaperone activities of PDI WT and S357E ( G , H ) or phosphorylated PDI WT and S357A by Fam20C for indicated times ( I Denatured and reduced GAPDH ( G , I ) and rhodanese ( H , were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . In ( I , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J ) . Denatured and reduced rhodanese J ) were used as substrates , respectively . The chaperone activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Reductase activities of PDI WT and S357E ( K ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Isomerase activities of PDI WT and S357E ( L ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * p < 0 . 01 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( M ) measured by insulin reduction assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times ( N ) measured by scrambled RNase A reactivation assay . The activity of PDI WT was taken as 100 % . Data information : All data were shown as mean ± SEM of three independent experiments . , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 4B The time course of the distance between the geometrical centers of domains a and a′ and the angle among domains b, b′ and a′ from the simulation of pS357-PDI (red) and PDI (black) (Yang et al., 2014).C (Left) ANS fluorescence spectra of PDI WT and S357E. Controls with ANS alone was shown. A.U., arbitrary units. (Right) Enhancement factor were quantified. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).D Limited proteolysis of PDI WT (Left) and S357E (Right) proteins by proteinase K (Pro K) for indicated times. Main digested fragments were indicated on the right margin. Band abb'x* is a digested product with cleavage after Met356 in the x-linker region (Wang et al., 2010).E shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 30 min, and in some aliquots of cells Tg was removed for 30 min. Cell lysates were subjected to proteinase K digestion for 3 min and immunoblotting analysis.F HepG2 cells expressing HA-tagged PDI and FLAG-tagged Fam20C with different ratios of plasmid concentrations were subjected to proteinase K digestion for 21 min and immunoblotting analysis.Chaperone activities of PDI WT and S357E (G, H) or phosphorylated PDI WT and S357A by Fam20C for indicated times (I Denatured and reduced GAPDH (G, I) and rhodanese (H, were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test). In (I , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Chaperone activities of phosphorylated PDI WT and S357A by Fam20C for indicated times J). Denatured and reduced rhodanese J) were used as substrates, respectively. The chaperone activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Reductase activities of PDI WT and S357E (K) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).Isomerase activities of PDI WT and S357E (L) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , **p < 0.01, ***p < 0.001 (two-tailed Student's t-test).Reductase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (M) measured by insulin reduction assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Isomerase activities of phosphorylated PDI WT and S357A by Fam20C for indicated times (N) measured by scrambled RNase A reactivation assay. The activity of PDI WT was taken as 100%. Data information: All data were shown as mean ± SEM of three independent experiments. , ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).


Figure 5B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER ( TMR - labeled , red ) . Endogenous PDI was immunostained ( green ) as an ER marker . Scale bar = 10 μm . C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) , followed by introduction of DMSO or 5 μM Tg for 30 min . Scale bar = 10 μm . D Confocal live cell imaging of WT and PDI KO HepG2 cells co - transfected with AgHaloER labeled by P1 ( green ) and TMR ( red ) and empty vector ( - ) , PDI WT , S357E or S357A . Scale bar = 10 μm . E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 ( green ) and TMR ( red ) . Scale bar = 10 μm . F ( Upper ) Cell viabilities of WT and PDI KO HepG2 cells co - transfected with AgHaloER and empty vector ( - ) , PDI WT , S357E or S357A . Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining . ( Lower ) Quantification of surviving fraction of cells . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 001 versus WT HepG2 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5B Fluorescent photomicrographs of HepG2 cells expressing AgHaloER (TMR-labeled, red). Endogenous PDI was immunostained (green) as an ER marker. Scale bar = 10 μm.C Confocal live cell imaging of HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red), followed by introduction of DMSO or 5 μM Tg for 30 min. Scale bar = 10 μm.D Confocal live cell imaging of WT and PDI KO HepG2 cells co-transfected with AgHaloER labeled by P1 (green) and TMR (red) and empty vector (-), PDI WT, S357E or S357A. Scale bar = 10 μm.E Confocal live cell imaging of shCtrl and shFAM20C HepG2 cells expressing AgHaloER labeled by P1 (green) and TMR (red). Scale bar = 10 μm.F (Upper) Cell viabilities of WT and PDI KO HepG2 cells co-transfected with AgHaloER and empty vector (-), PDI WT, S357E or S357A. Cells were treated by 5 μM Tg for indicated times and visualized by crystal violet staining. (Lower) Quantification of surviving fraction of cells. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus WT HepG2 (two-way ANOVA, the post hoc Tukey's HSD test).


Figure 6A ( Left ) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h . PDI KO was verified by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 ( two - tailed Student ' s t - test ) . B ( Left ) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h . PDI expression levels were shown by protein immunoblotting . ( Right ) Quantification of XBP1 mRNA splicing levels . Data were shown as mean ± SEM of three independent experiments . * p < 0 . 05 , * * p < 0 . 01 ( one - way ANOVA , the post hoc Tukey ' s HSD test ) . C Detection of IRE1α , PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min . Asterisk indicates unspecific background band . D HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting . E HepG2 cells expressing FLAG - tagged IRE1α were treated without or with 5 μM Tg for 1 h . An aliquot of Tg - treated cells was then washed with culture medium and chased for 4 h . FLAG - immunoprecipitates were analyzed by PDI and BiP immunoblotting . F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h . Endogenous IRE1α - immunoprecipitates were analyzed by PDI and BiP immunoblotting . G Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI in HepG2 cells expressing V5 - tagged Fam20C WT or DA . H Co - immunoprecipitation of FLAG - tagged IRE1α , HA - tagged PDI WT or S357A in HepG2 cells expressing V5 - tagged Fam20C . I Co - immunoprecipitation of HA - tagged PDI and FLAG - tagged IRE1αNLD in HepG2 cells . J Recombinant GST - PDI was phosphorylated by Fam20C protein for 2 h , and then used to pulldown His - IRE1αNLD protein , as visualized by Coomassie blue staining . Successful phosphorylation of GST - PDI was verified by pS357 - PDI immunoblotting . K Co - immunoprecipitation of FLAG - tagged IRE1αNLD WT or its mutant C109 / 148 / 332S with HA - tagged PDI ( Left ) or MYC - tagged P5 ( Right ) in HepG2 cells . L Co - immunoprecipitation of FLAG - tagged IRE1α and HA - tagged PDI WT or its binding mutant W128I / L403W in HepG2 cells expressing V5 - tagged Fam20C or not .

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

Figure 6A (Left) Detection of XBP1 mRNA splicing in WT and PDI KO HepG2 cells treated with or without 5 μM Tg for 1 h. PDI KO was verified by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05 (two-tailed Student's t-test).B (Left) Detection of XBP1 mRNA splicing in PDI KO HepG2 cells expressing PDI WT or its mutants treated with or without 5 μM Tg for 1 h. PDI expression levels were shown by protein immunoblotting. (Right) Quantification of XBP1 mRNA splicing levels. Data were shown as mean ± SEM of three independent experiments. *p < 0.05, **p < 0.01 (one-way ANOVA, the post hoc Tukey's HSD test).C Detection of IRE1α, PERK and ATF6 activation by immunoblotting in PDI KO HepG2 cells expressing PDI S537A or S357E treated with 5 μM Tg for 30 min. Asterisk indicates unspecific background band.D HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.E HepG2 cells expressing FLAG-tagged IRE1α were treated without or with 5 μM Tg for 1 h. An aliquot of Tg-treated cells was then washed with culture medium and chased for 4 h. FLAG-immunoprecipitates were analyzed by PDI and BiP immunoblotting.F shCtrl and shFAM20C HepG2 cells were treated without or with 5 μM Tg for 1 h. Endogenous IRE1α-immunoprecipitates were analyzed by PDI and BiP immunoblotting.G Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI in HepG2 cells expressing V5-tagged Fam20C WT or DA.H Co-immunoprecipitation of FLAG-tagged IRE1α, HA-tagged PDI WT or S357A in HepG2 cells expressing V5-tagged Fam20C.I Co-immunoprecipitation of HA-tagged PDI and FLAG-tagged IRE1αNLD in HepG2 cells.J Recombinant GST-PDI was phosphorylated by Fam20C protein for 2 h, and then used to pulldown His-IRE1αNLD protein, as visualized by Coomassie blue staining. Successful phosphorylation of GST-PDI was verified by pS357-PDI immunoblotting.K Co-immunoprecipitation of FLAG-tagged IRE1αNLD WT or its mutant C109/148/332S with HA-tagged PDI (Left) or MYC-tagged P5 (Right) in HepG2 cells.L Co-immunoprecipitation of FLAG-tagged IRE1α and HA-tagged PDI WT or its binding mutant W128I/L403W in HepG2 cells expressing V5-tagged Fam20C or not.


Figure 7B Sanger sequencing results of Pdi + / + ( WT ) and PdiS359A / S359A ( KI ) mice . C ( Left ) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm ( 50 ng / g , i . p . ) for indicated times . Each lane represents independent animals . ( Right ) Quantification of Xbp1 mRNA splicing levels at indicated times . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) . Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 2 μM Tg for 2 h ( D ) Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0 . 5 μg / ml Tm for 2 h ( E ) . Detection of Bip ( F ) , Pdi ( G ) , Erdj4 ( H ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . Detection of Edem1 ( I ) , Il6 ( J ) , Chop ( K ) mRNA levels by real - time PCR in the liver of the same animals after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . L Detection of serum alanine transaminase ( ALT ) levels of the same animals as in ( C ) after 24 h of Tm injection . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - way ANOVA , the post hoc Tukey ' s HSD test ) . M Hematoxylin and eosin staining of liver tissue the same animals as in ( C ) after 24 h of Tm injection ( magnification × 400 ) . Immune cell infiltration was indicated with a black arrow . Scale bar = 50 μm . Representative images were shown from five animals per group analyzed . N ( Left ) TUNEL assay of the same liver as in ( M ) . Scale bar = 40 μm ; Zoom box , scale bar = 5 μm . ( Right ) Quantification of apoptotic cells . 10 random fields were counted for each TUNEL - stained tissue sample . Data information : All data were shown as mean ± SEM from five biological replicates . , * p < 0 . 05 , * * * p < 0 . 001 ( two - tailed Student ' s t - test ) .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 7B Sanger sequencing results of Pdi+/+ (WT) and PdiS359A/S359A (KI) mice.C (Left) Detection of Xbp1 mRNA splicing in mice liver after injection of Tm (50 ng/g, i.p.) for indicated times. Each lane represents independent animals. (Right) Quantification of Xbp1 mRNA splicing levels at indicated times. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.2 μM Tg for 2 h (D)Detection of Xbp1 mRNA splicing in primary hepatocytes isolated from WT and KI mice treated with 0.5 μg/ml Tm for 2 h (E).Detection of Bip (F), Pdi (G), Erdj4 (H) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).Detection of Edem1 (I), Il6 (J), Chop (K) mRNA levels by real-time PCR in the liver of the same animals after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).L Detection of serum alanine transaminase (ALT) levels of the same animals as in (C) after 24 h of Tm injection. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-way ANOVA, the post hoc Tukey's HSD test).M Hematoxylin and eosin staining of liver tissue the same animals as in (C) after 24 h of Tm injection (magnification × 400). Immune cell infiltration was indicated with a black arrow. Scale bar = 50 μm. Representative images were shown from five animals per group analyzed.N (Left) TUNEL assay of the same liver as in (M). Scale bar = 40 μm; Zoom box, scale bar = 5 μm. (Right) Quantification of apoptotic cells. 10 random fields were counted for each TUNEL-stained tissue sample. Data information: All data were shown as mean ± SEM from five biological replicates. , *p < 0.05, ***p < 0.001 (two-tailed Student's t-test).


Figure 1Cryo - TEM images demonstrate the lipid bilayer EV purified from Molm - 14 and U - 937 cells . Scale bars are 100nm . Peripheral blood ( PB ) plasma of control animals contain no mGFP + foci ( top ) however Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) xenografts contain numerous mGFP - labeled EV ( green ) detectible without vesicle concentration . Scale bars are 5μm . Live - cell microscopy of KSL cells ( left panel ) LT - HSC cells ( right panel ) . Cells were sorted from control mice ( top ) , Molm - 14 - mGFP ( middle ) and U - 937 - mGFP ( bottom ) , stained with Cell Mask ( red ) and nuclear stain Hoechst ( blue ) ; imaged using the GE / API Deltavision ( DV ) widefield microscope ( 60X objective ) to show the uptake of the GFP + EV ( green ) . Scale bars are 5μm .

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 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

Figure 1Cryo-TEM images demonstrate the lipid bilayer EV purified from Molm-14 and U-937 cells. Scale bars are 100nm.Peripheral blood (PB) plasma of control animals contain no mGFP+ foci (top) however Molm-14-mGFP (middle) and U-937-mGFP (bottom) xenografts contain numerous mGFP-labeled EV (green) detectible without vesicle concentration. Scale bars are 5μm.Live-cell microscopy of KSL cells (left panel) LT-HSC cells (right panel). Cells were sorted from control mice (top), Molm-14-mGFP (middle) and U-937-mGFP (bottom), stained with Cell Mask (red) and nuclear stain Hoechst (blue); imaged using the GE/API Deltavision (DV) widefield microscope (60X objective) to show the uptake of the GFP+ EV (green). Scale bars are 5μm.


Figure 2Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : Molm - 14 - xenograft ( red , n = 10 ) vs . control ( black , n = 10 ) . Data were obtained from at least two independent experiments . Flow cytometric analysis showing the frequency of LK and KSL cells in lineage - negative cells ( left panel ) and LT - HSC in KSL ( right panel ) in : IF injection of EV from Molm - 14 cells ( red , n = 8 ) , AML plasma EV ( Orange , n = 6 ) and human CD34 EV ( Blue , n = 4 ) relative to the vehicle - injected contralateral femurs . Data were obtained from at least two independent experiments . Cell - cycle histograms of KSL ( upper panel ) and LT - HSC ( lower panel ) from Molm - 14 - xenografts or control using Hoechst - 33342 staining . Flow cytometric analysis of the Ki67 - ve percentage of LT - HSC representing the G0 phase of cell - cycle in : ( left panel ) Molm - 14 - xenografts ( red , n = 7 ) vs . control ( black , n = 9 ) ; ( right panel ) IF injection of Molm - 14 - EV , U - 937 EV , HL - 60 EV ( red , n = 5 , 4 , 4 ) vs . human CD34 EV ( blue , n = 3 ) vs . controls ( black ) . Flow cytometric analysis of intracellular P53 levels shown in histograms ( F ) of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs . Flow cytometric analysis of intracellular P53 levels shown in histograms of LT - HSC in : ( left panel ) Molm - 14 - xenograft , n = 6 red vs . non - engrafted control , n = 4 black , or ( right panel ) IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 5 , 3 ) or human CD34 cells ( blue , n = 6 ) normalized to vehicle - injected contralateral femurs .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 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 2Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: Molm-14-xenograft (red, n=10) vs. control (black, n=10). Data were obtained from at least two independent experiments.Flow cytometric analysis showing the frequency of LK and KSL cells in lineage-negative cells (left panel) and LT-HSC in KSL (right panel) in: IF injection of EV from Molm-14 cells (red, n=8), AML plasma EV (Orange, n=6) and human CD34 EV (Blue, n=4) relative to the vehicle-injected contralateral femurs. Data were obtained from at least two independent experiments.Cell-cycle histograms of KSL (upper panel) and LT-HSC (lower panel) from Molm-14-xenografts or control using Hoechst-33342 staining.Flow cytometric analysis of the Ki67-ve percentage of LT-HSC representing the G0 phase of cell-cycle in: (left panel) Molm-14-xenografts (red, n=7) vs. control (black, n=9); (right panel) IF injection of Molm-14-EV, U-937 EV, HL-60 EV (red, n=5,4,4) vs. human CD34 EV (blue, n=3) vs. controls (black).Flow cytometric analysis of intracellular P53 levels shown in histograms (F) of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.Flow cytometric analysis of intracellular P53 levels shown in histograms of LT-HSC in: (left panel) Molm-14-xenograft, n=6 red vs. non-engrafted control, n=4 black, or (right panel) IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,5,3) or human CD34 cells (blue, n=6) normalized to vehicle-injected contralateral femurs.


Figure 3A heatmap showing the highly - deregulated ribosomal proteins in AML - EV exposed cells . Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : Molm - 14 - xenografts , red n = 6 vs . non - engrafted controls , black n = 6 , Flow cytometric analysis showing the histograms and MFI of O - propagyl - puromysin ( OPP ) incorporation in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 5 , 43 ) , AML patients plasma ( Orange , n = 6 ) , or human CD34 + cells ( blue , n = 3 ) normalized to vehicle - injected contralateral femurs after subtracting the background fluorescence .

O B-PANEL_START 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 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

Figure 3A heatmap showing the highly-deregulated ribosomal proteins in AML-EV exposed cells.Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: Molm-14-xenografts, red n=6 vs. non-engrafted controls, black n=6,Flow cytometric analysis showing the histograms and MFI of O-propagyl-puromysin (OPP) incorporation in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=5,43), AML patients plasma (Orange, n=6), or human CD34+ cells (blue, n=3) normalized to vehicle-injected contralateral femurs after subtracting the background fluorescence.


Figure 4qRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : ( A ) Molm - 14 - xenografted miceqRT - PCR gene expression analysis showing the fold change of Pi3K / mTOR associated gene panel in KSL cells sorted from : IF injected mice with Molm - 14 - EV relative to their controls and normalized to Gapdh endogenous control . Flow cytometric analysis showing the histograms ( C ) Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : ( D ) Molm - 14 - xenograft ( red , n = 9 ) vs . non - engrafted control ( black , n = 7 ) , Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT - HSC in : IF injection of EV from Molm - 14 , U - 937 , HL - 60 ( red , n = 7 , 3 , 3 ) or human CD34 cells ( blue , n = 4 ) normalized to the control contralateral femur with subtraction of background fluorescence . Flow cytometric assessment of protein synthesis showing the MFI of : ( F ) OPP , or ( G ) pS6RP in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The results were calculated relative to control ( miR - scramble ) with the background fluorescence subtracted and performed with at least three independent experiments , in technical replicates . Flow cytometric analysis showing the MFI of Raptor in NIH - 3T3 cells 72 hours after transfection with the indicated miRNA mimics . The data were calculated relative to miR - scramble and presented as means bars ± SEM and the background fluorescence was subtracted . Performed with at least three independent experiments , in technical replicates . Dual Luciferase reporter assay . NIH - 3T3 was transfected with the miRNA mimics . Three hours later the cells were transfected with the Raptor 3 ′ UTR cloned into the psiCheck - 2 vector for a total of 48 hours . Data are presented as % RLU ( relative luciferase units ) of the miR - scramble control as means ± SEM from at least three independent experiments , performed in technical replicates . Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , ( J ) Cells were nucleofected with miR - Scramble ( CTRL ) or miR - 1246 mimic ( n = 3 ) for 72 hours , Cell - cycle flow cytometric analysis using Ki67 / Hoechst - 33342 staining of the percentage of LT - HSC in the G0 phase after nucleofection of cKit + cells using the AmaxaTM P3 Primary Cell 4D - Nucleoeftcor kit , Cells were nucleofected with Anti - miR - Scramble ( CTRL ) or Anti - miR - 1246 ( n = 5 ) and 1 - hour later co - treatment with Molm - 14 EVs for 72 hours .

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

Figure 4qRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: (A) Molm-14-xenografted miceqRT-PCR gene expression analysis showing the fold change of Pi3K/mTOR associated gene panel in KSL cells sorted from: IF injected mice with Molm-14-EV relative to their controls and normalized to Gapdh endogenous control.Flow cytometric analysis showing the histograms (C)Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: (D) Molm-14-xenograft (red, n=9) vs. non-engrafted control (black, n=7),Flow cytometric analysis showing the MFI of intracellular pS6KRP in LT-HSC in: IF injection of EV from Molm-14, U-937, HL-60 (red, n=7,3,3) or human CD34 cells (blue, n=4) normalized to the control contralateral femur with subtraction of background fluorescence.Flow cytometric assessment of protein synthesis showing the MFI of: (F) OPP, or (G) pS6RP in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The results were calculated relative to control (miR-scramble) with the background fluorescence subtracted and performed with at least three independent experiments, in technical replicates.Flow cytometric analysis showing the MFI of Raptor in NIH-3T3 cells 72 hours after transfection with the indicated miRNA mimics. The data were calculated relative to miR-scramble and presented as means bars ± SEM and the background fluorescence was subtracted. Performed with at least three independent experiments, in technical replicates.Dual Luciferase reporter assay. NIH-3T3 was transfected with the miRNA mimics. Three hours later the cells were transfected with the Raptor 3′UTR cloned into the psiCheck-2 vector for a total of 48 hours. Data are presented as %RLU (relative luciferase units) of the miR-scramble control as means ± SEM from at least three independent experiments, performed in technical replicates.Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, (J) Cells were nucleofected with miR-Scramble (CTRL) or miR-1246 mimic (n=3) for 72 hours,Cell-cycle flow cytometric analysis using Ki67/Hoechst-33342 staining of the percentage of LT-HSC in the G0 phase after nucleofection of cKit+ cells using the AmaxaTM P3 Primary Cell 4D-Nucleoeftcor kit, Cells were nucleofected with Anti-miR-Scramble (CTRL) or Anti-miR-1246 (n=5) and 1-hour later co-treatment with Molm-14 EVs for 72 hours.


Figure 5CFU - C assay showing the relative number of colonies ( left panel ) and the colony subtypes ( right panel ) from KSL cells derived from Molm - 14 - xenografted mice ( red , n = 6 ) vs . non - engrafted control mice ( black , n = 5 ) and in vitro cultured for 7 days in methylcellulose media . Experiments were performed in technical triplicates . " G " Granulocyte , " M " Monocyte , " E " Erythrocytes . LT - HSC assessment after repopulation , recipient female mice ( n = 4 per group ) were sacrificed after 21 days and their LT - HSC were compared using P53 , pS6RP and OPP flow cytometric assays . ( D - E ) Evaluation of the in vivo long - term repopulation capacity . Evaluation of the in vivo long - term repopulation capacity . For secondary transplantation ( n = 6 per group ) , PB samples were collected twice weekly for 12 weeks ( WK ) and the donor chimerism was measured by qPCR of Sex - determining Region Y ( SRY ) normalized to Gapdh gatekeeper as shown in the left panel . Sixteen weeks later , mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel . Tertiary transplantation , unfractionated BM cells from the secondary recipients ( n = 6 per group , 106 cells per mouse ) were injected into tertiary 150cGy sub - lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks .

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

Figure 5CFU-C assay showing the relative number of colonies (left panel) and the colony subtypes (right panel) from KSL cells derived from Molm-14-xenografted mice (red, n=6) vs. non-engrafted control mice (black, n=5) and in vitro cultured for 7 days in methylcellulose media. Experiments were performed in technical triplicates. "G" Granulocyte, "M" Monocyte, "E" Erythrocytes.LT-HSC assessment after repopulation, recipient female mice (n=4 per group) were sacrificed after 21 days and their LT-HSC were compared using P53, pS6RP and OPP flow cytometric assays. (D-E) Evaluation of the in vivo long-term repopulation capacity.Evaluation of the in vivo long-term repopulation capacity. For secondary transplantation (n=6 per group), PB samples were collected twice weekly for 12 weeks (WK) and the donor chimerism was measured by qPCR of Sex-determining Region Y (SRY) normalized to Gapdh gatekeeper as shown in the left panel. Sixteen weeks later, mice were sacrificed and BM male chimerism percentage was assessed as shown in the right panel. Tertiary transplantation, unfractionated BM cells from the secondary recipients (n=6 per group, 106 cells per mouse) were injected into tertiary 150cGy sub-lethally irradiated female recipients and blood SRY levels were assessed for 20 weeks.


Figure 6Serial replating CFU - C analysis presented as mean ± SEM showing : ( B ) the number of colonies per 1000 plated cells , and ( C ) the number of cells per colony , for each week time - point . DNA - damage assessment in KSL from primary recipients and after two turns of replating . ( D , F , H ) Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : ( D ) Primary recipient KSL , n = 211 and 321 , All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : ( E ) Primary recipient KSL , n = 6 Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W1 plating , n = 218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . Scale bars are 2μm . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W1 plating , n = 4 , Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . Number of γH2AX foci in control - derived and Molm - 14 - xenograft - derived : cells from W2 plating , n = 770 and 853 . All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst ( blue ) , imaged using the GE / API DV widefield microscope ( 60X objective ) to show the γH2AX foci ( green ) and counted using Imaris software . qRT - PCR gene expression analysis showing the fold - change of select DNA - damage response - genes panel in Molm - 14 - xenograft - derived : Cells from W2 plating n = 4 . Data indicated by bars of fold - change calculated relative to matching controls , normalized to the endogenous control Gapdh and presented as means ± SEM . umber of γH2AX foci , respectively , in control - and Molm - 14 - xenograft - derived : ( K ) KSL , n = 101 / 204 , and ( L ) LT - HSC , n = 33 / 53 . Images were processed similar to D - F . Scale bars are 3μm . Statistics : Student ' s t - test . ( * * P < 0 . 01 ) . qRT - PCR analysis of the DNA - damage - genes panel in Molm - 14 - xenograft - derived : ( M ) KSL , and ( N ) LT - HSC , relative to matched controls . Data depicted as fold - change relative to matching controls , normalized to the endogenous control Gapdh and presented as mean ± SEM .

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

Figure 6Serial replating CFU-C analysis presented as mean ± SEM showing: (B) the number of colonies per 1000 plated cells, and (C) the number of cells per colony, for each week time-point.DNA-damage assessment in KSL from primary recipients and after two turns of replating. (D, F, H) Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: (D) Primary recipient KSL, n=211 and 321, All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: (E) Primary recipient KSL, n=6 Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W1 plating, n=218 and 369 All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software. Scale bars are 2μm.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W1 plating, n=4, Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.Number of γH2AX foci in control-derived and Molm-14-xenograft-derived: cells from W2 plating, n=770 and 853. All cells were stained with phosphoH2axser139 and the nuclear stain Hoechst (blue), imaged using the GE/API DV widefield microscope (60X objective) to show the γH2AX foci (green) and counted using Imaris software.qRT-PCR gene expression analysis showing the fold-change of select DNA-damage response-genes panel in Molm-14-xenograft-derived: Cells from W2 plating n=4. Data indicated by bars of fold-change calculated relative to matching controls, normalized to the endogenous control Gapdh and presented as means ± SEM.umber of γH2AX foci, respectively, in control- and Molm-14-xenograft-derived: (K) KSL, n=101 / 204, and (L) LT-HSC, n=33 / 53. Images were processed similar to D-F. Scale bars are 3μm. Statistics: Student's t-test. (** P< 0.01).qRT-PCR analysis of the DNA-damage-genes panel in Molm-14-xenograft-derived: (M) KSL, and (N) LT-HSC, relative to matched controls. Data depicted as fold-change relative to matching controls, normalized to the endogenous control Gapdh and presented as mean ± SEM.


Figure 1B ) , C ) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively , in CKO cells compared to WT ( q = Benjamini - Hochberg corrected p - value , n = 24 time points over 3 days ) . Statistically significant changes ( q ≤ 0 . 05 ) are shown in red . Some proteins are labelled as space allows .

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Figure 1B), C) Volcano plots showing the fold change in average expression of all proteins and phosphopeptides respectively, in CKO cells compared to WT (q = Benjamini-Hochberg corrected p-value, n= 24 time points over 3 days). Statistically significant changes (q ≤ 0.05) are shown in red. Some proteins are labelled as space allows.


Figure 2A ) Fold - change in relative amplitude ( RA ) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis ( i . e . no RA cut - off ) . The mean fold change ( log ) in RA was an increase of 14 % in CKO cells compared to WT cells ( One sample t - test , * * * * p < 0 . 0001 ) . B ) For each genotype , all proteins were divided into 10 deciles of equal number , ranked by abundance . The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic . Linear regression lines are shown for each genotype , and the slopes were significantly non - zero ( F test , WT * * p = 0 . 0016 , CKO * * * * p < 0 . 0001 ) . The slopes were also significantly different to each other ( F test , p = 0 . 0001 ) . C ) Fold - change in relative amplitude ( RA ) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN . On average , the RA was increased in CKO cells compared to WT cells by 6 % ( One sample t - test , * p < 0 . 05 ) . D ) The same analysis in B ) was carried out for phosphopeptides . The slopes were significantly non - zero in CKO but not WT ( F test , WT p = 0 . 3 ( ns . : non significant ) , CKO * p = 0 . 04 ) . The slopes were also significantly different to each other ( F test , p < 0 . 0001 ) .

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

Figure 2A) Fold-change in relative amplitude (RA) was calculated for each of the proteins found to be rhythmic in both genotypes by RAIN analysis (i.e. no RA cut-off). The mean fold change (log) in RA was an increase of 14% in CKO cells compared to WT cells (One sample t-test, ****p<0.0001). B) For each genotype, all proteins were divided into 10 deciles of equal number, ranked by abundance. The mean abundance of each decile was plotted against the proportion of the decile that was rhythmic. Linear regression lines are shown for each genotype, and the slopes were significantly non-zero (F test, WT **p=0.0016, CKO ****p<0.0001). The slopes were also significantly different to each other (F test, p=0.0001). C) Fold-change in relative amplitude (RA) was calculated for each of the phosphopeptides found to be rhythmic in both genotypes by RAIN. On average, the RA was increased in CKO cells compared to WT cells by 6% (One sample t-test, *p<0.05). D) The same analysis in B) was carried out for phosphopeptides. The slopes were significantly non-zero in CKO but not WT (F test, WT p=0.3 (ns.: non significant), CKO *p=0.04). The slopes were also significantly different to each other (F test, p<0.0001).


Figure 3B ) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two - way ANOVA correcting for multiple comparisons , controlling for false discovery rate by the Benjamini , Krieger and Yekutieli method ( q = 0 . 05 ) . D - E ) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells ( D , p = 0 . 47 ) , but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts ( E , * * p = 0 . 0013 ) ; Kolmogorov - Smirmov test ( n = 4 ) . Continuous lines = median , dotted lines = quartiles .

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

Figure 3B) Bar chart reports the number and % of detected proteins showing a significant difference in abundance for the listed comparisons by two-way ANOVA correcting for multiple comparisons, controlling for false discovery rate by the Benjamini, Krieger and Yekutieli method (q=0.05).D-E) Proteins with significant abundance change over time had no significant difference in mean absolute fold change between WT and CKO cells (D, p=0.47), but significantly greater variance in absolute fold change between independent CKO biological replicates than between their WT counterparts (E, **p=0.0013); Kolmogorov-Smirmov test (n=4). Continuous lines = median, dotted lines = quartiles.


Figure 4A ) From the quantitative proteomics experiment , average abundance of catalytic proteasome subunits was calculated and normalised to WT means . Trypsin - like ( β2 ) , chymotrypsin - like ( β3 ) and caspase - like ( β1 ) catalytic subunits are shown . The average was calculated from all 24 time points of the proteomics experiment . Mean ± SD , 2 - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . B ) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome , with anti - histone H3 as loading control . C ) Quantification of the blots in B ) , using all replicates ( n = 3 ) , normalised to WT mean . Mean ± SD , one - tailed Student ' s t test with Welch correction - prior prediction from experiment in A ) that CKO abundance would be lower , * p ≤ 0 . 05 .

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

Figure 4A) From the quantitative proteomics experiment, average abundance of catalytic proteasome subunits was calculated and normalised to WT means. Trypsin-like (β2), chymotrypsin-like (β3) and caspase-like (β1) catalytic subunits are shown. The average was calculated from all 24 time points of the proteomics experiment. Mean±SD, 2-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.B) Representative Western blot using an antibody that recognises all 7 α subunits of the 20S proteasome, with anti-histone H3 as loading control. C) Quantification of the blots in B), using all replicates (n=3), normalised to WT mean. Mean±SD, one-tailed Student's t test with Welch correction - prior prediction from experiment in A) that CKO abundance would be lower, *p≤0.05.


Figure 5D - E ) From one time - course experiment , ions , cytosolic proteins and total protein were extracted in parallel samples . The presented experiment is representative of 3 separate time - course experiments that were carried out ( N = 3 ) . Representative experiment shown ( n = 4 technical replicates for [ K + ] , n = 3 technical replicates for the other traces ) . Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration . Mean ± SEM , p - values from RAIN , red lines are fits by a damped cosine compared with a straight line ( null hypothesis ) . Parallel PER2 : : LUC recordings were also performed and plotted as a phase marker . F ) Examples of key ion transporters are shown , as detected in the proteomics experiment . P values show the results of an F test comparing fits of damped cosine against straight line . All proteins except WT NKCC1 had RAIN p - values < 0 . 05 .

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

Figure 5D-E) From one time-course experiment, ions, cytosolic proteins and total protein were extracted in parallel samples. The presented experiment is representative of 3 separate time-course experiments that were carried out (N=3). Representative experiment shown (n=4 technical replicates for [K+], n=3 technical replicates for the other traces). Blue lines highlight the antiphasic relationship between oscillations in cytosolic protein and potassium concentration. Mean±SEM, p-values from RAIN, red lines are fits by a damped cosine compared with a straight line (null hypothesis). Parallel PER2::LUC recordings were also performed and plotted as a phase marker.F) Examples of key ion transporters are shown, as detected in the proteomics experiment. P values show the results of an F test comparing fits of damped cosine against straight line. All proteins except WT NKCC1 had RAIN p-values <0.05.


Figure 6A ) WT and CKO cells were treated with 500 nM tunicamycin ( TUN ) and lysed in RIPA buffer at time points between 0 - 24 hours afterwards . Western blots were carried out , probing for phosphorylated eIF2α , total eIF2α and actin . Representative blots shown , n = 4 . B ) Quantification of all replicates represented in ( A ) , where phosphorylated eIF2α is normalised to total eIF2α . Mean ± SD . 2 - way ANOVA ( TWA ) with Holm - Sidak ' s multiple comparisons . C ) Mice were housed in 12h : 12h light - dark cycles , with unrestricted feeding . Mouse lungs were collected 7 hours after transition to the light phase , 5 hours after intraperitoneal injection of mice with bortezomib ( BTZ , 2 . 5 mg / kg ) or vehicle ( Veh , 1 % DMSO in sterile PBS ) . Tissues were lysed in RIPA buffer and western blots were performed , probing for phosphorylated STAT3 , total STAT3 and actin . Male ( left ) and female ( right ) replicates shown , N = 2 mice for each condition . D ) Quantification of the blots shown in ( C ) , where phosphorylated STAT3 is normalised to total STAT3 . Mean ± SD . Multiple t - tests , corrected for multiple comparisons with the Holm - Sidak method , * p ≤ 0 . 05 , * * * p ≤ 0 . 001 .

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 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

Figure 6A) WT and CKO cells were treated with 500 nM tunicamycin (TUN) and lysed in RIPA buffer at time points between 0-24 hours afterwards. Western blots were carried out, probing for phosphorylated eIF2α, total eIF2α and actin. Representative blots shown, n=4. B) Quantification of all replicates represented in (A), where phosphorylated eIF2α is normalised to total eIF2α. Mean±SD. 2-way ANOVA (TWA) with Holm-Sidak's multiple comparisons.C) Mice were housed in 12h:12h light-dark cycles, with unrestricted feeding. Mouse lungs were collected 7 hours after transition to the light phase, 5 hours after intraperitoneal injection of mice with bortezomib (BTZ, 2.5 mg/kg) or vehicle (Veh, 1% DMSO in sterile PBS). Tissues were lysed in RIPA buffer and western blots were performed, probing for phosphorylated STAT3, total STAT3 and actin. Male (left) and female (right) replicates shown, N=2 mice for each condition. D) Quantification of the blots shown in (C), where phosphorylated STAT3 is normalised to total STAT3. Mean±SD. Multiple t-tests, corrected for multiple comparisons with the Holm-Sidak method, *p≤0.05, ***p≤0.001.


Figure 7A ) WT PER2 : : LUC cells were treated with epoxomicin or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . B ) Quantification of damping rate of the PER2 : : LUC recordings shown in A ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * p ≤ 0 . 01 , * * * * p ≤ 0 . 0001 . C ) WT PER2 : : LUC cells were treated with tunicamycin or vehicle control . Mean ( solid lines ) ± SD ( dotted lines ) . D ) Quantification of damping rate of the PER2 : : LUC recordings shown in C ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . E ) WT PER2 : : LUC cells were treated with radicicol or vehicle control . Blue arrows show the time points where drug was washed off . Mean ( solid lines ) ± SD ( dotted lines ) . F ) Quantification of damping rate of the PER2 : : LUC recordings shown in E ) , for the duration of drug treatment . Mean ± SD . One - way ANOVA with Holm - Sidak ' s multiple comparisons , * * * * p ≤ 0 . 0001 . I - J ) Representative double - plotted actograms of WT mice receiving drinking water with DMSO vehicle control ( N = 7 mice ) or 35 µg / mL ixazomib ( N = 8 mice ) . Two representative actograms are shown per condition . For the first 7 days the mice were in 12 : 12h light - dark cycles ( LD ) , and had drinking water supplemented with blackcurrant squash . Ixazomib / DMSO was added to this , and the lighting was changed to constant darkness ( DD ) . After 7 days the drug / vehicle was removed , and the mice remained in DD until the end of the experiment .

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

Figure 7A) WT PER2::LUC cells were treated with epoxomicin or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). B) Quantification of damping rate of the PER2::LUC recordings shown in A), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, **p≤0.01, ****p≤0.0001. C) WT PER2::LUC cells were treated with tunicamycin or vehicle control. Mean (solid lines) ± SD (dotted lines). D) Quantification of damping rate of the PER2::LUC recordings shown in C), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.E) WT PER2::LUC cells were treated with radicicol or vehicle control. Blue arrows show the time points where drug was washed off. Mean (solid lines) ± SD (dotted lines). F) Quantification of damping rate of the PER2::LUC recordings shown in E), for the duration of drug treatment. Mean±SD. One-way ANOVA with Holm-Sidak's multiple comparisons, ****p≤0.0001.I-J) Representative double-plotted actograms of WT mice receiving drinking water with DMSO vehicle control (N=7 mice) or 35 µg/mL ixazomib (N=8 mice). Two representative actograms are shown per condition. For the first 7 days the mice were in 12:12h light-dark cycles (LD), and had drinking water supplemented with blackcurrant squash. Ixazomib/DMSO was added to this, and the lighting was changed to constant darkness (DD). After 7 days the drug/vehicle was removed, and the mice remained in DD until the end of the experiment.


Figure 1A . Bclaf1 wild type ( WT ) and two Bclaf1 knockout HeLa cell lines ( KO - 1 and - 2 ) were pre - treated with DMSO or cycloheximide ( CHX , 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) together with CHX for 12 hours . Afterwards , the cells were subjected to Annexin V / 7AAD staining followed by flow cytometry analysis , all Annexin V positive cells were counted for analysis , or protein extractions followed by western blotting analysis . B , C . HeLa ( B ) or primary MEFs ( C ) were transfected with control ( siCtrl ) or two siRNAs against Bclaf1 ( siBclaf1 - 1 and - 2 ) and then treated and analyzed as described in A . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test . D , E . MEFs transfected with control ( siCtrl ) or siBclaf1 - 1 and / or siBclaf1 - 2 siRNAs were pre - treated with CHX ( 1 μg / ml ) / z - VAD ( 20 μM ) ( D ) or SM - 164 ( 100 nM ) / z - VAD ( 20 μM ) ( E ) for 30 min , and then added TNF ( 10 ng / ml ) for indicated times . The cells were subjected to Annexin V / 7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells ( up ) , or protein extractions and western blotting analysis at indicated time periods ( down ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1A. Bclaf1 wild type (WT) and two Bclaf1 knockout HeLa cell lines (KO-1 and -2) were pre-treated with DMSO or cycloheximide (CHX, 1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) together with CHX for 12 hours. Afterwards, the cells were subjected to Annexin V/7AAD staining followed by flow cytometry analysis, all Annexin V positive cells were counted for analysis, or protein extractions followed by western blotting analysis. B, C. HeLa (B) or primary MEFs (C) were transfected with control (siCtrl) or two siRNAs against Bclaf1 (siBclaf1-1 and -2) and then treated and analyzed as described in A. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.D, E. MEFs transfected with control (siCtrl) or siBclaf1-1 and/or siBclaf1-2 siRNAs were pre-treated with CHX (1 μg/ml)/z-VAD (20 μM) (D) or SM-164 (100 nM)/z-VAD (20 μM) (E) for 30 min, and then added TNF (10 ng/ml) for indicated times. The cells were subjected to Annexin V/7AAD staining after 9 hours followed by flow cytometry analysis for 7AAD positive and Annexin V negative cells (up), or protein extractions and western blotting analysis at indicated time periods (down). Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; **p<0.01; ****p<0.0001. One-way ANOVA test.


Figure 2A , B . Cell lysates of HeLa Bclaf1 WT and KO cells ( A ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( B ) treated with TNF ( 10 ng / ml ) for indicated time periods were analyzed by western blotting with the indicated antibodies . C , D . HeLa Bclaf1 WT and KO cells ( C ) , and HeLa cells transfected with siCtrl or siBclaf1 - 1 ( D ) were treated with TNF ( 10 ng / ml ) for the indicated time periods , and then subject to nucleus - cytoplasm fractionation as described in materials and methods followed by western blotting . Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions , 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 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

Figure 2A, B. Cell lysates of HeLa Bclaf1 WT and KO cells (A), and HeLa cells transfected with siCtrl or siBclaf1-1 (B) treated with TNF (10 ng/ml) for indicated time periods were analyzed by western blotting with the indicated antibodies.C, D. HeLa Bclaf1 WT and KO cells (C), and HeLa cells transfected with siCtrl or siBclaf1-1 (D) were treated with TNF (10 ng/ml) for the indicated time periods, and then subject to nucleus-cytoplasm fractionation as described in materials and methods followed by western blotting. Tubulin and histone H3 were used as loading controls for cytoplasmic and nuclear fractions, respectively.


Figure 3B . MEFs treated with TNF ( 10 ng / ml ) for 4 hours were subject to FADD immunoprecipitation , and then analyzed by western blotting . G , H . HeLa cells transfected with siRNAs against Bclaf1 or c - FLIP were pretreated with CHX ( 1 μg / ml ) for 30 min and then stimulated with TNF ( 10 ng / ml ) in the presence of CHX for 12 hours followed by Annexin V / 7AAD staining and flow cytometry analysis ( G ) , or protein extractions followed by western blotting analysis ( H ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * * p < 0 . 01 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

Figure 3B. MEFs treated with TNF (10 ng/ml) for 4 hours were subject to FADD immunoprecipitation, and then analyzed by western blotting.G, H. HeLa cells transfected with siRNAs against Bclaf1 or c-FLIP were pretreated with CHX (1 μg/ml) for 30 min and then stimulated with TNF (10 ng/ml) in the presence of CHX for 12 hours followed by Annexin V/7AAD staining and flow cytometry analysis (G), or protein extractions followed by western blotting analysis (H). Data information: Data are shown as mean ± SD. n=3 biological replicates. **p<0.01; ****p<0.0001. One-way ANOVA test.


Figure 4A - F . Bclaf1 WT , KO - 1 and - 2 HeLa cells ( A and D ) , as well as HeLa ( B and E ) and primary MEFs ( C and F ) which were transfected with siCtrl or siBclaf1 - 1 and - 2 were treated with TNF ( 10 ng / ml ) for 6 hours followed by western blotting analysis ( A - C ) , or for 4 hours followed by total RNA extraction and RT - PCR analysis ( D - F ) . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . H , I . CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa - Flag - Bclaf1 cells treated without ( H ) or with TNF ( I ) . The regions amplified by RT - PCR were delineated . Data information : Data are shown as mean ± SD . n = 3 biological replicates . * p < 0 . 05 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test .

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

Figure 4A-F. Bclaf1 WT, KO-1 and -2 HeLa cells (A and D), as well as HeLa (B and E) and primary MEFs (C and F) which were transfected with siCtrl or siBclaf1-1 and -2 were treated with TNF (10 ng/ml) for 6 hours followed by western blotting analysis (A-C), or for 4 hours followed by total RNA extraction and RT-PCR analysis (D-F). Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.H, I. CHIP analysis of the binding of Bclaf1 to the promoter of CFLAR was performed in HeLa-Flag-Bclaf1 cells treated without (H) or with TNF (I). The regions amplified by RT-PCR were delineated. Data information: Data are shown as mean ± SD. n=3 biological replicates. *p<0.05; ***p<0.001; ****p<0.0001. One-way ANOVA test.


Figure 5B . CFLAR promoter were co - transfected with Bclaf1 or / and p50 into HeLa - Bclaf1 - KO cells , and the luciferase activity was measured as described before . E , HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre - treated with DMSO or CHX ( 1 μg / ml ) for 30 min , and then treated with TNF ( 10 ng / ml ) for 12 hours . Cells were lysed and then analyzed by western blottingG . CHIP analysis of the CFLAR promoter in HeLa - Flag - Bclaf1 cells . Immunoprecipitation was performed using anti - Flag and control IgG antibodies followed by RT - PCR analysis . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant ; * p < 0 . 05 ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way or Two - way ANOVA test .

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Figure 5B. CFLAR promoter were co-transfected with Bclaf1 or/and p50 into HeLa-Bclaf1-KO cells, and the luciferase activity was measured as described before.E, HeLa cells were transfected with siCtrl or siRNA against Bclaf1 or p50 were pre-treated with DMSO or CHX (1 μg/ml) for 30 min, and then treated with TNF (10 ng/ml) for 12 hours. Cells were lysed and then analyzed by western blottingG. CHIP analysis of the CFLAR promoter in HeLa-Flag-Bclaf1 cells. Immunoprecipitation was performed using anti-Flag and control IgG antibodies followed by RT-PCR analysis. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant; *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. One-way or Two-way ANOVA test.


Figure 6A . HeLa - Flag - Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF ( 10 ng / ml ) for 2 hours were separated into the cytoplasmic and the nuclear fraction . Endogenous Flag - Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting . B . HeLa - Flag - Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF ( 10 ng / ml ) for 2 hours . The nuclear fraction was isolated and subjected to immunoprecipitation with an anti - Flag or control IgG antibody , followed by western blotting analysis . D . CFLAR FL promoter were co - transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa - Bclaf1 - KO cells . The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization . Data information : Data are shown as mean ± SD . n = 3 biological replicates . ns , not significant . One - way ANOVA test .

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Figure 6A. HeLa-Flag-Bclaf1 and Bclaf1 WT HeLa cells treated with or without TNF (10 ng/ml) for 2 hours were separated into the cytoplasmic and the nuclear fraction. Endogenous Flag-Bclaf1 complex in each fraction was immunoprecipitated with M2 beads and analyzed by western blotting.B. HeLa-Flag-Bclaf1 cells transfected with siCtrl or sip65 were treated with TNF (10 ng/ml) for 2 hours. The nuclear fraction was isolated and subjected to immunoprecipitation with an anti-Flag or control IgG antibody, followed by western blotting analysis.D. CFLAR FL promoter were co-transfected with p50 and Bclaf1 FL or its individual deletion mutant into HeLa-Bclaf1-KO cells. The experiments were performed in triplicate with a Renilla reporter in the transfection mixture for normalization. Data information: Data are shown as mean ± SD. n=3 biological replicates. ns, not significant. One-way ANOVA test.


Figure 7siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for Bclaf1 ( B ) and Hematoxylin - and - eosin staining ( C ) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis ( B ) . Hematoxylin - and - eosin staining showing the morphological change ( C ) . Villus height and crypt depth were measured ( D ) . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μm in B , 100 μm in C . siRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 ( E ) , phospholated RIPK3 ( F ) CC3 + and p - RIPK3 + positive cells in five fields per intestine were quantified . Data are shown as mean ± SD . n = 5 mice for each group . ns , not significant ; * * p < 0 . 01 ; * * * p < 0 . 001 ; * * * * p < 0 . 0001 . One - way ANOVA test . Scale bars : 50 μmsiRNAs against Bclaf1 and control siRNAs ( siCtrl ) mixed with in vivo - jetPEI ( Polyplus ) were injected into C57BL / 6 mice . The mice were then treated with mTNF for two hours before sacrifice . The small intestines were excised and processed for western blotting analysis ( G ) . HIEC - 6 cells were transfected siCtrl or siBclaf1 - 1 and - 2 . Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis ( I and J )

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Figure 7siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for Bclaf1 (B) and Hematoxylin-and-eosin staining (C) Bclaf1 knockdown efficiency is detected by immunohistochemical analysis (B). Hematoxylin-and-eosin staining showing the morphological change (C). Villus height and crypt depth were measured (D). Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μm in B, 100 μm in C.siRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for immunohistochemical staining for cleaved caspase 3 (E), phospholated RIPK3 (F) CC3+ and p-RIPK3+ positive cells in five fields per intestine were quantified. Data are shown as mean ± SD. n=5 mice for each group. ns, not significant; **p<0.01; ***p<0.001; ****p<0.0001. One-way ANOVA test. Scale bars: 50 μmsiRNAs against Bclaf1 and control siRNAs (siCtrl) mixed with in vivo-jetPEI (Polyplus) were injected into C57BL/6 mice. The mice were then treated with mTNF for two hours before sacrifice. The small intestines were excised and processed for western blotting analysis (G).HIEC-6 cells were transfected siCtrl or siBclaf1-1 and -2. Then the cells were treated with TNF for 24 hours were subjected for protein extractions and western blotting analysis (I and J)


Figure 3K . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media ( PM ) after isolation . L . Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media ( DM ) . hiPSC donor 1 . M - O . Quantification of the number of Pax7 ( M ) , MyoD ( N ) and Pax7 / MyoD double - positive ( O ) cells in hskMPs and eMPs in PM and after switching to DM . hiPSC donor 1 . P . Representative immunostaining for the muscle fiber marker myosin heavy chain ( MyHC ) of eMPs maintained in DM . Q . Quantification of the fusion index of hskMPs and eMPs in PM and DM . hiPSC donor 1 . U - Z , Representative immunostainings and quantification of eMPs in DM expressing Pax7 ( V ) , MyoD ( X ) , and MyoG ( Z ) , as well as the proliferation marker Ki67 . hiPSC donor 1 .

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Figure 3K. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn and maintained in proliferation media (PM) after isolation. L. Representative immunostainings for Pax7 and MyoD in eMPs plated on Vtn that were first maintained in PM and then switched to differentiation media (DM). hiPSC donor 1.M-O. Quantification of the number of Pax7 (M), MyoD (N) and Pax7/MyoD double-positive (O) cells in hskMPs and eMPs in PM and after switching to DM. hiPSC donor 1.P. Representative immunostaining for the muscle fiber marker myosin heavy chain (MyHC) of eMPs maintained in DM. Q. Quantification of the fusion index of hskMPs and eMPs in PM and DM. hiPSC donor 1.U-Z, Representative immunostainings and quantification of eMPs in DM expressing Pax7 (V), MyoD (X), and MyoG (Z), as well as the proliferation marker Ki67. hiPSC donor 1.


Figure 4A . Representative immunostainings for dystrophin in cross - sections of muscles of mdx mice after transplantation of hskMPs or eMPs . Scale bars in the two left - hand side images = 300 μm . Scale bars in inserts 1 and 2 = 50 μm . B . Number of dystrophin positive fibers in mdx muscles injected with vehicle ( Ctrl ) , eMPs and hskMPs . C . Maximal distance of clusters of dystrophin positive fibers in cross - sections of mdx muscles transplanted with eMPs and hskMPs . A - C . 10 days post - transplantation . hiPSC donor 1 .

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Figure 4A. Representative immunostainings for dystrophin in cross-sections of muscles of mdx mice after transplantation of hskMPs or eMPs. Scale bars in the two left-hand side images = 300 μm. Scale bars in inserts 1 and 2 = 50 μm. B. Number of dystrophin positive fibers in mdx muscles injected with vehicle (Ctrl), eMPs and hskMPs. C. Maximal distance of clusters of dystrophin positive fibers in cross-sections of mdx muscles transplanted with eMPs and hskMPs. A-C. 10 days post-transplantation. hiPSC donor 1.


Figure 1 ( a - c ) Low - magnification cryo - EM projection images of pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Different regions of the flagellum are annotated as follows : green - neck , yellow - midpiece , coral - principal piece , pink - endpiece . ( d - f ) Tomographic slices through cryo - FIB milled lamellae of pig ( d ) , horse ( e ) , and mouse ( f ) sperm . Transverse slices ( d ' - f ' ) show complete triplets in the pig ( d ' ) and the horse ( e ' ) , but not in the mouse ( f ' ) . Complete triplets are indicated by green arrowheads with black outlines , while degenerated triplets are indicated by white arrowheads with green outlines . Note the electron - dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece ( asterisks in d and d ' ) .

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Figure 1(a-c) Low-magnification cryo-EM projection images of pig (a), horse (b), and mouse (c) sperm. Different regions of the flagellum are annotated as follows: green - neck, yellow - midpiece, coral - principal piece, pink - endpiece. (d-f) Tomographic slices through cryo-FIB milled lamellae of pig (d), horse (e), and mouse (f) sperm. Transverse slices (d'-f') show complete triplets in the pig (d') and the horse (e'), but not in the mouse (f'). Complete triplets are indicated by green arrowheads with black outlines, while degenerated triplets are indicated by white arrowheads with green outlines. Note the electron-dense material in the lumen of the pig sperm proximal centriole that is continuous with the connecting piece (asterisks in d and d').


Figure 2 ( g - i ) Tomographic slices through cryo - FIB milled lamellae of the DC - to - axoneme transition in pig sperm show how the change in geometry ( g , white arrows and white dashed lines ) coincides with the appearance of axoneme accessory structures ( h , white arrows ) and with density in the A - tubule ( i , compare insets in white and black boxes ) . In ( i ) , the white and black arrows indicate where the cross - sections in white and black boxes were taken from .

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Figure 2(g-i) Tomographic slices through cryo-FIB milled lamellae of the DC-to-axoneme transition in pig sperm show how the change in geometry (g, white arrows and white dashed lines) coincides with the appearance of axoneme accessory structures (h, white arrows) and with density in the A-tubule (i, compare insets in white and black boxes). In (i), the white and black arrows indicate where the cross-sections in white and black boxes were taken from.


Figure 7 ( a - c ) Slices through defocus cryo - tomograms of the endpiece in pig ( a ) , horse ( b ) , and mouse ( c ) sperm . Insets show digital zooms ( black boxes ) and subtomogram averages ( white boxes ) of microtubule tips . Note the presence of a plug ( arrowhead ) extending into the lumen .

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Figure 7(a-c) Slices through defocus cryo-tomograms of the endpiece in pig (a), horse (b), and mouse (c) sperm. Insets show digital zooms (black boxes) and subtomogram averages (white boxes) of microtubule tips. Note the presence of a plug (arrowhead) extending into the lumen.


Figure 1A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup / Cdt1 RNAi , cyclin E RNAi , Cdk2 RNAi , Dacapo overexpression ( + Dacapo ) , and 1 mM HU . B Analysis of Mcm2 ‐ 7 nuclear chromatin association at different points in the cell cycle . Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2 ‐ 7 by Western blot . A non ‐ specific band is indicated by an asterisk ( * ) . C Quantification of the ratio of chromatin ‐ bound Mcm2 ‐ 7 relative to Orc2 ( log10 scale ) for a minimum of 11 replicates ( mean ± SD ) .

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Figure 1A FACS profiles of DNA content for cells arrested at different points in the cell cycle by Dup/Cdt1 RNAi, cyclin E RNAi, Cdk2 RNAi, Dacapo overexpression (+Dacapo), and 1 mM HU.B Analysis of Mcm2‐7 nuclear chromatin association at different points in the cell cycle. Nuclear chromatin fractions and cytoplasmic extracts were assayed for Orc2 and Mcm2‐7 by Western blot. A non‐specific band is indicated by an asterisk (*).C Quantification of the ratio of chromatin‐bound Mcm2‐7 relative to Orc2 (log10 scale) for a minimum of 11 replicates (mean ± SD).


Figure 2Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h ( top ) . Western blot analysis of Mcm2 ‐ 7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2 ‐ 7 , Orc2 , and Dacapo .

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Figure 2Cell cycle analysis by FACS of cells arrested in G1 by Dacapo overexpression followed by release back into the cell cycle for 13 h (top). Western blot analysis of Mcm2‐7 and Orc2 for the nuclear chromatin and cytoplasmic fractions of Mcm2‐7, Orc2, and Dacapo.


Figure 3B FACS profiles of DNA content for each cell population assayed . C Western blot analysis for Orc2 and Mcm2 ‐ 7 in the nuclear chromatin fraction and Orc2 , Mcm2 ‐ 7 , Dup / Cdt1 , and cyclin E in the cytoplasmic fraction for each condition assayed .

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Figure 3B FACS profiles of DNA content for each cell population assayed.C Western blot analysis for Orc2 and Mcm2‐7 in the nuclear chromatin fraction and Orc2, Mcm2‐7, Dup/Cdt1, and cyclin E in the cytoplasmic fraction for each condition assayed.


Figure 4A Genome ‐ wide analysis of ORC localization by ChIP ‐ chip . ORC enrichment from asynchronous cells is depicted for a 5 ‐ Mb section of chromosome 2L . B Genome ‐ wide analysis of Mcm2 ‐ 7 localization in early G1 by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from cyclin E RNAi ‐ depleted cells is depicted for a 5 ‐ Mb section of chromosome 2L . C Venn diagram depicting the overlap between ORC and Mcm2 ‐ 7 peaks .

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Figure 4A Genome‐wide analysis of ORC localization by ChIP‐chip. ORC enrichment from asynchronous cells is depicted for a 5‐Mb section of chromosome 2L.B Genome‐wide analysis of Mcm2‐7 localization in early G1 by ChIP‐chip. Mcm2‐7 enrichment from cyclin E RNAi‐depleted cells is depicted for a 5‐Mb section of chromosome 2L.C Venn diagram depicting the overlap between ORC and Mcm2‐7 peaks.


Figure 5A Genome ‐ wide analysis of Mcm2 ‐ 7 localization at the G1 / S transition by ChIP ‐ chip . Mcm2 ‐ 7 enrichment from HU ‐ arrested cells is depicted for a 5 ‐ Mb section of chromosome 2L . Inset : transcribed ( green ) and non ‐ transcribed ( red ) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom . B Bimodal distribution of Mcm2 ‐ 7 enrichment over transcribed and non ‐ transcribed genes . Histogram showing the distribution of probe scores found within transcribed ( green ) and non ‐ transcribed ( red ) genes . C " Meta " ‐ gene analysis of Mcm2 ‐ 7 enrichment for different deciles of gene expression and their aggregated probe intensities . D Mcm2 ‐ 7 is displaced from chromatin by DNA replication . Genome ‐ wide analysis of Mcm2 ‐ 7 localization in late S ‐ phase ( 6 h post HU release ) by ChIP ‐ chip . Mcm2 ‐ 7 enrichment is depicted for a 10 ‐ Mb section of chromosome 2L ( filled gray ) , replication timing profile ( black line ) and early ( yellow ) and late ( purple ) replication timing domains . E Box ‐ plots representing late S ‐ phase Mcm2 ‐ 7 ChIP signal found within early ( 246 ) or late ( 167 ) replication domains .

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Figure 5A Genome‐wide analysis of Mcm2‐7 localization at the G1/S transition by ChIP‐chip. Mcm2‐7 enrichment from HU‐arrested cells is depicted for a 5‐Mb section of chromosome 2L. Inset: transcribed (green) and non‐transcribed (red) genes are indicated above with genes on the positive strand on the top and those on the negative on the bottom.B Bimodal distribution of Mcm2‐7 enrichment over transcribed and non‐transcribed genes. Histogram showing the distribution of probe scores found within transcribed (green) and non‐transcribed (red) genes.C "Meta"‐gene analysis of Mcm2‐7 enrichment for different deciles of gene expression and their aggregated probe intensities.D Mcm2‐7 is displaced from chromatin by DNA replication. Genome‐wide analysis of Mcm2‐7 localization in late S‐phase (6 h post HU release) by ChIP‐chip. Mcm2‐7 enrichment is depicted for a 10‐Mb section of chromosome 2L (filled gray), replication timing profile (black line) and early (yellow) and late (purple) replication timing domains.E Box‐plots representing late S‐phase Mcm2‐7 ChIP signal found within early (246) or late (167) replication domains.


Figure 1A . Ovaries from control heterozygous Rpp3018 . 2 flies ( left ) , homozygous Rpp3018 . 2 or transheterozygous Rpp3018 . 2 / Rpp30PE flies ( middle ) and homozygous Rpp3018 . 2 carrying a copy of ubiRpp30GFP transgene ( right ) . Scale bar , 100µm . D . Ovaries were dissected , lysed and protein extracts analyzed by Western Blot using anti - Rpp30 , anti - GFP or anti - tubulin antibodies . Orange arrows indicate overexpressed and endogenous Rpp30 signals . Genotypes are numbered as 1 , 2 , 3 . E . Rpp30 - dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds : Left . Homozygous Rpp3018 . 2 ovariole expressing ubiquitous ubiRpp30GFP ( st , stage ) . Right . Transheterozygous Rpp3018 . 2 / Rpp30PE ovarioles overexpressing germline specific nanosGal4 , UASRpp30GFP . Note follicular somatic cells with no Rpp30GFP expression ( arrowhead ) . Magnifications show nuclear Rpp30 localization , Rpp30 perinuclear foci ( arrows ) , and Orb ( a specific oocyte marker ) . Scale bar , 10µm . F . flp / FRT clones mutant for Rpp3018 . 2specifically in the germline ( star ) and / or the follicular cells ( arrows ) are detected by the absence of GFP . Magnifications show mutant oocytes with Orb mislocalization next to the karyosome ( dotted circle ) instead of being localized to the posterior , as seen in stage 3 ( st . 3 ) wild - type clones . Scale bar , 10µm .

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Figure 1A. Ovaries from control heterozygous Rpp3018.2 flies (left), homozygous Rpp3018.2 or transheterozygous Rpp3018.2/Rpp30PE flies (middle) and homozygous Rpp3018.2 carrying a copy of ubiRpp30GFP transgene (right). Scale bar, 100µm.D. Ovaries were dissected, lysed and protein extracts analyzed by Western Blot using anti-Rpp30, anti- GFP or anti-tubulin antibodies. Orange arrows indicate overexpressed and endogenous Rpp30 signals. Genotypes are numbered as 1,2,3.E. Rpp30-dependent early oogenesis arrest is restored by expression of Rpp30GFP transgene in two different genetic backgrounds: Left. Homozygous Rpp3018.2 ovariole expressing ubiquitous ubiRpp30GFP (st, stage). Right. Transheterozygous Rpp3018.2/Rpp30PE ovarioles overexpressing germline specific nanosGal4,UASRpp30GFP. Note follicular somatic cells with no Rpp30GFP expression (arrowhead). Magnifications show nuclear Rpp30 localization, Rpp30 perinuclear foci (arrows), and Orb (a specific oocyte marker). Scale bar, 10µm.F. flp/FRT clones mutant for Rpp3018.2specifically in the germline (star) and/or the follicular cells (arrows) are detected by the absence of GFP. Magnifications show mutant oocytes with Orb mislocalization next to the karyosome (dotted circle) instead of being localized to the posterior, as seen in stage 3 (st.3) wild-type clones. Scale bar, 10µm.