( E ) TET1 expression in dentate gyrus region of hippocampus of DSCR1 mutants . TET1 is increased in DSCR1 knockout , while it is decreased in DSCR1 transgenic mice . Values are shown as mean ± , tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 , * * P < 0 . 01 .

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

(E) TET1 protein expression in the dentate gyrus region of hippocampus of DSCR1 mutants. TET1 is increased in DSCR1 knockout, while it is decreased in DSCR1 transgenic mice. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 3 animal for each condition, *P< 0.05, **P< 0.01.


( B ) 3 after lentivirus injection , neural progenitor are identified by double of GFP Sox2 in dentate gyrus region . arrow indicates Sox2 GFP double positive . Scale bar , 10 μm . ( C ) number of GFP Sox2 double positive is restored to that of wild type . Scale bar : 10μm . Values are shown as mean ± tested for statistical significance by paired t - . N = 3 for each condition , * P < 0 . 05 .

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

(B) 3 weeks after lentivirus injection, neural progenitor cells are identified by double staining of GFP and Sox2 in dentate gyrus region. The white arrow indicates Sox2 and GFP double positive cells. Scale bar, 10 μm. (C) The number of GFP and Sox2 double positive cells is restored to that of wild type. Scale bar: 10μm. Values are shown as mean ± SEM and tested for statistical significance by paired t-test. N = 3 animal for each condition, *P< 0.05.


( A ) Genotype of Ts65Dn / DSCR1 + / - mouse was confirmed by detecting markers for Ts65Dn DSCR1

(A) Genotype of Ts65Dn/DSCR1+/- mouse was confirmed by detecting markers for Ts65Dn and DSCR1 KO


( B ) Expression of DSCR1 mRNA in Ts65Dn mouse was increased about 2 . 5 folds compared to that of wild type . However , Ts65Dn / DSCR1 + / - mouse showed normal level of DSCR1 mRNA transcript . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 3 for each condition , * P < 0 . 05 .

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

(B) Expression of the DSCR1 mRNA in Ts65Dn mouse was increased about 2.5 folds compared to that of wild type. However, Ts65Dn/DSCR1+/- mouse showed normal level of DSCR1 mRNA transcript. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N=3 for each condition, *P< 0.05.


As expected , of TET1 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores TET1 mRNA expression . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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

As expected, the levels of TET1 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the TET1 mRNA expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


As expected , of miR - 124 are decreased in Ts65Dn mouse , however Ts65Dn / DSCR1 + / - mouse restores miR - 124 expression . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . ß - actin was used for normalization . N = 3 ( Diploid ) , 3 ( Ts65Dn ) , 4 ( Ts65Dn / DSCR1 + / - ) mice , * P < 0 . 05 , * * P < 0 . 01 .

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

As expected, the levels of miR-124 are decreased in Ts65Dn mouse, however Ts65Dn/DSCR1+/- mouse restores the miR-124 expression. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. ß-actin was used for normalization. N = 3 (Diploid), 3 (Ts65Dn), 4 (Ts65Dn/DSCR1+/-) mice, *P< 0.05, **P< 0.01.


( E , F ) number proliferating progenitor neurons identified by BrdU Ki - 67 double is restored in SGZ of Ts65Dn / DSCR1 + / - mouse compared to that of Ts65Dn mouse . box is magnified in lower panels : DAPI ( blue ) , Ki67 ( green ) , BrdU ( red ) . Arrow heads indicate BrdU Ki - 67 double positive in SGZ . Scale bars : 100 μm in large image 10 μm in magnified image . Each hippocampal section was 40 μm in thickness , total 6 hippocampi were used for . Values are shown as mean ± tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) , * P < 0 . 05 .

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

(E, F) The number proliferating progenitor neurons identified by BrdU and Ki-67 double staining is restored in the SGZ of Ts65Dn/DSCR1+/- mouse compared to that of Ts65Dn mouse. The white box area is magnified in the lower panels: DAPI (blue), Ki67 (green), and BrdU (red). Arrow heads indicate BrdU and Ki-67 double positive cells in the SGZ. Scale bars: 100 μm in the large image and 10 μm in magnified image. Each hippocampal section was 40 μm in thickness, and total 6 hippocampi were used for analysis. Values are shown as mean ± SEM and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals, *P< 0.05.


( G - J ) Ts65Dn has learning memory defects , but Ts65Dn / DSCR1 + / - mouse clearly rescues learning memory defects to control . Values are shown as mean ± , tested for statistical significance by one - way ANOVA followed by Bonferroni post hoc . N = 8 ( control ) , 5 ( Ts65Dn ) , 10 ( Ts65Dn / DSCR1 + / - ) , * P < 0 . 001 ( G , H ) . * P < 0 . 05 ( I ) , * * P < 0 . 01 ( I ) . ( J ) Open - field shows no defects in movement of tested . Values are shown as mean ± , N = 6 ( control ) , 5 ( Ts65Dn ) , 3 ( Ts65Dn / DSCR1 + / - ) .

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

(G-J) Ts65Dn has learning and memory defects, but Ts65Dn/DSCR1+/- mouse clearly rescues learning and memory defects to control levels. Values are shown as mean ± SEM, and tested for statistical significance by one-way ANOVA followed by Bonferroni post hoc test. N = 8 (control), 5 (Ts65Dn), 10 (Ts65Dn/DSCR1+/-) animals, *P< 0.001 (G, H). *P< 0.05 (I), **P< 0.01 (I). (J) Open-field analysis shows no defects in movement of tested animals. Values are shown as mean ± SEM, N = 6 (control), 5 (Ts65Dn), 3 (Ts65Dn/DSCR1+/-) animals.


( b ) Suppression of Htt103Q toxicity in yeast by Gpx1 , Hyr1 ( Gpx3 ) mGpx1 . viability of parental , Y258 , of overexpressing yeast GPxs was determined using spotting assays . Equal numbers of were serially diluted fivefold plated on medium containing glucose to assess numbers containing galactose ( GAL ) to induce expression of Htt103Q indicated ORF .

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

(b) Suppression of Htt103Q toxicity in yeast by Gpx1, Hyr1 (Gpx3) and mGpx1. The viability of the parental strain, Y258, and of cells overexpressing yeast GPxs was determined using spotting assays. Equal numbers of cells were serially diluted fivefold and plated on medium containing glucose to assess cell numbers and containing galactose (GAL) to induce the expression of Htt103Q and the indicated ORF.


a ) Total GPx activity was measured in mGpx1 - overexpressing ( mGpx1 ) or control ( WT ) lines with ( induced ) or without ( uninduced ) Htt103Q expression . Overexpression of mGpx1 increases total GPx activity in PC12 ( * * * * P 0 . 0001 compared to uninduced controls ; n = 7 per condition ) .

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

a) Total GPx activity was measured in mGpx1-overexpressing (mGpx1) or control (WT) cell lines with (induced) or without (uninduced) Htt103Q expression. Overexpression of mGpx1 increases total GPx activity in PC12 cells (****P 0.0001 compared to uninduced controls; n = 7 per condition).


( b ) of caspase - 3 / 7 activation in response to Htt103Q expression in overexpressing mGpx1 . Overexpression of mGpx1 reduces Htt103Q - mediated caspase - 3 / 7 activation ( * * * * P 0 . 0001 ) . Values represent of mGpx1 activity in expressing Htt103Q for 72 h compared to not expressing Htt103Q ( n = 12 ( WT ) ; n = 9 ( monomeric red ( mRFP ) ) ; n = 8 ( mGpx1 ) per condition ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of caspase-3/7 activation in response to Htt103Q expression in cells overexpressing mGpx1. Overexpression of mGpx1 reduces Htt103Q-mediated caspase-3/7 activation (****P 0.0001). Values represent the ratio of mGpx1 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q (n = 12 (WT); n = 9 (monomeric red fluorescent protein (mRFP)); n = 8 (mGpx1) per condition).


( c ) Effect of GPx mimetic ebselen on Htt103Q - mediated caspase activation . A single 10 μM dose of ebselen significantly reduced caspase - 3 / 7 activation in Htt103Q - expressing ( * * * * P 0 . 0001 ) . Values represent of caspase - 3 / 7 activity in expressing Htt103Q for 72 h compared to not expressing Htt103Q . Statistical was performed using an unpaired , two - Mann - Whitney ( n = 12 per condition ) .

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

(c) Effect of the GPx mimetic ebselen on Htt103Q-mediated caspase activation. A single 10 μM dose of ebselen significantly reduced caspase-3/7 activation in Htt103Q-expressing cells (****P 0.0001). Values represent the ratio of caspase-3/7 activity in cells expressing Htt103Q for 72 h compared to cells not expressing Htt103Q. Statistical analysis was performed using an unpaired, two-tailed Mann-Whitney test (n = 12 per condition).


( d ) mGpx1 overexpression ( * * * * P 0 . 0001 ) ebselen treatment ( * * * * P 0 . 0001 ) significantly reduce Htt103Q - mediated ROS production ( n = 9 per condition ) . Unless stated otherwise , statistical analyses were performed using one - way of variance ( ANOVA ) with post - hoc . NS , not significant . * * P 0 . 01 . data are shown as mean ± s . e . m .

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

(d) mGpx1 overexpression (****P 0.0001) and ebselen treatment (****P 0.0001) significantly reduce Htt103Q-mediated ROS production (n = 9 per condition). Unless stated otherwise, all statistical analyses were performed using one-way analysis of variance (ANOVA) with post-hoc tests. NS, not significant. **P 0.01. All data are shown as the mean ± s.e.m.


( a ) Pseudopupil from wild - type flies , Huntington ' s disease flies Huntington ' s disease flies overexpressing mGpx1 at day 10 . In wild - type flies , seven rhabdomeres are visible per ommatidium . Scale bar , ∼ 10

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

(a) Pseudopupil images from wild-type flies, Huntington's disease flies and Huntington's disease flies overexpressing mGpx1 at day 10. In wild-type flies, seven rhabdomeres are visible per ommatidium. Scale bar, ∼10 μM


( b ) of average number of rhabdomeres per ommatidium in Huntington ' s disease flies with without pan - neural mGpx1 overexpression at days 1 ( n = 14 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) , 7 ( n = 7 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) 10 ( n = 8 ( Htt93Q ) ; n = 9 ( Htt93Q + mGpx1 ) ) after eclosion . Significant effects of genotype ( F1 , 50 = 82 . 3 , P = 3 . 82 × 10 − 12 ) ( F2 , 50 = 94 . 3 , P = 1 . 06 × 10 − 17 ) were observed , as well as a significant interaction between genotype ( F2 , 50 = 7 . 76 , P = 0 . 00116 ) , indicating that mGpx1 reduces rate of neurodegeneration in Htt93Q flies . Post - hoc Newman - Keuls comparisons showed significant rescue of neurodegeneration in mGpx1 - expressing flies at days 1 ( * P 0 . 05 ) , 7 ( * * * P 0 . 001 ) 10 ( * * * P 0 . 001 ) . Browne - Forsythe analyses for equality of variance revealed a significant inequality of variance ( P = 0 . 02 ) due to a single group ( Htt93Q + mGpx1 , day 1 ) . nonparametric Mann - Whitney confirmed a difference at day 1 between two groups ( P = 0 . 03 ) , between Htt93Q + mGpx1 flies at days 1 7 ( P 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with and without pan-neural mGpx1 overexpression at days 1 (n = 14 (Htt93Q); n = 9 (Htt93Q + mGpx1)), 7 (n = 7 (Htt93Q); n = 9 (Htt93Q + mGpx1)) and 10 (n = 8 (Htt93Q); n = 9 (Htt93Q + mGpx1)) after eclosion. Significant effects of genotype (F1,50 = 82.3, P = 3.82 × 10−12) and age (F2,50 = 94.3, P = 1.06 × 10−17) were observed, as well as a significant interaction between genotype and age (F2,50 = 7.76, P = 0.00116), indicating that mGpx1 reduces the rate of neurodegeneration in Htt93Q flies. Post-hoc Newman-Keuls comparisons showed significant rescue of neurodegeneration in mGpx1-expressing flies at days 1 (*P 0.05), 7 (***P 0.001) and 10 (***P 0.001). Browne-Forsythe analyses for equality of variance revealed a significant inequality of variance (P = 0.02) due to a single group (Htt93Q + mGpx1, day 1). The nonparametric Mann-Whitney test confirmed a difference at day 1 between the two groups (P = 0.03), and between Htt93Q + mGpx1 flies at days 1 and 7 (P 0.0001).


( c ) Total locomotor activity was assessed as average number of beam crossings per 30 - min bin per fly . Htt93Qex1 - expressing flies ( n = 29 ) have reduced locomotor activity compared to controls ( * * * P 0 . 001 ; n = 30 ( WT ( w118 ) ) ; n = 32 ( mGpx1 ) ) , which is restored by mGpx1 ( * * P 0 . 01 ; n = 32 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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

(c) Total locomotor activity was assessed as the average number of beam crossings per 30-min bin per fly. Htt93Qex1-expressing flies (n = 29) have reduced locomotor activity compared to controls (***P 0.001; n = 30 (WT (w118)); n = 32 (mGpx1)), which is restored by mGpx1 (**P 0.01; n = 32). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( d ) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7 . Flies expressing Htt93Qex1 driven by Pdf - GAL4 ( n = 16 brain hemispheres ) showed loss of s - LNVs compared to Canton S ( CS ) control flies ( n = 13 ) , which was ameliorated by coexpression of mGpx1 ( * * * * P 0 . 0001 ; n = 16 ) . Comparisons were performed by Kruskal - Wallis one - way ANOVA .

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

(d) Neurodegeneration of Pdf clock neurons was assessed using confocal microscopy in adult flies at day 7. Flies expressing Htt93Qex1 driven by Pdf-GAL4 (n = 16 brain hemispheres) showed loss of s-LNVs compared to Canton S (CS) control flies (n = 13), which was ameliorated by coexpression of mGpx1 (****P 0.0001; n = 16). Comparisons were performed by Kruskal-Wallis one-way ANOVA.


( e ) of average number of rhabdomeres per ommatidium in Huntington ' s disease flies with ebselen or vehicle ( DMSO ; n = 10 ) treatment at day 7 after eclosion in newly emerged day 0 adult flies ( untreated control ; n = 10 ) . Significant treatment effects were observed , both with 100 μM ( * * P 0 . 01 ; n = 9 ) 300 μM ( * * * P 0 . 001 ; n = 9 ) doses of ebselen . data are shown as mean ± s . e . m .

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

(e) Quantification of the average number of rhabdomeres per ommatidium in Huntington's disease flies with ebselen or vehicle (DMSO; n = 10) treatment at day 7 after eclosion and in newly emerged day 0 adult flies (untreated control; n = 10). Significant treatment effects were observed, both with the 100 μM (**P 0.01; n = 9) and 300 μM (***P 0.001; n = 9) doses of ebselen. All data are shown as the mean ± s.e.m.


PC12 were treated with either 10 μM ebselen or 10 mM N - acetylcysteine ( NAc ) for 24 h , with addition of 400 nM bafilomycin A1 for final 4 h . Autophagy was induced in indicated samples by addition of 200 nM rapamycin for 24 h . ( a ) Autophagy was determined by measuring amount of LC3 - II by immunoblot . ( b ) Densitometric of bands was performed using ImageJ , amounts of LC3 - II are expressed relative to untreated control . statistical analyses were performed using one - way ANOVA with post - hoc ( * * * * P 0 . 0001 ) . data are shown as mean ± s . e . m . ; n = 7 per condition .

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

PC12 cells were treated with either 10 μM ebselen or 10 mM N-acetylcysteine (NAc) for 24 h, with the addition of 400 nM bafilomycin A1 for the final 4 h. Autophagy was induced in the indicated samples by the addition of 200 nM rapamycin for 24 h. (a) Autophagy was determined by measuring the amount of LC3-II by immunoblot analysis. (b) Densitometric analysis of the bands was performed using ImageJ, and the amounts of LC3-II are expressed relative to the untreated control. All statistical analyses were performed using one-way ANOVA with post-hoc tests (****P 0.0001). All data are shown as the mean ± s.e.m.; n = 7 per condition.


A , Genome browser of ChIP - seq for SAGA before after induction of ER stress at ER stress response genes CHOP , ERP70 , GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 GSE49952 ) .

A, Genome browser images of ChIP-seq for SAGA proteins before and after induction of ER stress at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


C , Mean ChIP - seq signal for USP22 , GCN5 , ATXN7L3 surrounding gene promoters from B . Nonparametric Mann - Whitney of 7 bins ( grey shade ) surrounding maximal peak in Thaps treated conditions are reported .

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

C, Mean ChIP-seq signal for USP22, GCN5, and ATXN7L3 surrounding gene promoters from B. Nonparametric Mann-Whitney analysis of 7 bins (grey shade) surrounding maximal peak in Thaps treated conditions are reported.


A , HCT116 were treated with 100 nM Thapsigargin for 24 hrs . were stained with dye recognizing Caspases 3 7 imaged every 4 hours . Scale bar = 400 µm . B , of apoptotic populations as indicated by Cas3 / 7 from A . Three independent experiments are represented as mean ± , with significance calculated by Two - way ANOVA between conditions over time ( f ( 3 ) = 50 . 530 , p < 0 . 001 ) ; significant pairwise comparisons by Tukey ' s HSD post - hoc assessment are indicated by * . C

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

A, HCT116 cells were treated with 100 nM Thapsigargin for 24 hrs. Cells were stained with dye recognizing cleaved Caspases 3 and 7 and imaged every 4 hours. Scale bar = 400 µm. B, Quantification of apoptotic populations as indicated by Cas3/7 fluorescence from A. Three independent experiments are represented as mean ± SEM, with significance calculated by Two-way ANOVA between conditions over time (f(3)=50.530, p<0.001); significant pairwise comparisons by Tukey's HSD post-hoc assessment are indicated by *. C


A , HCT116 were treated with 100 nM Thapsigargin for indicated times in presence or absence of shRNA targeting USP22 . were harvested whole - lysate was subjected to immunoblotting with indicated antibodies .

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

A, HCT116 cells were treated with 100 nM Thapsigargin for the indicated times in the presence or absence of shRNA targeting USP22. Cells were harvested and whole-cell lysate was subjected to immunoblotting with the indicated antibodies.


A , Genome browser of ChIP - seq for Pol II Ub - H2B before after induction of ER stress , with without USP22 knockdown , at ER stress response genes CHOP , ERP70 , GRP78 . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 GSE49952 ) .

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

A, Genome browser images of ChIP-seq for Pol II and Ub-H2B before and after induction of ER stress, with and without USP22 knockdown, at ER stress response genes CHOP, ERP70, and GRP78. ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B , Metagene profiles for Pol II ( upper panel ) Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , USP22 - Bound genes . mRNA analyses of select Bound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± , with significance measured by Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 C , Metagene profiles for Pol II ( upper panel ) Ub - H2B ( middle panel ) ChIP - seq for USP22 - dependent , SAGA - Unbound genes . Nonparametric Mann - Whitney analyses of 7 bins ( grey shade ) surrounding TSS ( TSS ) midpoint of profiles ( ) in Thaps treated conditions are reported . mRNA of select Unbound gene transcripts are presented ( lower panel ) . Three independent experiments are represented as mean ± , with significance measured by Student ' s t - , * * p < 0 . 01 , * * * p < 0 . 005 .

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

B, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, USP22-Bound genes. mRNA analyses of select Bound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005 C, Metagene profiles for Pol II (upper panel) and Ub-H2B (middle panel) ChIP-seq for USP22-dependent, SAGA-Unbound genes. Nonparametric Mann-Whitney analyses of 7 bins (grey shade) surrounding TSS (TSS) and the midpoint of the profiles (Body) in Thaps treated conditions are reported. mRNA analysis of select Unbound gene transcripts are presented (lower panel). Three independent experiments are represented as mean ± SEM, with significance measured by Student's t-test, **p<0.01, ***p<0.005.


C , ChIP - qPCR for total Pol II , pSer5 Pol II , pSer2 Pol II at ERP70 , GRP78 , a gene desert ( Prom = gene promoter , Coding = downstream coding region - see Appendix Table S1 for primer sequences ) . Three independent experiments are represented as mean ± . with significance measured by Student ' s t - , * p < 0 . 05 , * * p < 0 . 01 , * * * p < 0 . 005 .

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

C, ChIP-qPCR for total RNA Pol II, pSer5 Pol II, and pSer2 Pol II at ERP70, GRP78, and a gene desert (Prom = gene promoter, Coding = downstream coding region - see Appendix Table S1 for primer sequences). Three independent experiments are represented as mean ± SEM. with significance measured by Student's t-test, *p<0.05, **p<0.01, ***p<0.005.


H , An aliquot of from D ‑ G were harvested for whole - lysate subjected to immunoblotting with indicated antibodies .

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

H, An aliquot of cells from D‑G were harvested for whole-cell lysate and subjected to immunoblotting with the indicated antibodies.


B , HCT116 were treated for 2 hrs with 100 nM Thapsigargin in presence or absence of shRNA targeting USP22 . were harvested , fractionated , fractions were subjected to immunoblotting with indicated antibodies .

O O O O O O O O O O O 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, HCT116 cells were treated for 2 hrs with 100 nM Thapsigargin in the presence or absence of shRNA targeting USP22. Cells were harvested, fractionated, and fractions were subjected to immunoblotting with the indicated antibodies.


C , In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24 . HCT116 were treated with MG132 in presence or absence of shRNA targeting USP22 . Lysates were generated using buffer containing protease cocktail inhibitor , pan - DUB inhibitor PR619 , JAMM protease inhibitor o - phenanthroline . Ubiquitylated were purified on ubiquitin binding resin eluates were either undigested ( lanes 1 2 ) or digested with USP2 to strip polyubiquitin ( lanes 3 4 ) reduce target to unit . Digestion reactions were subjected to immunoblotting with indicated antibodies .

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

C, In vitro deubiquitylation UbiTest of endogenously ubiquitylated MED24. HCT116 cells were treated with MG132 in the presence or absence of shRNA targeting USP22. Lysates were generated using buffer containing protease cocktail inhibitor, pan-DUB inhibitor PR619, and the JAMM protease inhibitor o-phenanthroline. Ubiquitylated proteins were purified on ubiquitin binding resin and eluates were either undigested (lanes 1 and 2) or digested with USP2 to strip polyubiquitin (lanes 3 and 4) and reduce target proteins to unit length. Digestion reactions were subjected to immunoblotting with the indicated antibodies.


A , Genome browser image of ChIP - seq for middle tail Mediator subunits H3K4me3 HiChIP tracks before after induction of ER stress at ER stress response gene BHLHE40 . H3K4me1 H3K27ac tracks are from ENCODE Consortium ( GEO accessions GSM945858 GSM945853 ) . ATF4 / Xbp1s binding sites are from publicly available datasets ( GEO accessions GSE69304 GSE49952 ) .

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

A, Genome browser image of ChIP-seq for middle and tail Mediator subunits and H3K4me3 HiChIP tracks before and after induction of ER stress at ER stress response gene BHLHE40. H3K4me1 and H3K27ac tracks are from the ENCODE Consortium (GEO accessions GSM945858 and GSM945853). ATF4/Xbp1s binding sites are from publicly available datasets (GEO accessions GSE69304 and GSE49952).


B - F Live confocal of ddaC clones expressing mCD8 : : GFP driven by ppk - Gal4 at WP stage or 16 h APF . l ( 3 ) 810 l ( 3 ) 924 mutant ddaC clones ( C , E ) displayed dendrite arborization defects at WP stage pruning defects at 16 h APF . ( D , F ) Introduction of one genomic construct of msps , HN267 / g - msps , rescued dendrite pruning defects in msps810 msps924 clones . Red arrowheads point to ddaC somas .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-F Live confocal images of ddaC clones expressing mCD8::GFP driven by ppk-Gal4 at WP stage or 16 h APF. l(3)810 and l(3)924 mutant ddaC clones (C, E) displayed dendrite arborization defects at WP stage and pruning defects at 16 h APF. (D, F) Introduction of one genomic construct of msps, HN267/g-msps, rescued dendrite pruning defects in msps810 and msps924 clones. Red arrowheads point to the ddaC somas.


G Percentages of ddaC clones showing severing defects at 16 h APF .

G Percentages of ddaC clones showing severing defects at 16 h APF.


H Quantitative of unpruned dendrite lengths at 16 h APF .

H Quantitative analysis of unpruned dendrite lengths at 16 h APF.


A Pan - neuronal driver elav - Gal4 was used to co - express GFP - Msps TACC in postmitotic neurons . elav - Gal4 driven mCD8 : : GFP expression was used as a control . TACC was pulled down by GFP - Msps but not by mCD8 : : GFP . Alpha - tubulin was used as a loading probing control . Neither mCD8 : : GFP nor GFP - Msps could pull down alpha - tubulin .

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

A Pan-neuronal driver elav-Gal4 was used to co-express GFP-Msps and TACC in postmitotic neurons. elav-Gal4 driven mCD8::GFP expression was used as a control. TACC was pulled down by GFP-Msps but not by mCD8::GFP. Alpha-tubulin was used as a loading and probing control. Neither mCD8::GFP nor GFP-Msps could pull down alpha-tubulin.


B Endogenous Msps were co - immunoprecipitated with Venus - TACC by GFP beads but not by control IgG beads . Alpha - tubulin was used as loading probing control .

O O O O O O O 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 Endogenous Msps proteins were co-immunoprecipitated with Venus-TACC by GFP beads but not by control IgG beads. Alpha-tubulin was used as loading and probing control.


C Endogenous Msps or TACC was pulled down by their respective antibodies , other was simultaneously detected in immunoprecipitated contents . Alpha - tubulin was used as loading probing control .

C Endogenous Msps or TACC was pulled down by their respective antibodies, and the other protein was simultaneously detected in the immunoprecipitated contents. Alpha-tubulin was used as loading and probing control.


D - G Confocal of ddaC neurons expressing control RNAi ( D ) , msps RNAi ( E ) , tacc RNAi ( F ) αTub84B RNAi ( G ) that were immunostained for TACC at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at top right corner .

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

D-G Confocal images of ddaC neurons expressing control RNAi (D), msps RNAi (E), tacc RNAi (F) and αTub84B RNAi (G) that were immunostained for TACC at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


H Quantitative of normalized TACC in ddaC somas .

H Quantitative analysis of normalized TACC fluorescence intensity in ddaC somas.


I - L Confocal of ddaC neurons expressing control RNAi ( I ) , msps RNAi ( J ) , tacc RNAi ( K ) αTub84B RNAi ( L ) that were immunostained for Msps at wL3 stage . ddaC somas are labeled by dashed lines . ddaC neurons are identified by ppk - Gal4 driven mCD8 : : GFP ( green channel ) expression , as shown at top right corner .

I-L Confocal images of ddaC neurons expressing control RNAi (I), msps RNAi (J), tacc RNAi (K) and αTub84B RNAi (L) that were immunostained for Msps at wL3 stage. ddaC somas are labeled by dashed lines. ddaC neurons are identified by ppk-Gal4 driven mCD8::GFP (green channel) expression, as shown at the top right corner.


M Quantitative of normalized Msps in ddaC somas .

M Quantitative analysis of normalized Msps fluorescence intensity in ddaC somas.


N , O expression of Msps TACC in brain tissues . ( N ) Larval brains of msps810 / mspsP transheterozygotes at early wL3 stage were immunoblotted probed with α - Msps α - TACC antibody . Actin blotting was used as loading control . dot plot on right depicts quantitative of total TACC in control msps transheterozygous larvae .

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

N, O Protein expression of Msps and TACC in brain tissues. (N) Larval brains of msps810/mspsP transheterozygotes at early wL3 stage were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading control. The dot plot on the right depicts the quantitative analysis of total TACC levels in control and msps transheterozygous larvae.


O expression of Msps TACC in brain tissues . ( O ) Adult brains of tacc59 / tacc74 transheterozygotes were immunoblotted probed with α - Msps α - TACC antibody . Actin blotting was used as loading probing control . dot plot on right displays quantitative of total Msps level in control tacc transheterozygotes .

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

O Protein expression of Msps and TACC in brain tissues. (O) Adult brains of tacc59/ tacc74 transheterozygotes were immunoblotted and probed with α-Msps and α-TACC antibody. Actin blotting was used as loading and probing control. The dot plot on the right displays the quantitative analysis of total Msps level in control and tacc transheterozygotes.


A - C Live confocal of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . While expression of control RNAi in ddaC neurons ( A ) did not affect dendrite morphogenesis nor pruning processes , knockdown of tacc using RNAi # 1 ( B ) or tacc59 / tacc74 transheterozygous mutant ( C ) severely perturbed dendrite arborization as well as pruning . Red arrowheads point to ddaC somas .

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

A-C Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. While expression of control RNAi in ddaC neurons (A) did not affect the dendrite morphogenesis nor the pruning processes, knockdown of tacc using RNAi #1 (B) or tacc59/tacc74 transheterozygous mutant (C) severely perturbed the dendrite arborization as well as pruning. Red arrowheads point to the ddaC somas.


D Percentages of ddaC neurons showing severing defects in ( A - C ) at 16 h APF .

D Percentages of ddaC neurons showing severing defects in (A-C) at 16 h APF.


E Quantitative of unpruned dendrite lengths in ( A - C ) at 16 h APF .

E Quantitative analysis of unpruned dendrite lengths in (A-C) at 16 h APF.


F - H Live confocal of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown of tacc msps via co - expressing tacc RNAi msps RNAi ( H ) in ddaC neurons did not enhance pruning defects , compared to those neurons co - expressing control RNAi msps RNAi ( F ) . Red arrowheads point to ddaC somas .

F-H Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown of tacc and msps via co-expressing tacc RNAi and msps RNAi (H) in the ddaC neurons did not enhance the pruning defects, compared to those neurons co-expressing control RNAi and msps RNAi (F). Red arrowheads point to the ddaC somas.


I Percentages of ddaC neurons showing severing defects in ( F - H ) at 16 h APF .

I Percentages of ddaC neurons showing severing defects in (F-H) at 16 h APF.


J Quantitative of unpruned dendrite lengths in ( F - H ) at 16 h APF .

J Quantitative analysis of unpruned dendrite lengths in (F-H) at 16 h APF.


Confocal of ddaC neurons expressing UAS - mCD8 : : GFP , UAS - Nod - lacZ or UAS - Kin - lacZ immunostained for β - galactosidase at wL3 stages . ( A - C ) Wild - type ddaC neurons ( A ) displayed normal distribution of Nod - lacZ signals in dendrites . In contrast , in msps810 mutants ( B ) tacc59 / tacc74 mutants ( C ) , ddaC neurons exhibited altered Nod - lacZ distribution patterns , with highly enriched stainings in somas decreased signals in dendrites .

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

Confocal images of ddaC neurons expressing UAS-mCD8::GFP, UAS-Nod-lacZ or UAS-Kin-lacZ and immunostained for β-galactosidase at wL3 stages. (A-C) Wild-type ddaC neurons (A) displayed normal distribution of Nod-lacZ signals in dendrites. In contrast, in msps810 mutants (B) and tacc59/tacc74 mutants (C), ddaC neurons exhibited altered Nod-lacZ distribution patterns, with highly enriched stainings in the somas and decreased signals in the dendrites.


Confocal of ddaC neurons ( D - F ) Kin - lacZ signals were present in axons but not detectable in dendrites of control ddaC neurons ( D ) , whereas part of Kin - lacZ were mislocalized to dendrites in msps RNAi ( E ) tacc RNAi ( F ) ddaC neurons . Asterisks indicate location of ddaC somas . arrows indicate location of axons . Curly brackets mark dendritic regions where of Nod - lacZ was measured . arrowheads point to dendritically localized Kin - lacZ signals .

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

Confocal images of ddaC neurons (D-F) Kin-lacZ signals were present in axons but not detectable in dendrites of the control ddaC neurons (D), whereas part of the Kin-lacZ were mislocalized to dendrites in msps RNAi (E) and tacc RNAi (F) ddaC neurons. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured. White arrowheads point to dendritically localized Kin-lacZ signals.


G Percentages of ddaC neurons showing defective Nod - lacZ distribution in mutants or RNAi - expressing neurons .

G Percentages of ddaC neurons showing defective Nod-lacZ distribution in mutants or RNAi-expressing neurons.


H Quantitative of normalized Nod - lacZ for 20 μm of major dendrite located 40 μm away from soma .

H Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 40 μm away from the soma.


I Percentages of ddaC neurons showing defective Kin - lacZ distribution .

I Percentages of ddaC neurons showing defective Kin-lacZ distribution.


A - F Representative kymographs depicting EB1 comet movement patterns in proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates direction towards somas , vertical arrow indicates time . EB1 : : GFP expression was driven under Gal44 - 77 . In control ddaC neurons ( A , D ) , EB1 comets move predominantly in retrograde direction . EB1 comets were largely diminished in msps810 / mspsP mutant neurons ( B ) . Interestingly , in msps810 / mspsP18 mutants ( C ) , tacc RNAi ( E ) tacc59 / tacc74 mutant ( F ) , increased anterograde comets were detected in ddaC dendrites .

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

A-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. EB1::GFP expression was driven under Gal44-77. In the control ddaC neurons (A, D), EB1 comets move predominantly in retrograde direction. EB1 comets were largely diminished in msps810/mspsP mutant neurons (B). Interestingly, in msps810/mspsP18 mutants (C), tacc RNAi (E) and tacc59/tacc74 mutant (F), increased anterograde comets were detected in ddaC dendrites.


G - J Quantitative of percentages of anterograde EB1 : : GFP comets , track , number ( per 30 µm ) velocity of comets in each neuron imaged .

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

G-J Quantitative analysis of the percentages of anterograde EB1::GFP comets, the track length, the number (per 30 µm) and the velocity of the comets in each neuron imaged.


A - F Confocal of ddaC neurons expressing UAS - mCD8 : : GFP ( Green channel ) , UAS - Nod - lacZ immunostained for β - galactosidase at wL3 stages . Simultaneous knockdown of klp10A msps ( B ) significantly restored Nod - lacZ localization in dendrites , compared to msps control RNAi ( A ) . ddaC neurons with colchicine treatment ( D ) or with Kat - 60 overexpression ( F ) exhibited perturbed Nod - lacZ distribution with highly enriched in soma decreased signals in dendrites . Asterisks indicate location of ddaC somas . arrows indicate location of axons . Curly brackets mark dendritic regions where of Nod - lacZ was measured .

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

A-F Confocal images of ddaC neurons expressing UAS-mCD8::GFP (Green channel), UAS-Nod-lacZ and immunostained for β-galactosidase at wL3 stages. Simultaneous knockdown of klp10A and msps (B) significantly restored Nod-lacZ localization in the dendrites, compared to msps and control RNAi (A). ddaC neurons with colchicine treatment (D) or with Kat-60 overexpression (F) exhibited perturbed Nod-lacZ distribution with highly enriched staining in the soma and decreased signals in the dendrites. Asterisks indicate the location of ddaC somas. White arrows indicate the location of axons. Curly brackets mark the dendritic regions where fluorescence intensity of Nod-lacZ was measured.


G - L Live confocal of ddaC neurons visualized by ppk - Gal4 driven mCD8 : : GFP expression at WP or 16 h APF . Simultaneous knockdown klp10A msps ( H ) almost fully rescued dendrite morphological defects pruning defects compared with msps , control RNAi neurons . Wild type larvae fed with colchicine ( J ) showed pruning defects in comparison with those feed with DMSO ( I ) . ddaC neurons with Kat - 60 overexpression ( L ) exhibited abnormal dendrite arborization pruning defects . Red arrowheads point to ddaC somas .

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

G-L Live confocal images of ddaC neurons visualized by ppk-Gal4 driven mCD8::GFP expression at WP or 16 h APF. Simultaneous knockdown klp10A and msps (H) almost fully rescued the dendrite morphological defects and the pruning defects compared with the msps, control RNAi neurons. Wild type larvae fed with colchicine (J) showed pruning defects in comparison with those feed with DMSO (I). ddaC neurons with Kat-60 overexpression (L) exhibited abnormal dendrite arborization and pruning defects. Red arrowheads point to the ddaC somas.


M , N Quantitative of normalized Nod - lacZ for 20 μm of major dendrite located 55 μm or 40 μm away from soma respectively .

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

M, N Quantitative analysis of normalized Nod-lacZ intensity for 20 μm of major dendrite located 55 μm or 40 μm away from the soma respectively.


O Percentages of ddaC neurons showing severing defects at 16 h APF .

O Percentages of ddaC neurons showing severing defects at 16 h APF.


P Quantitative of unpruned dendrite lengths at 16 h APF .

P Quantitative analysis of unpruned dendrite lengths at 16 h APF.


- F Representative kymographs depicting EB1 comet movement patterns in proximal dendrites of ddaC neurons at 96 h AEL . Horizontal arrow indicates direction towards somas , vertical arrow indicates time . ( B ) RNAi knockdown of klp10A in msps810 / mspsP18 mutants restored retrograde movement pattern of EB1 comets . MT depolymerization or severing induced by Colchicine treatment ( D ) or overexpression of Kat - 60 ( F ) led to mixed MT orientation in ddaC neurons .

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

-F Representative kymographs depicting EB1 comet movement patterns in the proximal dendrites of ddaC neurons at 96 h AEL. Horizontal arrow indicates the direction towards the somas, and vertical arrow indicates the time. (B) RNAi knockdown of klp10A in msps810/mspsP18 mutants restored the retrograde movement pattern of EB1 comets. MT depolymerization or severing induced by Colchicine treatment (D) or overexpression of Kat-60 (F) led to mixed MT orientation in ddaC neurons.


G - J Quantitative of percentages of anterograde EB1 comets , track , number ( per 30 µm ) velocity of EB1 comets in each neuron imaged .

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

G-J Quantitative analysis of the percentages of anterograde EB1 comets, the track length, number (per 30 µm) and the velocity of EB1 comets in each neuron imaged.


( A ) 2D STED of RPE1 co - stained with indicated antibodies . Scale bar : 200 nm .

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

(A) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies. Scale bar: 200 nm.


( B ) Dot plots showing average pixel intensities at parent centrioles expressed as PIDD1 / ODF2 in indicated lines genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed for each condition , a . u . = arbitrary units . Unpaired Mann - Whitney ( * * * * P < 0 . 0001 ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) Dot plots showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/ODF2 fluorescence ratio in the indicated cell lines and genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed for each condition, a.u. = arbitrary units. Unpaired Mann-Whitney test (****P < 0.0001).


( C ) Representative from indicated lines co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(C) Representative fluorescence micrographs from the indicated cell lines co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Co - localization between ODF2 PIDD1 ( n = 11 ) , FBF1 PIDD1 ( n = 21 ) , ANKRD26 PIDD1 ( n = 17 ) assessed on value represents fraction PIDD1 objects touching either ODF2 , FBF1 or ANKRD26 objects .

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

(D) Co-localization between ODF2 and PIDD1 (n = 11), FBF1 and PIDD1 (n = 21), and ANKRD26 and PIDD1 (n = 17) assessed on images The value represents the fraction PIDD1 objects touching either ODF2, FBF1 or ANKRD26 objects.


( E ) 2D STED of RPE1 co - stained with indicated antibodies , scale bar : 200 nm .

(E) 2D STED micrographs of RPE1 cells co-stained with the indicated antibodies, scale bar: 200 nm.


( F ) Representative of RPE1 of indicated genotypes co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of RPE1 cells of the indicated genotypes co-stained with indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( G ) Dot plot showing average pixel intensities at parent centrioles expressed as PIDD1 / CEP128 in RPE1 of indicated genotypes . Mean values ( red lines ) ± s . e . m . are reported . N ≥ 50 centrosomes were assessed from as many for each condition , a . u . = arbitrary units . Statistical significance was assessed by Kruskal - Wallis , comparing each sample to wild type ( * * * * P < 0 . 0001 ) .

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

(G) Dot plot showing the average pixel intensities at individual parent centrioles expressed as the PIDD1/CEP128 fluorescence ratio in RPE1 of the indicated genotypes. Mean values (red lines) ± s.e.m. are reported. N ≥ 50 centrosomes were assessed from as many individual cells for each condition, a.u. = arbitrary units. Statistical significance was assessed by Kruskal-Wallis test, comparing each sample to the wild type (****P < 0.0001).


( B ) Representative of RPE1 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . were co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

O O O O O O O O O O O O O O O O O O O O O O O O O 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) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( C ) Dot plot showing average pixel intensities of Myc signal at parent centrioles in RPE1 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units . Kruskal - Wallis ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

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

(C) Dot plot showing the average pixel intensities of the Myc signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( D ) Dot plot showing average pixel intensities of PIDD1 signal at parent centrioles in RPE1 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units . Kruskal - Wallis ( * * * * P < 0 . 0001 ; n . s . = non - significant ) .

(D) Dot plot showing the average pixel intensities of the PIDD1 signal at individual parent centrioles in RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. Kruskal-Wallis test (****P < 0.0001; n.s. = non-significant).


( E ) Representative of RPE1 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . were co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

(E) Representative fluorescent micrographs of RPE1 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). Cells were co-stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) RPE1 of indicated genotypes were treated either with DMSO or with DHCB for 24h . A fraction of DHCB treated were released into fresh medium for other 24h ( release ) . Samples were subjected to immunoblotting ; n = 3 independent experiments .

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

(A) RPE1 cells of the indicated genotypes were treated either with DMSO or with DHCB for 24h. A fraction of DHCB treated cells were released into fresh medium for other 24h (release). Samples were subjected to immunoblotting; n = 3 independent experiments.


( B ) of RPE1 of indicated genotypes , either treated for 24h with DHCB or with vehicle alone ( DMSO ) . Blow - up without Hoechst 33342 is magnified 2 . 5X . Scale bar : 5 μm . ( C ) Immunofluorescence of RPE1 as in ( B ) were used to visually assess of presenting one or two nuclei . N = 3 , ≥ 50 from each independent experiment . Mean values ± s . e . m . are reported . increase in number of binucleated between wild type sample other genotypes was assessed ( ANOVA ; * P < 0 . 05 ) . ( D ) Immunofluorescence of RPE1 as in ( B ) were used to visual score number of centrosomes per by counting number of γ - tubulin - positive centrioles . N = 3 , ≥ 50 from each independent experiment . Mean values ± s . e . m . are reported . increase in number of with > 2 centrosomes between wild type sample other genotypes was assessed ( ANOVA ; * * P < 0 . 01 ) .

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

(B) Fluorescence micrographs of RPE1 cells of the indicated genotypes, either treated for 24h with DHCB or with vehicle alone (DMSO). Blow-up without Hoechst 33342 is magnified 2.5X. Scale bar: 5 μm. (C) Immunofluorescence micrographs of RPE1 as in (B) were used to visually assess the percentage of cells presenting one or two nuclei. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of binucleated cells between the wild type sample and all the other genotypes was assessed (ANOVA test; *P < 0.05). (D) Immunofluorescence micrographs of RPE1 as in (B) were used to visual score the number of centrosomes per cell by counting the number of γ-tubulin-positive centrioles. N = 3, ≥ 50 cells from each independent experiment. Mean values ± s.e.m. are reported. The increase in the number of cells with > 2 centrosomes between the wild type sample and all the other genotypes was assessed (ANOVA test; **P < 0.01).


( E ) Quantitative assessment of fraction of RPE1 undergoing cytokinesis failure upon DHCB treatment , inferred on basis of increase of ploidies ≥ 4C ( upper panel ) , of fraction of abovementioned undergoing genome reduplication upon release after DHCB treatment ( lower panel ) . values of biological replicates , their mean standard deviations are reported .

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

(E) Quantitative assessment of the fraction of RPE1 cells undergoing cytokinesis failure upon DHCB treatment, inferred on the basis of the increase of ploidies ≥ 4C (upper panel), and of the fraction of the abovementioned cells undergoing genome reduplication upon release after DHCB treatment (lower panel). Individual values of biological replicates, their mean and standard deviations are reported.


( F ) RPE1 were either left untransduced ( mock ) or transduced with lentiviral vectors expressing indicated Myc - ANKRD26 constructs . Subsequently , were treated either with DMSO or ZM447439 for 24h subjected to immunoblotting ; n = 3 independent experiments .

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

(F) RPE1 cells were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated Myc-ANKRD26 constructs. Subsequently, cells were treated either with DMSO or ZM447439 for 24h and subjected to immunoblotting; n = 3 independent experiments.


( B ) A549 of indicated genotypes were either left untransduced ( mock ) or transduced with lentiviral vectors expressing indicated PIDD1 - V5 derivatives subjected to immunoblotting . N = 2 independent experiments .

O O O O O O O O O O O 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) A549 cells of the indicated genotypes were either left untransduced (mock) or transduced with lentiviral vectors expressing the indicated PIDD1-V5 derivatives and subjected to immunoblotting. N = 2 independent experiments.


( C ) of A549 of indicated genotypes either left untransduced ( mock ) , or transduced with PIDD1 - V5 lentiviral vectors co - stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(C) Fluorescence micrographs of A549 cells of the indicated genotypes either left untransduced (mock), or transduced with PIDD1-V5 lentiviral vectors and co- stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( D ) Dot plot showing average V5 pixel intensities at parent centrioles in A549 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units . ( E ) Dot plot showing average V5 pixel intensities at parent centrioles in A549 of indicated genotypes , either transduced with indicated lentiviral vectors or left untransduced ( mock ) . N > 50 centrosomes were assessed for each condition in as many ; a . u . = arbitrary units .

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

(D) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units. (E) Dot plot showing the average V5 pixel intensities at individual parent centrioles in A549 cells of the indicated genotypes, either transduced with the indicated lentiviral vectors or left untransduced (mock). N > 50 centrosomes were assessed for each condition in as many individual cells; a.u. = arbitrary units.


( F ) Representative of A549 of indicated genotypes . were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors expressing PIDD1 non - cleavable derivative ( PIDD1S446A - S588A ) or truncations thereof . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors expressing the PIDD1 non-cleavable derivative (PIDD1S446A-S588A) or truncations thereof. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) A549 of indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1 - V5 in its wild type form . were treated either with DMSO or with ZM447439 for 24h subjected to immunoblotting . N = 2 independent experiments .

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

(A) A549 cells of the indicated genotypes were either left untransduced or transduced with a lentiviral vector expressing PIDD1-V5 in its wild type form. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( B , C ) A549 of indicated genotypes were either left untransduced ( mock ) , or transduced with lentiviral vectors expressing PIDD1 - V5 in its wild type form or carrying indicated point mutations . were treated either with DMSO or with ZM447439 for 24h subjected to immunoblotting . N = 2 independent experiments .

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

(B,C) A549 cells of the indicated genotypes were either left untransduced (mock), or transduced with lentiviral vectors expressing PIDD1-V5 in its wild type form or carrying the indicated point mutations. Cells were treated either with DMSO or with ZM447439 for 24h and subjected to immunoblotting. N = 2 independent experiments.


( D ) of A549 of indicated genotypes . were either left untransduced ( mock ) or transduced with PIDD1 - V5 lentiviral vectors carrying indicated point mutations , stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(D) Fluorescence micrographs of A549 cells of the indicated genotypes. Cells were either left untransduced (mock) or transduced with PIDD1-V5 lentiviral vectors carrying the indicated point mutations, and stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


( A ) Representative across indicated cycle phases from RPE1 stably expressing CETN1 - GFP . Centrosomal antigens were stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2 . 5X . Scale bar : 5 μm .

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

(A) Representative fluorescence micrographs across the indicated cell cycle phases from RPE1 cells stably expressing CETN1-GFP. Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2.5X. Scale bar: 5 μm.


centrioles subjected to measurement in ( B ) are labelled with numbers . ( B ) of PIDD1 average pixel at centrioles across indicated cycle phases of RPE1 stained N > 50 centrioles were assessed for each phase , a . u . = arbitrary units .

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

The centrioles subjected to fluorescence intensity measurement in (B) are labelled with numbers. (B) Quantification of the PIDD1 average pixel intensity at individual centrioles across the indicated cell cycle phases of RPE1 cells stained N > 50 centrioles were assessed for each phase, a.u. = arbitrary units.


( C ) of RPE1 stably expressing CETN1 - GFP treated either with DMSO or with DHCB for 24h . Blow - ups 33342 are magnified 2 . 5X . Scale bar : 5 μm . ( D ) Dot plot showing PIDD1 average pixel intensities at parent centrioles calculated from as in ( C ) . Mean values ( red lines ) ± s . e . m . are reported . N > 50 centrosomes were assessed for each condition , a . u . = arbitrary units .

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

(C) Fluorescence micrographs of RPE1 cells stably expressing CETN1-GFP treated either with DMSO or with DHCB for 24h. Blow-ups 33342 are magnified 2.5X. Scale bar: 5 μm. (D) Dot plot showing PIDD1 average pixel intensities at individual parent centrioles calculated from images as in (C). Mean values (red lines) ± s.e.m. are reported. N > 50 centrosomes were assessed for each condition, a.u. = arbitrary units.


( E ) Movie stills of a representative RPE1 stably expressing CETN1 - GFP treated with DHCB subjected to time - lapse video microscopy in presence of SiR - . Time is expressed in minutes , relative to anaphase onset . dashed indicates plasma of of interest , arrowheads indicate centrosomes ' position .

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

(E) Movie stills of a representative RPE1 cell stably expressing CETN1-GFP treated with DHCB and subjected to time-lapse video microscopy in the presence of SiR-DNA. Time is expressed in minutes, relative to anaphase onset. The dashed line indicates the plasma membrane of the cell of interest, arrowheads indicate the centrosomes' position.


( F ) Centrosomal over time in RPE1 stably expressing CETN1 - GFP treated with DHCB . Time zero corresponds to frame preceding anaphase onset . Coloured dots ( lower panel ) summarize clustering time for each , mean ± standard deviation in black . Data calculated from four - dimensional imaging as in ( E ) . N = 10 .

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

(F) Centrosomal distance over time in RPE1 cells stably expressing CETN1-GFP and treated with DHCB. Time zero corresponds to the frame preceding anaphase onset. Coloured dots (lower panel) summarize the clustering time for each cell, mean ± standard deviation in black. Data calculated from four-dimensional imaging as in (E). N = 10 cells.


( A ) Dot plot showing between parent centrioles pairs in A549 following indicated treatments ( ZM = ZM447439 ; REV = reversine ) . Nocodazole concentrations are 0 . 03 μM , 0 . 1 μM , 0 . 33 μM , 1 μM , 3 . 3 μM . Median ( red ) 95 % confidence interval thereof ( black ) are shown . N > 50 were analysed . ( B ) Immunoblot of A549 subjected to indicated treatments for 24h as in ( A ) . N = 3 independent experiments . ( C ) content of A549 subjected to indicated treatments as in ( A ) either for 24h ( left panels ) or 48h ( right panels ) . N = 2 independent experiments .

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

(A) Dot plot showing the distance between parent centrioles pairs in A549 cells following the indicated treatments (ZM = ZM447439; REV = reversine). Nocodazole concentrations are 0.03 μM, 0.1 μM, 0.33 μM, 1 μM, 3.3 μM. Median (red) and 95% confidence interval thereof (black) are shown. N > 50 cells were analysed. (B) Immunoblot of A549 cells subjected to the indicated treatments for 24h as in (A). N = 3 independent experiments. (C) DNA content analysis of A549 cells subjected to the indicated treatments as in (A) either for 24h (left panels) or 48h (right panels). N = 2 independent experiments.


(E) Immunoblots of RPE1 cells synchronized


( F ) Representative of RPE1 synchronized Centrosomal antigens were stained with indicated antibodies . Blow - ups without Hoechst 33342 are magnified 2X . Scale bar : 5 μm .

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

(F) Representative fluorescence micrographs of RPE1 cells synchronized Centrosomal antigens were stained with the indicated antibodies. Blow-ups without Hoechst 33342 are magnified 2X. Scale bar: 5 μm.


( A ) A549 of indicated genotypes were treated for 24h as indicated ( CPT = camptothecin , ZM = ZM447439 ) . Samples were subjected to immunoblotting ; n = 3 independent experiments . ( B ) A549 treated as in ( A ) were subjected to microscopy centrosome abundance was assessed by visually scoring γ - tubulin - positive centrioles per . N = 3 , ≥ 50 from each independent experiment .

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

(A) A549 cells of the indicated genotypes were treated for 24h as indicated (CPT = camptothecin, ZM = ZM447439). Samples were subjected to immunoblotting; n = 3 independent experiments. (B) A549 cells treated as in (A) were subjected to fluorescence microscopy and centrosome abundance was assessed by visually scoring γ-tubulin-positive centrioles per cell. N = 3, ≥ 50 cells from each independent experiment.


( C ) RT - qPCR of PIDD1 mRNA expression in A549 upon treatment with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . average fold of induction ± standard deviation is shown . N = 3 biological replicates with two technical replicates each . Comparisons were performed between treatments of every genotype corresponding treatment of wild type ( WT )

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

(C) RT-qPCR analysis of PIDD1 mRNA expression in A549 cells upon treatment with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. The average fold of induction ± standard deviation is shown. N = 3 biological replicates with two technical replicates each. Comparisons were performed between treatments of every genotype and the corresponding treatment of wild type (WT) cells


with increasing doses of Nutlin 3a ( i . e . 3 . 3 μM or 10 μM ) using two independent probes . ( D ) Immunoblot of samples treated N = 3 independent experiments .

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

with increasing doses of Nutlin 3a (i.e. 3.3 μM or 10 μM) using two independent probes. (D) Immunoblot analysis of samples treated N = 3 independent experiments.


( A ) Recombinant c10orf76 directly binds to PI4KB in vitro . His - pulldown assays of baculovirus / Sf9 produced 6xHis - c10orf76 ( 3 μM ) were carried out with untagged PI4KB ( 2 . 5 μM ) .

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

(A) Recombinant c10orf76 directly binds to PI4KB in vitro. His-pulldown assays of baculovirus/Sf9 produced 6xHis-c10orf76 (3 μM) were carried out with untagged PI4KB (2.5 μM).


( B ) PI4KB c10orf76 form a stable . of c10orf76 - PI4KB eluted from a S200 superdex 10 / 300 GL increase gel filtration column ( GE ) at a volume consistent with a heterodimer ( 169 kDa ) , while c10orf76 alone eluted at a volume consistent with a monomer ( 79 kDa ) . Lines with MW values indicate elution of MW standards ( 158 kDa aldolase , 75 kDa conalbumin ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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) PI4KB and c10orf76 form a stable complex. The complex of c10orf76-PI4KB eluted from a S200 superdex 10/300 GL increase gel filtration column (GE) at a volume consistent with a heterodimer (169 kDa), while c10orf76 alone eluted at a volume consistent with a monomer (79 kDa). Lines with MW values indicate elution of MW standards (158 kDa aldolase, 75 kDa conalbumin).


( C ) PI4KB is potently inhibited by c10orf76 in a dose - dependent manner in vitro . Kinase assays of PI4KB ( 20 nM ) in presence of varying concentrations of c10orf76 ( 1 . 6 nM - 1 μM ) were carried out on pure PI lipid vesicles ( 0 . 5 mg / L ) in presence of 100 μM ATP . data was normalized to kinase activity of PI4KB alone . IC50 values were determined by one binding site , nonlinear regression ( curve fit ) using Graphpad . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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

(C) PI4KB is potently inhibited by c10orf76 in a dose-dependent manner in vitro. Kinase assays of PI4KB (20 nM) in the presence of varying concentrations of c10orf76 (1.6 nM-1 μM) were carried out on pure PI lipid vesicles (0.5 mg/L) in the presence of 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. IC50 values were determined by one binding site, nonlinear regression (curve fit) using Graphpad. Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB is potently inhibited by c10orf76 on pure PI vesicles vesicles mimicking Golgi composition . Kinase assays of PI4KB c10orf76 were carried out on lipid substrate composed of pure PI vesicles ( 0 . 5 mg / mL ) with 100 μM ATP , Golgi mimic vesicles ( 0 . 5 mg / ml , 10 % PS , 20 % PI , 25 % PE , 45 % PC ) with 10 μM ATP . PI4KB was present at 20 300 nM in PI Golgi substrate assays respectively , with c10orf76 present at 500 nM in both experiments . data is normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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

(D) PI4KB is potently inhibited by c10orf76 on pure PI vesicles and vesicles mimicking Golgi composition. Kinase assays of PI4KB and c10orf76 were carried out on lipid substrate composed of pure PI vesicles (0.5 mg/mL) with 100 μM ATP, and Golgi mimic vesicles (0.5 mg/ml, 10% PS, 20% PI, 25% PE, 45% PC) with 10 μM ATP. PI4KB was present at 20 and 300 nM in the PI and Golgi substrate assays respectively, with c10orf76 present at 500 nM in both experiments. The data is normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Changes in deuterium incorporation PI4KB in presence of c10orf76 showed a profound ordering of kinase domain N - lobe linker smaller changes in helical domain C - lobe of kinase domain . sum of difference mapped as difference in number of deuterons incorporated for PI4KB ( 400 nM ) in presence absence of c10orf76 ( 400 nM ) over time points ( 3s at 1 oC ; 3s , 30s , 300s at 23 oC ) . Each dot represents a peptide graphed on x - axis according to central residue . red boxes highlight key regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , unpaired two - student t - p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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

(E) Changes in deuterium incorporation PI4KB in the presence of c10orf76 showed a profound ordering of the kinase domain N-lobe linker and smaller changes in the helical domain and C-lobe of the kinase domain. The sum of the difference mapped as the difference in number of deuterons incorporated for PI4KB (400 nM) in the presence and absence of c10orf76 (400 nM) over all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC). Each dot represents a peptide graphed on the x-axis according to the central residue. The red boxes highlight key regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Changes in deuterium incorporation of c10orf76 in presence of PI4KB . H / D exchange reactions displayed as sum of difference in HDX in number of deuterons for c10orf76 ( 400 nM ) in presence of PI4KB ( 400 nM ) at time points ( 3s at 1 oC ; 3s , 30s , 300s at 23 oC ) analyzed . Red boxes highlight regions that showed significant changes ( > 7 % decrease in exchange , > 0 . 5 Da difference , unpaired two - student t - p < 0 . 05 ) . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Changes in the deuterium incorporation of c10orf76 in the presence of PI4KB. H/D exchange reactions displayed as the sum of the difference in HDX in the number of deuterons for c10orf76 (400 nM) in the presence of PI4KB (400 nM) at all time points (3s at 1 oC; 3s, 30s, and 300s at 23 oC) analyzed. Red boxes highlight regions that showed significant changes (>7% decrease in exchange, >0.5 Da difference, and unpaired two-tailed student t-test p<0.05). Error bars represent standard deviation of independent technical replicates (n=3).


( H ) PI4KB N - lobe linker undergoes a disorder - to - order transition upon binding c10orf76 . Selected peptides ( including , domain information , numbering ) of both PI4KB c10orf76 displayed as % deuteration incorporation over time . Error bars represent standard deviation of independent technical replicates ( n = 3 ) , are typically smaller than of point on graph .

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(H) The PI4KB N-lobe linker undergoes a disorder-to-order transition upon binding c10orf76. Selected peptides (including the sequence, domain information, and numbering) of both PI4KB and c10orf76 displayed as the % deuteration incorporation over time. Error bars represent standard deviation of independent technical replicates (n=3), and are typically smaller than the size of the point on the graph.


( B ) N - lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA . Recombinant PKA at different concentrations ( 0 , 7 , 34 , 168 , or 840 ng ) was incubated with recombinant ( E . coli ) wild - type PI4KB ( 20 μg ) for 1 hour with 200 μM ATP amount of phosphorylation was followed using mass spectrometry . Relative abundance of Ser496 phosphorylated PI4KB was calculated using relative ( total ) of phosphorylated vs non - phosphorylated peptide ( 486 - 506 ) .

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(B) The N-lobe kinase linker of PI4KB can be efficiently phosphorylated by PKA. Recombinant PKA at different concentrations (0, 7, 34, 168, or 840 ng) was incubated with recombinant (E. coli) wild-type PI4KB (20 μg) for 1 hour with 200 μM ATP and the amount of phosphorylation was followed using mass spectrometry. Relative abundance of Ser496 phosphorylated PI4KB was calculated using the relative intensity (total area) of the phosphorylated vs non-phosphorylated peptide (486-506).


( C ) PI4KB phosphorylation by PKA alters affinity for c10orf76 . kinase activity of different variants of PI4KB ( 15 nM ) was measured in presence of varying amounts of c10orf76 ( 23 nM - 1 . 5μM ) with 100 % PI lipid substrate ( 0 . 5 mg / L ) 100 μM ATP . data was normalized to kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(C) PI4KB phosphorylation by PKA alters the affinity for c10orf76. The kinase activity of different variants of PI4KB (15 nM) was measured in the presence of varying amounts of c10orf76 (23 nM-1.5μM) with 100% PI lipid substrate (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone Error bars represent standard deviation of independent technical replicates (n=3).


( D ) PI4KB has same kinase activity when Ser496 is phosphorylated or mutated to alanine . Kinase of PI4KB non - phosphorylated , phos - Ser496 or S496A ( 15 nM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . data was normalized to kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

(D) PI4KB has the same kinase activity when Ser496 is phosphorylated or mutated to alanine. Kinase assay of PI4KB non-phosphorylated, phos-Ser496 or S496A (15 nM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( E ) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76 . His - pulldown assays of 6xHis - c10orf76 ( 3 μM ) with wild - type or RL494EA PI4KB ( 1 - 2 μM ) .

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(E) Engineered RL494EA PI4KB mutant shows decreased binding to c10orf76. His-pulldown assays of 6xHis-c10orf76 (3 μM) with wild-type or RL494EA PI4KB (1-2 μM).


( F ) RL494EA PI4KB activity is not inhibited by c10orf76 . Kinase assays of either wild type or mutant RL494EA PI4KB ( 40 nM ) were carried out with varying concentrations of c10orf76 ( 3 . 9 nM - 2μM ) with 100 % PI lipid vesicles ( 0 . 5 mg / L ) 100 μM ATP . data was normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(F) RL494EA PI4KB activity is not inhibited by c10orf76. Kinase assays of either wild type or mutant RL494EA PI4KB (40 nM) were carried out with varying concentrations of c10orf76 (3.9 nM-2μM) with 100% PI lipid vesicles (0.5 mg/L) and 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( G ) Wild - type PI4KB RL494EA PI4KB mutant have same lipid kinase activity . Kinase assays of either wild - type mutant PI4KB ( 10 nM ) were carried out with 100 % PI lipid vesicles ( 0 . 5 mg / L ) , 100 μM ATP , PI4KB ( 300 nM ) on Golgi - mimic vesicles ( 0 . 5 mg / mL ) with 10 μM ATP . data was normalized to kinase activity of WT PI4KB . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

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(G) Wild-type PI4KB and RL494EA PI4KB mutant have the same lipid kinase activity. Kinase assays of either wild-type and mutant PI4KB (10 nM) were carried out with 100% PI lipid vesicles (0.5 mg/L), 100 μM ATP, and PI4KB (300 nM) on Golgi-mimic vesicles (0.5 mg/mL) with 10 μM ATP. The data was normalized to the kinase activity of WT PI4KB. Error bars represent standard deviation of independent technical replicates (n=3).


( H ) FLH409AAA - c10orf76 mutant shows decreased affinity for PI4KB . His - pulldown assays of 6xHis - c10orf76 ( 1 μM ) with wild - type PI4KB ( 1 μM ) . Samples washed a total of 4 times .

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(H) FLH409AAA-c10orf76 mutant shows decreased affinity for PI4KB. His-pulldown assays of 6xHis-c10orf76 (1 μM) with wild-type PI4KB (1 μM). Samples washed a total of 4 times.


( I ) Kinase shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced . Kinase of PI4KB ( 40 nM ) a curve of c10orf76 ( 3 . 9 nM - 2μM ) on pure PI lipid vesicles ( 0 . 5 mg / L ) with 100 μM ATP . data was normalized to kinase activity of PI4KB alone . Error bars represent standard deviation of independent technical replicates ( n = 3 ) .

(I) Kinase assay shows FLH409AAA c10orf76 inhibition of PI4KB is greatly reduced. Kinase assay of PI4KB (40 nM) and a concentration curve of c10orf76 (3.9 nM-2μM) on pure PI lipid vesicles (0.5 mg/L) with 100 μM ATP. The data was normalized to the kinase activity of PI4KB alone. Error bars represent standard deviation of independent technical replicates (n=3).


( A ) Transfections of HEK293 revealed that both wild - type GFP - PI4KB RL494EA GFP - PI4KB localize to Golgi .

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(A) Transfections of HEK293 cells revealed that both wild-type GFP-PI4KB and RL494EA GFP-PI4KB localize to the Golgi.


( B ) WT c10orf76 also localized to Golgi , however , PI4KB binding deficient mutant of c10orf76 ( FLH409AAA ) predominantly localized to cytosol .

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(B) WT c10orf76 also localized to the Golgi, however, the PI4KB binding deficient mutant of c10orf76 (FLH409AAA) predominantly localized to the cytosol.


( D ) Mitochondria recruitment experiment with wild - type PI4KB c10orf76 . Left : AKAP1 - FRB - CFP is localized to mitochondria before ( top ) 5 minutes after rapamycin ( 100 nM ) treatment ( bottom ) . Middle : mRFP - FKBP12 - PI4KB at high expression level is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 at a high expression level is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) .

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(D) Mitochondria recruitment experiment with wild-type PI4KB and c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after rapamycin (100 nM) treatment (bottom). Middle: mRFP-FKBP12-PI4KB at high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 at a high expression level is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom).


( F ) Mitochondria recruitment experiment with mutant PI4KB WT c10orf76 . Left : AKAP1 - FRB - CFP is localized to mitochondria before ( top ) 5 minutes after ( bottom ) rapamycin treatment . Middle : mRFP - FKBP12 - PI4KB ( RL494EA ) is located in cytosol before rapamycin ( top ) translocates to mitochondria after rapamycin induction ( bottom ) . Right : eGFP - c10orf76 is located in cytosol before ( top ) after ( bottom ) rapamycin induction .

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(F) Mitochondria recruitment experiment with mutant PI4KB and WT c10orf76. Left: AKAP1-FRB-CFP is localized to the mitochondria before (top) and 5 minutes after (bottom) rapamycin treatment. Middle: mRFP-FKBP12-PI4KB(RL494EA) is located in the cytosol before rapamycin (top) and translocates to the mitochondria after rapamycin induction (bottom). Right: eGFP-c10orf76 is located in the cytosol before (top) and after (bottom) rapamycin induction.


( A HAP1 were fixed stained with antibodies examining PI4P PI4KB Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(A HAP1 cells were fixed and stained with antibodies examining PI4P and PI4KB Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


B ) HAP1 were fixed stained with antibodies examining Golgi markers ( B ) . ( C ) Western blot of GBF1 , PI4KB B - Actin in both wild - type c10orf76 KO HAP1 . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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B) HAP1 cells were fixed and stained with antibodies examining Golgi morphology markers (B). (C) Western blot of GBF1, PI4KB and B-Actin levels in both wild-type and c10orf76 KO HAP1 cells. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( D ) Markers of Arf1 activation - coatomer COPIα / γ βCOP act as a readout for GTP - bound Arf1 , while native coatomer was detected with CM1 antibody . Data information : Nuclei were stained with DAPI ( blue ) . Scale bars represent 10 μm .

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(D) Markers of Arf1 activation - the coatomer proteins COPIα/γ and βCOP act as a readout for GTP-bound Arf1, while the native coatomer was detected with the CM1 antibody. Data information: Nuclei were stained with DAPI (blue). Scale bars represent 10 μm.


( A ) Viral infection assays determining viral titers by end - point titration at 0 hours 8 hours in HAP1 wild - type or c10orf76 knockout . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Titre values for c10orf76 knockout at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours wild - type titres at 8 hours using a one - way ANOVA . * * , P < 0 . 01 ; N . S . , P > 0 . 05 .

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(A) Viral infection assays determining viral titers by end-point titration at 0 hours and 8 hours in HAP1 wild-type or c10orf76 knockout cells. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Titre values for c10orf76 knockout cells at 8 hours were statistically evaluated compared to c10orf76 knockout titres at 0 hours and wild-type titres at 8 hours using a one-way ANOVA. **, P<0.01; N.S., P>0.05.


( B ) Viral infection assays determining titers by end point titration at 8 hours in HeLa wild - type PI4KB knockout upon transfection of wild - type PI4KB , - disrupting RL494EA PI4KB mutant or kinase dead D674A PI4KB mutant . Left : Coxsackievirus A10 infection . Middle : Poliovirus - 1 infection . Right : Coxsackievirus B3 infection . Values were statistically evaluated compared to GalT control using a one - way ANOVA . * * , P < 0 . 01 ; * , P < 0 . 05 ; N . S . , P > 0 . 05 .

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(B) Viral infection assays determining virus titers by end point titration at 8 hours in HeLa wild-type and PI4KB knockout cells upon transfection of wild-type PI4KB, the complex-disrupting RL494EA PI4KB mutant or the kinase dead D674A PI4KB mutant. Left: Coxsackievirus A10 infection. Middle: Poliovirus-1 infection. Right: Coxsackievirus B3 infection. Values were statistically evaluated compared to the GalT control using a one-way ANOVA. **, P<0.01; *, P<0.05; N.S., P>0.05.


Overall structure of 2019 - nCoV RBD bound with ACE2 . ACE2 is colored green . 2019 - nCoV RBD is colored cyan RBM is colored red . Disulfide bonds in 2019 - nCoV RBD are shown as stick indicated by yellow arrows . N - terminal helix of ACE2 responsible for binding is labeled .

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Overall structure of 2019-nCoV RBD bound with ACE2. ACE2 is colored green. 2019-nCoV RBD core is colored cyan and RBM is colored red. Disulfide bonds in the 2019-nCoV RBD are shown as stick and indicated by yellow arrows. The N-terminal helix of ACE2 responsible for binding is labeled.


Alignment of 2019 - nCoV RBD ( in cyan RBM in red ) SARS - CoV RBD ( in orange RBM in blue ) structures .

Alignment of the 2019-nCoV RBD (core in cyan and RBM in red) and SARS-CoV RBD (core in orange and RBM in blue) structures.


Structural alignment of 2019 - nCoV RBD / ACE2 SARS - CoV RBD / ACE2 complexes . 2019 - nCoV RBD is colored cyan red , its interacting ACE2 is colored green . SARS - CoV RBD is colored orange blue , its interacting ACE2 is colored salmon . PDB code for SARS - CoV RBD / ACE2 : 2AJF .

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Structural alignment of 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 complexes. 2019-nCoV RBD is colored cyan and red, its interacting ACE2 is colored green. SARS-CoV RBD is colored orange and blue, its interacting ACE2 is colored salmon. The PDB code for SARS-CoV RBD/ACE2 complex: 2AJF.


Contacting residues shown as stick at 2019 - nCoV RBD / ACE2 SARS - CoV RBD / ACE2 interfaces . Positions in both RBDs involved in ACE2 binding are indicated by red labels .

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Contacting residues shown as stick at the 2019-nCoV RBD/ACE2 and SARS-CoV RBD/ACE2 interfaces. Positions in both RBDs involved in ACE2 binding are indicated by red labels.


( B - K ' ) Drosophila posterior midguts containing Su ( H ) - Gal4 > UAS - CD8 : GFP ( Su ( H ) > GFP ) with sucrose ( Suc , control ) ( B - C ' ) , P . e for 36 h ( D - F ' ) , Ecc15 for 36 h ( G - I ' ) DSS for 36 h ( J - K ' ) were immunostained for GFP ( green ) , PH3 ( red ) DAPI ( blue ) . ( C - C ' , E - E ' , H - H ' K - K ' ) Magnification of selected areas containing PH3 + GFP ─ from B , D , G J . EB close to PH3 + ( arrow ) in C - C ' is out of focus . ( F - F ' I - I ' ) Magnification of selected areas containing PH3 + EBs ( GFP + ) from D G . arrowheads red arrows indicate PH3 in GFP ─ GFP + , respectively . ( L M ) of PH3 GFP ─ ISCs ( L ) PH3 + GFP + EBs ( M ) with given treatments . n = 21 guts for each treatment .

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(B-K') The Drosophila posterior midguts containing Su(H)-Gal4>UAS-CD8:GFP (Su(H)>GFP) with sucrose (Suc, control) (B-C'), P.e for 36 h (D-F'), Ecc15 for 36 h (G-I') and DSS for 36 h (J-K') were immunostained for GFP (green), PH3 (red) and DAPI (blue). (C-C', E-E', H-H' and K-K') Magnification of selected areas containing PH3+GFP─ cells from B, D, G and J. The EB cell close to the PH3+ cell (white arrow) in C-C' is out of focus. (F-F' and I-I') Magnification of selected areas containing PH3+ EBs (GFP+) from D and G. White arrowheads and red arrows indicate PH3 in GFP─ and GFP+ cells, respectively. (L and M) Quantification of PH3 GFP─ ISCs (L) and PH3+GFP+ EBs (M) with given treatments. n = 21 guts for each treatment.