( B ) STING Tyr - phosphorylation was inhibited by gefitinib . MEF were pre - treated with gefitinib before cGAMP - stimulation for 60 min . lysates were immuno - precipitated with anti - pTyr antibody then analyzed by anti - STING Western blot . NTC : no treatment control .

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(B) STING Tyr-phosphorylation was inhibited by gefitinib. MEF cells were pre-treated with gefitinib before cGAMP-stimulation for 60 min. Cell lysates were immuno-precipitated with anti-pTyr antibody and then analyzed by anti-STING Western blot. NTC: no treatment control.


( C ) STING Tyr - phosphorylation required EGFR . Raw 264 . 7 expressing EGFR shRNA ( KD ) or non - target shRNA ( CO ) were transfected with cGAMP for 2 hours ; lysates were analyzed by Western blot before or after immunoprecipitation with anti - pTyr antibodies .

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(C) STING Tyr-phosphorylation required EGFR. Raw 264.7 cells expressing EGFR shRNA (KD) or non-target shRNA (CO) were transfected with cGAMP for 2 hours; cell lysates were analyzed by Western blot before or after immunoprecipitation with anti-pTyr antibodies.


( D ) cGAMP stimulation triggered EGFR - binding to STING . Raw 264 . 7 were transfected with cGAMP for 1h ; lysates were immunoprecipitated with anti - STING antibody analyzed by Western blot .

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(D) cGAMP stimulation triggered EGFR-binding to STING. Raw 264.7 cells were transfected with cGAMP for 1h; cell lysates were immunoprecipitated with anti-STING antibody and analyzed by Western blot.


( E ) STING co - localized with EGFR after cGAMP stimulation . STING - / - MEF were transfected with STING - GFP ( Green ) for 24hours stimulated with cGAMP for 1h , were fixed labeled with EGFR ( Red ) Calnexin ( Purple ) antibodies . Co - localization was analyzed by Fuji co - localized dots are shown in . Scale bars represent 10 µ m .

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(E) STING co-localized with EGFR after cGAMP stimulation. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours and stimulated with cGAMP for 1h, cells were fixed and labeled with EGFR (Red) and Calnexin (Purple) antibodies. Co-localization was analyzed by Fuji and co-localized dots are shown in white. Scale bars represent 10 µ m.


( F ) STING - EGFR interaction was transient . Raw 264 . 7 lysates were prepared at indicated time points after cGAMP transfection , immunoprecipitated with anti - EGFR antibodies subjected to Western blot .

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(F) STING-EGFR interaction was transient. Raw 264.7 cell lysates were prepared at indicated time points after cGAMP transfection, immunoprecipitated with anti-EGFR antibodies and subjected to Western blot.


( G ) Gefitinib did not block , but prolonged STING - EGFR interaction . Raw 264 . 7 were pre - treated with gefitinib or DMSO for 1 hour before cGAMP stimulation . lysates were prepared at indicated time points , immuno - precipitated with anti - STING antibody subjected to Western blot .

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(G) Gefitinib did not block, but prolonged STING-EGFR interaction. Raw 264.7 cells were pre-treated with gefitinib or DMSO for 1 hour before cGAMP stimulation. Cell lysates were prepared at indicated time points, immuno-precipitated with anti-STING antibody and subjected to Western blot.


( H ) Poly I : C did not activate EGFR . STING - / - MEF were transfected with cGAMP or Poly ( I : C ) ; after 3h , p - EGFR were detected by Western blot .

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(H) Poly I:C did not activate EGFR. STING-/- MEF cells were transfected with cGAMP or Poly (I:C); after 3h, p-EGFR were detected by Western blot.


( I ) Kinetics of EGFR activation . Raw 264 . 7 were treated with cGAMP for indicated time . lysates were analyzed for p - EGFR via Western blot .

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(I) Kinetics of EGFR activation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were analyzed for p-EGFR via Western blot.


( J ) STING - bound EGFR was phosphorylated . Raw 264 . 7 were stimulated for 1h , lysates were immunoprecipitated with anti - STING antibody subjected to Western blot .

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(J) STING-bound EGFR was phosphorylated. Raw 264.7 cells were stimulated for 1h, cell lysates were immunoprecipitated with anti-STING antibody and subjected to Western blot.


( K ) Kinetics of phosphorylation . Raw 264 . 7 were treated with cGAMP for indicated time . lysates were subjected to Western blot using indicated antibodies .

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(K) Kinetics of phosphorylation. Raw 264.7 cells were treated with cGAMP for the indicated time. Cell lysates were subjected to Western blot using the indicated antibodies.


( L ) Poly I : C did not trigger Tyr or Ser phosphorylation of STING . HeLa were transfected with cGAMP or Poly ( I : C ) ; after 3h , lysates were analyzed by Western blot using indicated antibodies .

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(L) Poly I:C did not trigger Tyr or Ser phosphorylation of STING. HeLa cells were transfected with cGAMP or Poly (I:C); after 3h, cell lysates were analyzed by Western blot using the indicated antibodies.


( A ) In absence of EGFR , after cGAMP stimulation , STING translocated out of ER . STING - / - MEF were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h ; were fixed labeled with Calreticulin ( ER marker , Red ) antibody . Co - localization ( Col ) is indicated by dots .

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(A) In the absence of EGFR, after cGAMP stimulation, STING translocated out of the ER. STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h; cells were fixed and labeled with Calreticulin (ER marker, Red) antibody. Co-localization (Col) is indicated by white dots.


STING - / - MEF were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by dots . ( B ) STING was present in ERGIC of stimulated in presence or absence of gefitinib . , were labeled with P58 ( ERGIC marker , Red ) antibody .

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STING-/- MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (B) STING was present in the ERGIC of stimulated cells in the presence or the absence of gefitinib. , the cells were labeled with P58 (ERGIC marker, Red) antibody.


MEF were transfected with STING - GFP ( Green ) for 24h , pretreated with gefitinib 1h transfected with cGAMP for 2 . 5h Co - localization ( Col ) is indicated by dots . ( C ) STING translocated to autophagosomes in absence of EGFR . were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( E ) In gefitinib - treated , STING was detected in autophagosomes as early as 0 . 5h . procedures were as in C .

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MEF cells were transfected with STING-GFP (Green) for 24h, pretreated with gefitinib 1h and transfected with cGAMP for 2.5h Co-localization (Col) is indicated by white dots. (C) STING translocated to the autophagosomes in the absence of EGFR. the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (E) In gefitinib-treated cells, STING was detected in the autophagosomes as early as 0.5h. The procedures were as in C.


Co - localization ( Col ) is indicated by dots . STING translocated to autophagosomes in absence of EGFR . , were labeled with LC3 ( Autophagosome marker , Red ) antibody . ( D ) No STING was detected in autophagosomes even after 3h after stimulation if EGFR was not inhibited .

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Co-localization (Col) is indicated by white dots. STING translocated to the autophagosomes in the absence of EGFR. , the cells were labeled with LC3 (Autophagosome marker, Red) antibody. (D) No STING was detected in the autophagosomes even after 3h after stimulation if EGFR was not inhibited.


( F ) STING translocation to late endosomes required EGFR activity . STING - / - MEF were transfected with STING - GFP ( Green ) for 24hours , pretreated with gefitinib for 1h transfected with cGAMP for indicated time periods ; were fixed labeled with CD63 ( late endosome marker , Red ) antibody .

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(F) STING translocation to late endosomes required EGFR activity. STING-/- MEF cells were transfected with STING-GFP (Green) for 24hours, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time periods; cells were fixed and labeled with CD63 (late endosome marker, Red) antibody.


( A ) EGFR phosphorylates STING in vitro . Purified STING - GFP was incubated with increasing amounts of EGFR for in vitro kinase assays ; products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . substrate was analyzed , before incubations , by same method ( lower panel ) .

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(A) EGFR phosphorylates STING in vitro. Purified STING-GFP was incubated with increasing amounts of EGFR for in vitro kinase assays; the products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel).


products were analyzed on phos - tag gel by Western blot using GFP antibody ( upper panel ) . substrate was analyzed , before incubations , by same method ( lower panel ) . ( B ) Y245F STING mutant is not a substrate of EGFR . WT indicated mutants of STING - GFP were purified used as substrates for

products were analyzed on phos-tag gel by Western blot using GFP antibody (upper panel). The substrate was analyzed, before the assay incubations, by the same method (lower panel). (B) Y245F STING mutant is not a substrate of EGFR. WT and the indicated mutants of STING-GFP were purified and used as substrates for EGFR


( C ) LC - MS / MS of STING identifies phosphorylated Y245 . STING - GFP was purified from cGAMP - stimulated , subjected to protease digestion LC - MS / MS . Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified . MS / MS spectra for phosphorylated peptide VYSNSIpYELLENGQR is shown . This doubly charged peptide has an observed m / z of 932 . 9266 Da is within - 0 . 96 ppm of expected mass . mass difference between y8 y9 ions is consistent with phosphorylation at Y245 .

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(C) LC-MS/MS analysis of STING identifies phosphorylated Y245. STING-GFP was purified from cGAMP-stimulated cells, subjected to protease digestion and LC-MS/MS analysis. Phosphopeptides corresponding to 239VYSNSIpYELLENGQR253 containing pY245 were identified. The MS/MS spectra for the phosphorylated peptide VYSNSIpYELLENGQR is shown. This doubly charged peptide has an observed m/z of 932.9266 Da and is within -0.96 ppm of the expected mass. The mass difference between the y8 and y9 ions is consistent with phosphorylation at Y245.


( E ) Gefitinib pretreatment inhibits phosphorylation of Y245 . STING - GFP - 293XL were pretreated with gefitinib then treated with cGAMP . LC - MS / MS was done , as above , for detecting pY245 ( n = 3 ) .

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(E) Gefitinib pretreatment inhibits phosphorylation of Y245. STING-GFP-293XL cells were pretreated with gefitinib and then treated with cGAMP. LC-MS/MS analysis was done, as above, for detecting pY245 (n=3).


( F ) EGFR is required for STING Tyr phosphorylation . STING - GFP was expressed in 293XL in which EGFR expression had been knocked down . STING phosphorylation was analyzed by LC - MS /

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(F) EGFR is required for STING Tyr phosphorylation. STING-GFP was expressed in 293XL cells in which EGFR expression had been knocked down. STING phosphorylation was analyzed by LC-MS/MS


( G ) Y245F STING mutant cannot trigger IFNβ mRNA induction . was isolated from , expressing WT or Y245F STING , 3h after stimulation subjected to qRT - PCR ( n = 3 ) .

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(G) Y245F STING mutant cannot trigger IFNβ mRNA induction. RNA was isolated from cells, expressing WT or Y245F STING, 3h after stimulation and subjected to qRT-PCR analysis (n=3).


( H ) Y245F STING is phosphorylated on S366 after stimulation of . Phosphorylation of S366 was detected in expressing WT or mutant STING after stimulation for indicated time .

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(H) Y245F STING is phosphorylated on S366 after stimulation of cells. Phosphorylation of S366 was detected in cells expressing the WT or the mutant STING after stimulation for the indicated time.


( I ) TBK1 , but not IRF3 , can bind to Y245F STING be phosphorylated in cGAMP - stimulated . Lysates from expressing WT or mutant STING were analyzed by Western blot after ( upper panel ) or before ( lower panel ) GFP pull down .

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(I) TBK1, but not IRF3, can bind to Y245F STING and be phosphorylated in cGAMP-stimulated cells. Lysates from cells expressing the WT or the mutant STING were analyzed by Western blot after (upper panel) or before (lower panel) GFP pull down.


( J ) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL expressing WT or mutant STING .

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(J) Y245F mutant induced more LC3 lipidation after 1h cGAMP treatment of 293XL cells expressing WT or mutant STING.


( K ) Inhibition of EGFR by gefitinib induced more LC3II after 1 . 5h cGAMP treatment of L929 .

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(K) Inhibition of EGFR by gefitinib induced more LC3II after 1.5h cGAMP treatment of L929 cells.n


( A ) STING - / - MEF were transfected with mSTING WT - GFP Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h transfected with cGAMP for indicated time ; were fixed labeled with LC3 ( autophagosome marker , Red ) antibody . Co - localization ( Col ) is indicated by dots .

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(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with LC3 (autophagosome marker, Red) antibody. Co-localization (Col) is indicated by white dots.


( A ) STING - / - MEF were transfected with mSTING WT - GFP Y244F - GFP ( Green ) for 24h , pretreated with gefitinib for 1h transfected with cGAMP for indicated time ; were fixed labeled with CD63 ( late endosome marker ) .

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(A) STING-/- MEF cells were transfected with mSTING WT-GFP and Y244F-GFP (Green) for 24h, pretreated with gefitinib for 1h and transfected with cGAMP for the indicated time; cells were fixed and labeled with CD63 (late endosome marker).


A Whole mount immunofluorescence of a P8 Syne4 - / - organ of Corti showing intact hair , as labelled with myosin VIIa . B Whole mount immunofluorescence of WT Syne4 - / - organ of Corti from 8 , 16 , 32 kHz regions at P8 P14 .

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A Whole mount immunofluorescence of a P8 Syne4-/- organ of Corti showing intact hair cells, as labelled with myosin VIIa. B Whole mount immunofluorescence of WT and Syne4-/- organ of Corti from the 8, 16, and 32 kHz regions at P8 and P14.


C Inner outer hair of Syne4 - / - organ of Corti at P8 , P10 , P12 , P14 .

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C Inner and outer hair cell counts of Syne4-/- organ of Corti at P8, P10, P12, and P14.


D FM1 - 43 uptake performed on P8 + 1 DIV ( days - in - vitro ) WT Syne4 - / - organ of Corti . Top shows OHC plane bottom shows plane .

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D FM1-43 uptake performed on P8+1 DIV (days-in-vitro) WT and Syne4-/- organ of Corti. Top shows OHC plane and bottom shows IHC plane.


B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV . GFP at P1 showing complete transduction of both inner outer HC . Myosin VIIa was used to label hair . C Examples of 8 , 16 , 32 kHz regions of an organ of Corti of a mouse injected with AAV . GFP . Top shows OHC plane , bottom shows plane , right shows YZ orthogonal projection . Black asterisks show bright Deiters . D of GFP of inner outer HC from 3 injected mice , normalized to average of HC in a control , un - injected mouse .

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B Whole mount immunofluorescence of a P9 organ of Corti of a mouse injected with AAV.GFP at P1 showing complete transduction of both inner and outer HC. Myosin VIIa was used to label the hair cells. C Examples of 8, 16, and 32 kHz regions of an organ of Corti of a mouse injected with AAV.GFP. Top shows OHC plane, bottom shows IHC plane, right shows YZ orthogonal projection. Black asterisks show bright Deiters cells. D Quantification of GFP intensity of inner and outer HC from 3 injected mice, normalized to the average intensity of HC in a control, un-injected mouse.


E Transduction rates of AAV9 - PHP . B at 8 , 16 , 32kHz regions on GFP . A total of 162 841 OHC were analyzed from 3 injected mice 1 control littermate .

E Transduction rates of AAV9-PHP.B at 8, 16, and 32kHz regions based on GFP fluorescence. A total of 162 IHC and 841 OHC were analyzed from 3 injected mice and 1 control littermate.


F for FLAG at at P14 of organ of Corti of a mouse injected at P1 with AAV . Syne4 .

F Staining for FLAG at at P14 of the organ of Corti of a mouse injected at P1 with AAV.Syne4.


G of FLAG DAPI along a centered at envelope . Eight OHC were measured .

G Quantification of FLAG and DAPI fluorescence intensity along a line centered at the nuclear envelope. Eight OHC were measured.


H 3D surface projection of two adjacent OHC from a mouse injected with AAV . Syne4 . In left myosin VIIa DAPI signals were removed to only show FLAG .

H 3D surface projection of two adjacent OHC from a mouse injected with AAV.Syne4. In the left cell myosin VIIa and DAPI signals were removed to only show FLAG staining.


A Whole - mount immunofluorescence of 8 kHz region from P14 organ of Corti from WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 . B 3D surface projection of OHC at P14 from 8kHz region of WT , Syne4 - / - mice Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , arrows denote cuticular plate .

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A Whole-mount immunofluorescence of the 8 kHz region from P14 organ of Corti from WT, Syne4-/-, and Syne4-/- mice injected with AAV.Syne4. B 3D surface projection of IHC and OHC at P14 from the 8kHz region of WT, Syne4-/- mice and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate.


C Image of nucleus position of OHC . show an arc fitted through apical surface , nucleus , basal end of in 3D . X , Y , Z axes show position in μm .

C Image analysis of nucleus position of IHC and OHC. Images show an arc fitted through the apical surface, nucleus, and basal end of the cell in 3D. X, Y, and Z axes show position in μm.


D of from cuticular plate . A total of 45 135 OHC from 3 WT mice , 45 81 OHC from 3 Syne4 - / - mice , 30 90 OHC from 2 Syne4 - / - mice injected with AAV . Syne4 were measured .

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D Quantification of nuclear distance from the cuticular plate. A total of 45 IHC and 135 OHC from 3 WT mice, 45 IHC and 81 OHC from 3 Syne4-/- mice, 30 IHC and 90 OHC from 2 Syne4-/- mice injected with AAV.Syne4 were measured.


A Representative example of ABR traces obtained at 4w from a WT , Syne4 - / - mouse , Syne4 - / - mouse injected with AAV . Syne4 in response to an 18 kHz stimulus .

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A Representative example of ABR traces obtained at 4w from a WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4 in response to an 18 kHz stimulus.


B ABR thresholds at 4w of WT , Syne4 - / - , Syne4 - / - mice injected with AAV . Syne4 , Syne4 - / - mice injected with AAV . GFP , n = 7 for WT , n = 8 for Syne4 - / - , n = 20 for Syne4 - / - + AAV . Syne4 , n = 6 for Syne4 - / - + AAV . GFP . C of P1 - N1 amplitude delta from ( B ) , n = 4 for WT , n = 4 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 . D of P1 latency from ( B ) , n = 4 for WT , n = 2 for Syne4 - / - , n = 7 for Syne4 - / - + AAV . Syne4 .

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B ABR thresholds at 4w of WT, Syne4-/-, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=7 for WT, n=8 for Syne4-/-, n=20 for Syne4-/- + AAV.Syne4, n=6 for Syne4-/- + AAV.GFP. C Quantification of P1-N1 amplitude delta from (B), n=4 for WT, n=4 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4. D Quantification of P1 latency from (B), n=4 for WT, n=2 for Syne4-/-, n=7 for Syne4-/- + AAV.Syne4.


E DPOAE thresholds at 4w , n = 6 for WT , n = 5 for Syne4 - / - , n = 6 for Syne4 - / - + AAV . Syne4 . F ABR threshold at 12w , n = 6 for WT , n = 8 for Syne4 - / - , n = 16 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP .

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E DPOAE thresholds at 4w, n=6 for WT, n=5 for Syne4-/-, n=6 for Syne4-/- + AAV.Syne4. F ABR threshold at 12w, n=6 for WT, n=8 for Syne4-/-, n=16 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP.


A Whole mount immunofluorescence at 12w of organ of Corti of a Syne4 - / - mouse injected with AAV . Syne4 mouse injected with AAV . Syne4 . B Whole mount immunofluorescence at 12w of 12 kHz region of WT , Syne4 - / - mouse , Syne4 - / - mouse injected with AAV . Syne4 .

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A Whole mount immunofluorescence at 12w of the organ of Corti of a Syne4-/- mouse injected with AAV.Syne4 mouse injected with AAV.Syne4. B Whole mount immunofluorescence at 12w of the 12 kHz region of WT, Syne4-/- mouse, and Syne4-/- mouse injected with AAV.Syne4.


C OHC , n = 3 for WT , n = 4 for Mut , n = 6 for Mut + AAV . Syne4 . D Correlation between HC ABR threshold at 12kHz .

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C IHC and OHC counts, n=3 for WT, n=4 for Mut, n=6 for Mut + AAV.Syne4. D Correlation between HC count and ABR threshold at 12kHz.


E 3D surface projection of an OHC at 12w from 12kHz region of WT Syne4 - / - mice injected with AAV . Syne4 . Open arrows denote basal end , arrows denote cuticular plate . F Image of nucleus position of OHC . G of from cuticular plate . A total of 30 OHC from 3 WT mice 38 OHC from 4 Syne4 - / - mice injected with AAV . Syne4 were measured .

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E 3D surface projection of an OHC at 12w from the 12kHz region of WT and Syne4-/- mice injected with AAV.Syne4. Open arrows denote basal end, white arrows denote cuticular plate. F Image analysis of nucleus position of OHC. G Quantification of nuclear distance from the cuticular plate. A total of 30 OHC from 3 WT mice and 38 OHC from 4 Syne4-/- mice injected with AAV.Syne4 were measured.


B , C of activity level cumulative freezing duration at 12w during seconds 90 - 210 of day 2 in WT , Syne4 - / - mice , Syne4 - / - mice injected with AAV . Syne4 , Syne4 - / - mice injected with AAV . GFP , n = 14 for WT , n = 16 for Syne4 - / - , n = 9 for Syne4 - / - + AAV . Syne4 , n = 5 for Syne4 - / - + AAV . GFP . ( C ) Activity level . ( B ) Cumulative freezing duration .

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B, C Quantification of activity level and cumulative freezing duration at 12w during seconds 90-210 of day 2 in WT, Syne4-/- mice, Syne4-/- mice injected with AAV.Syne4, and Syne4-/- mice injected with AAV.GFP, n=14 for WT, n=16 for Syne4-/-, n=9 for Syne4-/- + AAV.Syne4, n=5 for Syne4-/- + AAV.GFP. (C) Activity level. (B) Cumulative freezing duration.


F - I ChIP assays with antibodies against H3K9me3 , H4K20me3 , H3K4me3 H3K9Acetyl were performed as in ( D ) . Only ' C ' position signals were showed normalized to Input .

F-I ChIP assays with antibodies against H3K9me3, H4K20me3, H3K4me3 and H3K9Acetyl were performed as in (D). Only 'C' position signals were showed and normalized to Input.


M ChIP assays with antibodies against CDK9 Pho - Pol II were performed in siNC siCHAF1A J - Lat 10 . 6 . ChIP signals in each group were normalized to Input .

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M ChIP assays with antibodies against CDK9 and Pho-Pol II were performed in siNC and siCHAF1A J-Lat 10.6 cells. ChIP signals in each group were normalized to Input.


siRNAs targeting each CAF - 1 subunits corresponding enriched genes were transfected into TZM - bl lines respectively . fold change of luciferase expression of each groups was normalized to siNC .

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siRNAs targeting each CAF-1 subunits and corresponding enriched genes were transfected into TZM-bl cell lines respectively. The fold change of luciferase expression of each groups was normalized to siNC.


C , D ImmunoFISH of CAF - 1 bodies HIV - 1 genomic within J - Lat 10 . 6 . Naïve were treated with DMSO . Activated were acquired by treating with TNFα .

C, D ImmunoFISH images of CAF-1 bodies and HIV-1 genomic DNA within J-Lat 10.6. Naïve cells were treated with DMSO. Activated cells were acquired by treating with TNFα.


E RFP - tagged CHAF1A GFP - tagged PML component SP100 were co - overexpressed in HEK293T imaged by SIM . F GFP - tagged CHAF1A RFP - tagged BRD4 were co - overexpressed in HEK293T imaged by SIM .

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E RFP-tagged CHAF1A and GFP-tagged PML body component SP100 were co-overexpressed in HEK293T cells and imaged by SIM. F GFP-tagged CHAF1A and RFP-tagged BRD4 were co-overexpressed in HEK293T cells and imaged by SIM.


SUV39H1 , HP1α , DNMT1 , MBD1 , RING1B , EZH2 were co - overexpressed with CHAF1A in HEK293T respectively . SIM imaging was performed for each combination .

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SUV39H1, HP1α, DNMT1, MBD1, RING1B, EZH2 were co-overexpressed with CHAF1A in HEK293T cells respectively. SIM imaging was performed for each combination.


E GFP - tagged CHAF1A - N - NLS CHAF1A - C were imaged with RFP - tagged CHAF1A respectively . N : CHAF1A N - terminal . NLS : localization signal of CHAF1A , which was presented within C - terminal . C : CHAF1A C - terminal .

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E GFP-tagged CHAF1A-N-NLS and CHAF1A-C were imaged with RFP-tagged CHAF1A respectively. N: CHAF1A N-terminal. NLS: nuclear localization signal of CHAF1A, which was presented within C-terminal. C: CHAF1A C-terminal.


F Upper panel showed WT-CHAF1A bodies. Lower panel showed dLLPS-CHAF1A which was CHAF1A-Q34A-I116A-K395D-R402D-K409D.


G - I GFP - tagged CHAF1A - IDR CHAF1A - mIDR were purified in vitro . Ten micromole of each was incubated with gradient droplet formation buffer . droplets of CHAF1A - IDR appeared in NaCl of 125 mM or lower ( G ) . No CHAF1A - mIDR droplet was detected within any NaCl ( I ) . volumes of CHAF1A - IDR droplets increased with increase of ( H ) . Left subpanels within each panel represented of droplets in different buffers . Right subpanels within each panel represented statistical results of droplets areas numbers .

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G-I GFP-tagged CHAF1A-IDR and CHAF1A-mIDR were purified in vitro. Ten micromole of each protein was incubated with gradient droplet formation buffer. The droplets of CHAF1A-IDR appeared in NaCl concentration of 125 mM or lower (G). No CHAF1A-mIDR droplet was detected within any NaCl concentration (I). The volumes of CHAF1A-IDR droplets increased with the increase of protein concentration (H). Left subpanels within each panel represented images of protein droplets in different buffers. Right subpanels within each panel represented statistical analysis results of droplets areas and numbers.


B First panel : co - localization of PCNA CHAF1A . Second panel : distribution of PCNA in absence of endogenous CHAF1A . Third panel : distribution of PCNA in presence of CHAF1A - dPIP1 mutant absence of endogenous CHAF1A . Fourth panel : co - localization of PCNA over - expressed DNMT1 in absence of endogenous CHAF1A .

B First panel: the co-localization of PCNA and CHAF1A. Second panel: the distribution of PCNA in the absence of endogenous CHAF1A. Third panel: the distribution of PCNA in the presence of CHAF1A-dPIP1 mutant and the absence of endogenous CHAF1A. Fourth panel: the co-localization of PCNA and over-expressed DNMT1 in the absence of endogenous CHAF1A.


C Upper two panels : distribution of HP1α in presence or absence of CHAF1A . Lower two panels : distribution of HP1α in presence of CHAF1A - dHP1BD or CHAF1A - DS2 - dHP1BD . Both of lower two panels were in absence of endogenous CHAF1A .

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C Upper two panels: the distribution of HP1α in the presence or absence of CHAF1A. Lower two panels: the distribution of HP1α in the presence of CHAF1A-dHP1BD or CHAF1A-DS2-dHP1BD. Both images of lower two panels were in the absence of endogenous CHAF1A.


distribution of SUV39H2 ( E ) MBD1 ( F ) in presence or absence of CHAF1A .

The distribution of SUV39H2 (E) and MBD1 (F) in the presence or absence of CHAF1A.


G distribution of SUMO paralogs in absence of CHAF1A .

G The distribution of SUMO paralogs in the absence of CHAF1A.


A Flow cytometry figures of CAF - 1 rescue experiment . First figure : Wildtype J - Lat 10 . 6 . Second figure : CHAF1A knock - out J - Lat 10 . 6 . Third figure : CHAF1A - KO J - Lat 10 . 6 was re - introduced with wildtype CHAF1A . Fourth figure : CHAF1A - KO J - Lat 10 , 6 was re - introduced with dLLPS - CHAF1A which harbored five mutations . percentages of GFP - positive were labeled in top right corner of each figure .

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A Flow cytometry figures of CAF-1 body rescue experiment. First figure: Wildtype J-Lat 10.6. Second figure: CHAF1A knock-out J-Lat 10.6. Third figure: CHAF1A-KO J-Lat 10.6 was re-introduced with wildtype CHAF1A. Fourth figure: CHAF1A-KO J-Lat 10,6 was re-introduced with dLLPS-CHAF1A which harbored five mutations. The percentages of GFP-positive cells were labeled in the top right corner of each figure.


D amounts of intracellular HIV - 1 RNAs of latently infected CD4 + T which were isolated from PBMCs of HIV - 1 - infected individuals . αCD3 / αCD28 / IL - 2 treatment was performed as positive control . shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A . shluc lentiviruses treatment was performed as negative control . SAHA was added as LRA supplement .

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D The amounts of intracellular HIV-1 RNAs of latently infected primary CD4+ T cells which were isolated from PBMCs of HIV-1-infected individuals. αCD3/αCD28/IL-2 treatment was performed as positive control. shCHAF1A lentiviruses were packaged to knock down endogenous CHAF1A. shluc lentiviruses treatment was performed as negative control. SAHA was added as LRA supplement.


A Representative of fly eyes expressing ( CGG ) 90 - EGFP under a GMR - GAL4 driver , with various belle disruptions .

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A Representative photographs of fly eyes expressing (CGG)90-EGFP under a GMR-GAL4 driver, with various belle disruptions.


B Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye with belle disruptions ( Mann - Whitney U with Bonferonni corrections for multiple comparisons ; n = 35 - 77 / genotype ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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B Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with belle disruptions (Mann-Whitney U test with Bonferonni corrections for multiple comparisons; n=35-77/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; Tub5 - GS ( Log - rank Mantel - Cox with Bonferroni corrections for multiple comparisons ; n = 110 - 219 / genotype ) flies with belle knockdown . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; Tub5-GS (Log-rank Mantel-Cox test with Bonferroni corrections for multiple comparisons; n=110-219/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


Longevity assays of ( CGG ) 90 - EGFP ; ElaV - GS ( n = 147 - 299 / genotype ) flies with belle knockdown . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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Longevity assays of (CGG)90-EGFP; ElaV-GS (n=147-299/genotype) flies with belle knockdown. Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


F Quantitation of FMRpolyG - EGFP band density , normalized to β tubulin band density , from blots in E ( Student ' s t ; n = 4 - 5 / genotype ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . data in panels are presented as mean ± SD ( compiled from ≥ 3 replicates ) .

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F Quantitation of FMRpolyG-EGFP band density, normalized to β tubulin band density, from blots in E (Student's t test; n=4-5/genotype). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All data in all panels are presented as mean ± SD (compiled from ≥3 replicates).


G Abundance of ( CGG ) 90 - EGFP mRNA normalized to RPL32 mRNA , following belle knockdown , determined by qRT - PCR ( n = 8 / genotype ) .

G Abundance of (CGG)90-EGFP mRNA normalized to RPL32 mRNA, following belle knockdown, determined by qRT-PCR (n=8/genotype).


H Western blot of AUG - driven EGFP in EGFP ; Tub5 - GS flies with without belle knockdown .

H Western blot of AUG-driven EGFP in EGFP; Tub5-GS flies with and without belle knockdown.


I Quantitation of EGFP band density , normalized to β tubulin band density , from blot in H ( n = 4 / genotype ) .

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I Quantitation of EGFP band density, normalized to β tubulin band density, from blot in H (n=4/genotype).


A Dose - response curves showing effects of two independent anti - DDX3X siRNAs on expression of AUG - NL - 3xF ( top ) ( CGG ) 100 + 1 NL - 3xF ( bottom ) reporters . Plasmid - reporters were transfected into HeLa 24 hours after knockdown , reporter expression was quantified by luminescence . Nanoluciferase ( NL ) luminescence has been normalized to luminescence from firefly luciferase ( FF ) , which was co - transfected , in order to control for transfection variability . Asterisks refer to comparisons between anti - DDX3X siRNAs siRNAs against EGFP ( siEGFP ; two - way ANOVA with Dunnett ' s multiple comparisons ; n = 12 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Dose-response curves showing the effects of two independent anti-DDX3X siRNAs on the expression of AUG-NL-3xF (top) and (CGG)100 +1 NL-3xF (bottom) reporters. Plasmid-based reporters were transfected into HeLa cells 24 hours after knockdown, and reporter expression was quantified by luminescence. Nanoluciferase (NL) luminescence has been normalized to luminescence from firefly luciferase (FF), which was co-transfected, in order to control for transfection variability. Asterisks refer to comparisons between anti-DDX3X siRNAs and siRNAs against EGFP (siEGFP; two-way ANOVA with Dunnett's multiple comparisons test; n=12/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 1 NL - 3xF expression ( normalized to FF ) with without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 17 - 30 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

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(CGG)n +1 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


( CGG ) n + 2 NL - 3xF expression ( normalized to FF ) with without DDX3X knockdown across a range of CGG repeat sizes . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 17 - 30 / condition ) . Data Information : For panels , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD ( compiled from ≥ 3 replicates ) .

(CGG)n +2 NL-3xF expression (normalized to FF) with and without DDX3X knockdown across a range of CGG repeat sizes. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter (two-way ANOVA with Tukey's multiple comparisons test; n=17-30/condition). Data Information: For all panels, * P≤0.05, ** P≤0.01, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD (compiled from ≥3 replicates).


Western blots of FMRpolyG - NL - 3xF products with without DDX3X knockdown across a range of repeat sizes .

Western blots of FMRpolyG-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


Western blots of FMRpolyA - NL - 3xF products with without DDX3X knockdown across a range of repeat sizes .

Western blots of FMRpolyA-NL-3xF products with and without DDX3X knockdown across a range of repeat sizes.


A Expression of in vitro transcribed AUG , + 1 ( CGG ) 100 , + 2 ( CGG ) 100 NL - 3xF RNAs following DDX3X knockdown in HeLa , expressed as NL luminescence normalized to FF luminescence . ( Student ' s t with Bonferroni corrections for multiple comparisons ; n = 21 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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A Expression of in vitro transcribed AUG, +1 (CGG)100, and +2 (CGG)100 NL-3xF RNAs following DDX3X knockdown in HeLa cells, expressed as NL luminescence normalized to FF luminescence. (Student's t test with Bonferroni corrections for multiple comparisons; n=21/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


B Abundance of reporter mRNAs following DDX3X knockdown plasmid - reporter transfection , determined by qRT - PCR . ( n = 7 / condition . ) This panel depicts data as means ± .

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B Abundance of reporter mRNAs following DDX3X knockdown and plasmid-reporter transfection, determined by qRT-PCR. (n=7/condition.) This panel depicts data as means ± SEM.


C Expression of AUG - NL - 3xF + 1 ( CGG ) 100 NL - 3xF in in vitro translation extracts , collected from HeLa treated with siRNAs against EGFP or DDX3X ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 4 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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C Expression of AUG-NL-3xF and +1 (CGG)100 NL-3xF in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X (two-way ANOVA with Tukey's multiple comparisons test; n=4/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


D Enrichment of HSPA1A + 1 ( CGG ) 100 NL - 3xF mRNA following anti - DDX3X RIP , relative to incubation with isotype control IgG . MALAT , in contrast , is not enriched ( Student ' s t , n = 3 ) .

D Enrichment of HSPA1A and +1 (CGG)100 NL-3xF mRNA following anti-DDX3X RIP, relative to incubation with isotype control IgG. MALAT RNA, in contrast, is not enriched (Student's t test, n=3).


E Expression of + 1 + 2 ( CGG ) 100 NL - 3xF plasmid reporters with without an AUG inserted 5 ' to CGG repeat , with without DDX3X knockdown . Black asterisks refer to comparisons between siDDX3X - siEGFP - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing either + 1 or + 2 ( CGG ) 100 NL - 3xF those expressing respective AUG - driven variant ( two - way ANOVA with Tukey ' s multiple comparisons ; n = 11 - 12 / condition ) . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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E Expression of +1 and +2 (CGG)100 NL-3xF plasmid reporters with and without an AUG inserted 5' to the CGG repeat, with and without DDX3X knockdown. Black asterisks refer to comparisons between siDDX3X- and siEGFP-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing either +1 or +2 (CGG)100 NL-3xF and those expressing the respective AUG-driven variant (two-way ANOVA with Tukey's multiple comparisons test; n=11-12/condition). Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


F Expression of NL - 3xF plasmids with initiator AUG codons mutated to near - AUG codons , with without DDX3X knockdown ( two - way ANOVA with Dunnett ' s multiple comparisons ; n = 18 - 24 / condition ) . Black asterisks refer to comparisons between siEGFP - treated siDDX3X - treated ; orange asterisks refer to comparisons between siDDX3X - treated expressing AUG - NL - 3xF those expressing a different reporter ; asterisks refer to comparisons between siDDX3X - treated expressing + 1 ( CGG ) 100 NL - 3xF those expressing a different reporter . Data Information : For panels , ns = non - significant , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . panels depict data as means ± SD , unless indicated otherwise ( compiled from ≥ 3 replicates ) .

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F Expression of NL-3xF plasmids with initiator AUG codons mutated to near-AUG codons, with and without DDX3X knockdown (two-way ANOVA with Dunnett's multiple comparisons test; n=18-24/condition). Black asterisks refer to comparisons between siEGFP-treated and siDDX3X-treated cells; orange asterisks refer to comparisons between siDDX3X-treated cells expressing AUG-NL-3xF and those expressing a different reporter; white asterisks refer to comparisons between siDDX3X-treated cells expressing +1 (CGG)100 NL-3xF and those expressing a different reporter. Data Information: For all panels, ns=non-significant, *** P≤0.001, **** P≤0.0001 for the specified statistical test. All panels depict data as means ± SD, unless indicated otherwise (compiled from ≥3 replicates).


G Expression of in vitro transcribed near - AUG reporter RNAs in in vitro translation extracts , collected from HeLa treated with siRNAs against EGFP or DDX3X . ( n = 4 / group ) .

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G Expression of in vitro transcribed near-AUG reporter RNAs in in vitro translation extracts, collected from HeLa cells treated with siRNAs against EGFP or DDX3X. (n=4/group).


A Representative of GMR - GAL4 ; ( CGG ) 90 - EGFP fly eyes expressing manipulations of eIF4B eIF4H . ( B ) Quantitation of GMR - GAL4 , ( CGG ) 90 - EGFP eye with eIF4B / H manipulations ( Mann - Whitney U with Bonferroni corrections for multiple comparisons ; n = 26 - 55 / genotype ) . Data Information : For panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for specified statistical . panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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A Representative photographs of GMR-GAL4; (CGG)90-EGFP fly eyes expressing manipulations of eIF4B and eIF4H. (B) Quantitation of GMR-GAL4, (CGG)90-EGFP eye phenotypes with eIF4B/H manipulations (Mann-Whitney U test with Bonferroni corrections for multiple comparisons; n=26-55/genotype). Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


C , D Expression of plasmid - AUG - NL + 1 ( CGG ) 100 NL - 3xF reporters ( C ) , or co - transfected AUG - FF reporters ( D ) , following knockdown of EIF4B or EIF4H . Black asterisks refer to comparisons between siEGFP - siEIF4B / H - treated ; pink blue asterisks refer to comparisons between siEIF4B - ( pink ) or siEIF4H - ( blue ) treated expressing AUG - NL - 3xF those expressing + 1 ( CGG ) 100 ( two - way ANOVA with Tukey ' s multiple comparisons , n = 9 / condition ) .

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C, D Expression of plasmid-based AUG-NL and +1 (CGG)100 NL-3xF reporters (C), or co-transfected AUG-FF reporters (D), following knockdown of EIF4B or EIF4H. Black asterisks refer to comparisons between siEGFP- and siEIF4B/H-treated cells; pink and blue asterisks refer to comparisons between siEIF4B- (pink) or siEIF4H- (blue) treated cells expressing AUG-NL-3xF and those expressing +1 (CGG)100 (two-way ANOVA with Tukey's multiple comparisons test, n=9/condition).


E Expression of plasmid - AUG - NL - 3xF ( CGG ) 100 + 1 NL - 3xF reporters with without over - expression of EIF4B , EIF4H , or both ( two - way ANOVA with Dunnett ' s multiple comparisons ; n = 20 / condition ) . Asterisks refer to comparisons between over - expressing either EGFP or EIF4B , EIF4H , or EIF4B EIF4H expressing same reporter . Data Information : For panels , ns = non - significant , * P ≤ 0 . 05 , * * P ≤ 0 . 01 , * * * * P ≤ 0 . 0001 for specified statistical . panels present data as means ± SD ( compiled from ≥ 3 replicates ) .

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E Expression of plasmid-based AUG-NL-3xF and (CGG)100 +1 NL-3xF reporters with and without over-expression of EIF4B, EIF4H, or both (two-way ANOVA with Dunnett's multiple comparisons test; n=20/condition). Asterisks refer to comparisons between cells over-expressing either EGFP or EIF4B, EIF4H, or EIF4B and EIF4H and expressing the same reporter. Data Information: For all panels, ns=non-significant, * P≤0.05, ** P≤0.01, **** P≤0.0001 for the specified statistical test. All panels present data as means ± SD (compiled from ≥3 replicates).


A Expression of plasmid - NL - 3xF reporters in HEK293 with without over - expression of EIF1 ( two - way ANOVA with Sidak ' s multiple comparisons ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected EIF1 - transfected ; green asterisks refer to comparisons between EIF1 - transfected expressing AUG - NL - 3xF those expressing a different reporter . For panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . Bars represent mean ± ( compiled from ≥ 3 replicates ) .

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A Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF1 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF1-transfected cells; green asterisks refer to comparisons between EIF1-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


B Expression of plasmid - NL - 3xF reporters in HEK293 with without over - expression of EIF5 ( two - way ANOVA with Sidak ' s multiple comparisons ; n = 9 - 12 / condition ) . Black asterisks refer to comparisons between Empty Vector - transfected EIF5 - transfected ; pink asterisks refer to comparisons between EIF5 - transfected expressing AUG - NL - 3xF those expressing a different reporter . For panels , * * * P ≤ 0 . 001 , * * * * P ≤ 0 . 0001 for specified statistical . Bars represent mean ± ( compiled from ≥ 3 replicates ) .

B Expression of plasmid-based NL-3xF reporters in HEK293 cells with and without over-expression of EIF5 (two-way ANOVA with Sidak's multiple comparisons test; n=9-12/condition). Black asterisks refer to comparisons between Empty Vector-transfected and EIF5-transfected cells; pink asterisks refer to comparisons between EIF5-transfected cells expressing AUG-NL-3xF and those expressing a different reporter. For all panels, *** P≤0.001, **** P≤0.0001 for the specified statistical test. Bars represent mean ± SEM (compiled from ≥3 replicates).


A Sample collected by automated longitudinal microscopy , demonstrating automated determination of .

A Sample micrographs collected by automated longitudinal fluorescence microscopy, demonstrating the automated determination of cell death.


B Anti - DDX3X western blot of B35 transfected with either of two independent anti - DDX3X LNAs or a control LNA .

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B Anti-DDX3X western blot of B35 cells transfected with either of two independent anti-DDX3X LNAs or a control LNA.


C Expression of EGFP in rat neurons transfected with ( CGG ) 100 ( + 1 ) EGFP either anti - DDX3X LNAs or a control ( one - way ANOVA with Tukey ' s multiple comparisons ; n = 2408 - 5689 / condition ) . graphs depict pooled data , normalized first within replicate . Data Information : For panels , * * * * P ≤ 0 . 0001 for specified statistical ( compiled from ≥ 3 replicates ) .

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C Expression of EGFP in primary rat neurons transfected with (CGG)100 (+1) EGFP and either anti-DDX3X LNAs or a control (one-way ANOVA with Tukey's multiple comparisons test; n=2408-5689 cells/condition). All graphs depict pooled data, normalized first within the replicate. Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


D , E Transfection of anti - DDX3X LNA # 1 ( D ) or # 2 ( E ) reduced cumulative risk of in ( CGG ) 100 ( + 1 ) EGFP - expressing neurons ( Cox proportional hazard ; n = 2408 - 3676 / condition ) . Data Information : For panels , * * * * P ≤ 0 . 0001 for specified statistical ( compiled from ≥ 3 replicates ) .

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D, E Transfection of anti-DDX3X LNA #1 (D) or #2 (E) reduced the cumulative risk of death in (CGG)100 (+1) EGFP-expressing neurons (Cox proportional hazard analysis; n=2408-3676 cells/condition). Data Information: For all panels, **** P≤0.0001 for the specified statistical test (compiled from ≥3 replicates).


A Immunofluorescence of MMS22L foci formation at etoposide ( ETP ) - induced DSBs during S , M G1 phases of cycle in HeLa . Scale bar : 5 μm . B of a representative experiment from ( A ) shows number of MMS22L foci per nucleus during indicated cycle phases ( n = 2 ; nnuclei ≥ 60 ) .

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A Immunofluorescence images of MMS22L foci formation at etoposide (ETP)-induced DSBs during S, M and G1 phases of the cell cycle in HeLa cells. Scale bar: 5 μm.B Quantification of a representative experiment from (A) shows number of MMS22L foci per nucleus during indicated cell cycle phases (n = 2; nnuclei ≥ 60).


C Immunofluorescence of CPT - induced MMS22L , RPA2 , BRCA2 RAD51 foci in U2OS . Scale bar : 5 μm . D of a representative experiment ( n = 3 ) from ( C ) shows of γH2AX , RPA2 , BRCA2 RAD51 foci co - localizing with MMS22L foci ( nnuclei = 30 ; nuclei with ≥ 25 MMS22L foci were analyzed ) .

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C Immunofluorescence images of CPT-induced MMS22L, RPA2, BRCA2 and RAD51 foci in U2OS cells. Scale bar: 5 μm.D Quantification of a representative experiment (n = 3) from (C) shows percentage of γH2AX, RPA2, BRCA2 and RAD51 foci co-localizing with MMS22L foci (nnuclei = 30; nuclei with ≥ 25 MMS22L foci were analyzed).


A Electrophoretic mobility shift monitoring MMS22L - TONSL binding to ssDNA without RPA with RPA - coated ssDNA . B of data as in ( A ) shows MMS22L - TONSL binding to ssDNA with or without RPA , n = 2 ; error bars , .

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A Electrophoretic mobility shift assay monitoring MMS22L-TONSL binding to ssDNA without RPA and with RPA-coated ssDNA.B Quantification of data as in (A) shows percent MMS22L-TONSL binding to ssDNA with or without RPA, n = 2; error bars, SEM.


C Schematic ( left ) western blots ( right ) of streptavidin NiNTA pulldowns of his - tagged MMS22L - TONSL , RPA biotinylated ssDNA .

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C Schematic (left) and western blots (right) of streptavidin and NiNTA pulldowns of his-tagged MMS22L-TONSL, RPA and biotinylated ssDNA.


D Yeast two - hybrid shows effect of deletion of 384 - 522 of MMS22L on interaction with RPA . interaction between indicated MMS22L variants fused to LexA - binding domain RPA1 fused to GAL4 activating domain ( GAD ) is assessed by survival on media containing 3 - aminotriazole ( 3 - AT ) in absence of histidine by β - galactosidase activity ( X - Gal ) assays .

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D Yeast two-hybrid analysis shows the effect of deletion of the amino acids 384-522 of MMS22L on the interaction with RPA. The interaction between indicated MMS22L variants fused to LexA DNA-binding domain and RPA1 fused to GAL4 activating domain (GAD) is assessed by survival on media containing 3-aminotriazole (3-AT) in the absence of histidine and by β-galactosidase activity (X-Gal) assays.


E of MMS22L foci formed in U2OS stably expressing wild - type ( WT ) or Δ384 - 522 variants of siRNA - resistant HSS - MMS22L , depleted of endogenous MMS22L by RNAi treated with 50 nM CPT for 18 hours . Scale bar : 5 μm .

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E Images of MMS22L foci formed in U2OS cells stably expressing wild-type (WT) or Δ384-522 variants of siRNA-resistant HSS-MMS22L, depleted of endogenous MMS22L by RNAi and treated with 50 nM CPT for 18 hours. Scale bar: 5 μm.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . A Abundance of MMS22L - TONSL at active HU - stalled replication forks , fully replicated .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.A Abundance of MMS22L-TONSL at active and HU-stalled replication forks, and fully replicated DNA.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . B , C MMS22L dynamics at stalled replication forks . shows average relative abundance of indicated detected in immunoblots , n = 3 except of time - point 5 h , n = 1 ; error bars , .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.B, C MMS22L dynamics at stalled replication forks. Quantification shows average relative abundance of indicated proteins detected in immunoblots, n = 3 except of time-point 5 h, n = 1; error bars, SEM.


( A - E ) iPOND assays in Hela . Experiment schematic ( top ) resulting immunoblots ( bottom ) are shown in ( A , B , D ) quantifications are shown in ( C , E ) with antibodies used for immunoblots indicated throughout . D , E Effect of MMS22L depletion on RAD51 at stalled replication forks . shows average relative RAD51 abundance , n = 2 ; error bars , .

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(A-E) iPOND assays in Hela cells. Experiment schematic (top) and resulting immunoblots (bottom) are shown in (A, B, D) and quantifications are shown in (C, E) with antibodies used for immunoblots indicated throughout.D, E Effect of MMS22L depletion on RAD51 levels at stalled replication forks. Quantification shows average relative RAD51 abundance, n = 2; error bars, SEM.


A Representative immunofluorescence of foci induced by short CPT treatment ( 50 nM , 1 h ) in U2OS treated with indicated siRNAs showing effect of MMS22L depletion on RAD51 foci . Scale bar : 5 μm . B Boxplot of representative experiment in ( A ) ( n = 3 ; nnuclei ≥ 78 ) . Boxes indicate 25 - 75 percentile whiskers 10 - 90 percentile ; horizontal lines mark medians . Nuclei with ≥ 25 γH2AX foci ( marker of replication stress sites ) were analyzed . Statistical : Mann - Whitney U ; * * * P ≤ 0 . 0001 .

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A Representative immunofluorescence images of foci induced by short CPT treatment (50 nM, 1 h) in U2OS cells treated with indicated siRNAs showing the effect of MMS22L depletion on the RAD51 foci. Scale bar: 5 μm.B Boxplot quantification of representative experiment in (A) (n = 3; nnuclei ≥ 78). Boxes indicate the 25-75 percentile and whiskers the 10-90 percentile; horizontal lines mark
the medians. Nuclei with ≥ 25 γH2AX foci (marker of replication stress sites) were analyzed. Statistical analysis: Mann-Whitney U test; *** P ≤ 0.0001.


C , D Electron of a representative reversed fork ( C ) a fork with extended ssDNA region at junction ( D ) ( arrow ) . P , parental duplex ; R , regressed arm . E Frequency of reversed replication forks detected by EM . Similar results were obtained in one independent experiment ; n = 2 . F Distribution of ssDNA at replication forks isolated from U2OS transfected with indicated siRNAs treated with 25 nM CPT for 1h . In brackets , total number of analyzed molecules is given . Statistical according to Mann - Whitney U ; * * P ≤ 0 . 01 , n = 2 .

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C, D Electron micrographs of a representative reversed fork (C) and a fork with extended ssDNA region at the junction (D) (arrow). P, parental duplex; R, regressed arm.E Frequency of reversed replication forks detected by EM. Similar results were obtained in one independent experiment; n = 2.F Distribution of ssDNA length at replication forks isolated from U2OS cells transfected with the indicated siRNAs and treated with 25 nM CPT for 1h. In brackets, the total number of analyzed molecules is given. Statistical analysis according to Mann-Whitney U test; ** P ≤ 0.01, n = 2.


A Amylose pulldown of MBP ( control ) or MBP - tagged MMS22L - TONSL RAD51 . Eluate were detected by silver . MT : MBP - MMS22L - GST - TONSL . B Quantitative of RAD51 binding to MMS22L - TONSL such as shown in Figure EV6A . Averages shown , n = 2 ; error bars , .

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A Amylose pulldown of MBP (control) or MBP-tagged MMS22L-TONSL and RAD51. Eluate proteins were detected by silver staining. MT: MBP-MMS22L-GST-TONSL.B Quantitative analysis of RAD51 binding to MMS22L-TONSL such as shown in Figure EV6A. Averages shown, n = 2; error bars, SEM.


C , D Yeast two - hybrid of interactions between LexA binding domain - tagged MMS22L indicated fused to GAL4 activating domain ( GAD ) assessed by survival on media containing 3 - AT in absence of histidine by X - Gal assays .

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C, D Yeast two-hybrid analysis of interactions between LexA DNA binding domain-tagged MMS22L and indicated proteins fused to GAL4 activating domain (GAD) assessed by survival on media containing 3-AT in the absence of histidine and by X-Gal assays.


E , F Streptavidinpulldowns of indicated recombinant biotinylated ssDNA . in input eluates were detected by western blotting .

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E, F Streptavidinpulldowns of indicated recombinant proteins and biotinylated ssDNA. The proteins in the input and eluates were detected by western blotting.


G Streptavidinpulldown of HSS - tagged MMS22L variants stably integrated overexpressed in U2OS . Input co - precipitated were detected by immunoblotting .

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G Streptavidinpulldown of HSS-tagged MMS22L variants stably integrated and overexpressed in U2OS cells. Input and co-precipitated proteins were detected by immunoblotting.


H Representative immunofluorescence of CPT - induced foci in stable U2OS lines depleted of endogenous MMS22L overexpressing indicated siRNA - resistant variants of HSS - MMS22L treated with 50 nM CPT for 1 hour . Scale bar : 5 μm .

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H Representative immunofluorescence images of CPT-induced foci in stable U2OS cell lines depleted of endogenous MMS22L and overexpressing indicated siRNA-resistant variants of HSS-MMS22L treated with 50 nM CPT for 1 hour. Scale bar: 5 μm.


I - K of effect of indicated MMS22L mutations on RAD51 foci in U2OS treated with 50 nM CPT for 1 h ( I ; n = 3 ) , 50 nM CPT for 18 h ( J ; n = 3 ) or 5 µM ETP for 1 h followed by 3 hours incubation without drug ( K ; n = 3 ) . Graphs represent averaged median values ; error bars indicate . Statistical according to one - way ANOVA with Bonferroni post - ; * * * P ≤ 0 . 001 ; * * P ≤ 0 . 01 ; ns , not significant . Representative immunofluorescence are shown in ( H ) , Figures EV6G EV6H , respectively .

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I-K Quantification of the effect of indicated MMS22L mutations on RAD51 foci in U2OS cells treated with 50 nM CPT for 1 h (I; n = 3), 50 nM CPT for 18 h (J; n = 3) or 5 µM ETP for 1 h followed by 3 hours incubation without the drug (K; n = 3). Graphs represent averaged median values; error bars indicate SEM. Statistical analysis according to one-way ANOVA with Bonferroni post-test; *** P ≤ 0.001; ** P ≤ 0. 01; ns, not significant. Representative immunofluorescence images are shown in (H), Figures EV6G and EV6H, respectively.


B - D Strand exchange with ssDNA RAD51 , with ( C ) or without ( B ) addition of MMS22L - TONSL ( 75 nM ) . D shows averages , n = 2 ; error bars , .

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B-D Strand exchange assay with ssDNA and RAD51, and with (C) or without (B) the addition of MMS22L-TONSL (75 nM).D Quantification shows averages, n = 2; error bars, SEM.


E Averages of quantified strand exchange assays with ssDNA , RAD51 ( 270 nM ) varying concentrations of MMS22L - TONSL or MMS22L . n = 2 ; error bars , .

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E Averages of quantified strand exchange assays with ssDNA, RAD51 (270 nM) and varying concentrations of MMS22L-TONSL or MMS22L. n = 2; error bars, SEM.


F of electrophoretic mobility shift assays with dsDNA or ssDNA , RAD51 MMS22L - TONSL or MMS22L . Averages shown , n = 2 ; error bars , .

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F Quantification of electrophoretic mobility shift assays with dsDNA or ssDNA, RAD51 and MMS22L-TONSL or MMS22L. Averages shown, n = 2; error bars, SEM.


H A representative immunoblot showing effect of increasing amounts of dsDNA on RAD51 binding to ssDNA ( 1 nM ) .

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H A representative immunoblot showing the effect of increasing amounts of dsDNA on RAD51 binding to ssDNA (1 nM).


I A representative immunoblot showing effects of MMS22L - TONSL MMS22L titration into reactions containing RAD51 , ssDNA ( 1 nM ) dsDNA ( 10 nM ) , as indicated .

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I A representative immunoblot showing effects of MMS22L-TONSL and MMS22L titration into reactions containing RAD51, ssDNA (1 nM) and dsDNA (10 nM), as indicated.


( A ) After 4 h of starvation in 1 % K ‐ acetate medium in presence of PMSF , autophagic vesicles can be visualized in vacuoles of wild ‐ type pep4prb1 using Normaski optics . In aut2Δ aut7Δ no autophagic vesicles can be seen . Bar represents 20 μm .

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(A) After 4 h of starvation in 1% K‐acetate medium in the presence of PMSF, autophagic vesicles can be visualized in vacuoles of wild‐type and pep4prb1 cells using Normaski optics. In aut2Δ and aut7Δ cells no autophagic vesicles can be seen. Bar represents 20 μm.


( B ) Total breakdown of starved on nitrogen ‐ free medium is significantly reduced in aut2Δ aut7Δ compared with wild ‐ type . pep4Δ ( pra1Δ ) are almost completely impaired in starvation ‐ induced vacuolar breakdown . were ‐ labeled with [ 35S ] methionine chased on non ‐ radioactive starvation medium . Aliquots were taken at indicated times acid soluble small peptides generated by proteolysis were determined according to Straub et al . ( 1997 ) .

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(B) Total protein breakdown of cells starved on nitrogen‐free medium is significantly reduced in aut2Δ and aut7Δ cells compared with wild‐type cells. pep4Δ (pra1Δ) cells are almost completely impaired in starvation‐induced vacuolar protein breakdown. Cells were pulse‐labeled with [35S]methionine and chased on non‐radioactive starvation medium. Aliquots were taken at the indicated times and acid soluble small peptides generated by proteolysis were determined according to Straub et al. (1997).


( C ) Survival during starvation . Like pep4Δ ( pra1Δ ) , which are almost completely impaired in vacuolar proteolysis , aut2Δ aut7Δ exhibit a significantly reduced survival rate during starvation on nitrogen ‐ free medium as compared with wild ‐ type . Aliquots of incubated in 1 % K ‐ acetate were plated growing colonies were determined according to Straub et al . ( 1997 ) .

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(C) Survival during starvation. Like pep4Δ (pra1Δ) cells, which are almost completely impaired in vacuolar proteolysis, aut2Δ and aut7Δ cells exhibit a significantly reduced survival rate during starvation on nitrogen‐free medium as compared with wild‐type cells. Aliquots of cells incubated in 1% K‐acetate were plated and growing colonies were determined according to Straub et al. (1997).


Maturation of proaminopeptidase I is impaired in aut2 ‐ 1 ( A ) , aut2Δ ( B ) aut7Δ ( C ) . Further details are outlined in text . Crude extracts were prepared from starved 4 h on 1 % K ‐ acetate .

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Maturation of proaminopeptidase I is impaired in aut2‐1 (A), aut2Δ (B) and aut7Δ (C) cells. Further details are outlined in the text. Crude extracts were prepared from cells starved 4 h on 1% K‐acetate.


( A ) Detection of - interactions using two ‐ hybrid system . Intense indicates an interaction between Aut2p Aut7p , Aut2p Tub1p , Aut2p Tub2p . AUT7 , TUB1 TUB2 are cloned in ‐ frame with GAL4 ‐ activator domain . AUT2 AUT7 were fused to GAL4 ‐ binding domain ( see Materials methods ) .

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(A) Detection of protein-protein interactions using the two‐hybrid system. Intense staining indicates an interaction between Aut2p and Aut7p, Aut2p and Tub1p, and Aut2p and Tub2p. AUT7, TUB1 and TUB2 are cloned in‐frame with the GAL4‐activator domain. AUT2 and AUT7 were fused to the GAL4‐binding domain (see Materials and methods).


( B ) Affinity chromatography confirms - interaction between Aut2p Aut7p . A fusion of Aut2p with 26 kDa glutathione ‐ S ‐ transferase ( GST ) domain from Schistosoma japonicum ( see Materials methods ) was bound to glutathione - Sepharose 4B . This GST - Aut2p column was incubated with a crude extract from aut7Δ cen Met25 : : AUT7 - GFP . non ‐ bound supernatant ( S ) fraction ( lane 1 ) was taken away . Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non ‐ specifically bound . After final 500 mM NaCl wash ( W , lane 2 ) , bound were eluted with Laemmli buffer ( E , lane 3 ) . Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP . Using Aut2p - GST , a clear band corresponding to Aut7p - GFP is seen in eluate fraction ( lane 3 , arrowhead ) . As a control , GST alone was attached to column no band was detectable ( lane 6 ) .

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(B) Affinity chromatography confirms protein-protein interaction between Aut2p and Aut7p. A fusion protein of Aut2p with the 26 kDa glutathione‐S‐transferase (GST) domain from Schistosoma japonicum (see Materials and methods) was bound to glutathione-Sepharose 4B. This GST-Aut2p column was incubated with a crude extract from aut7Δ cen Met25::AUT7-GFP cells. The non‐bound supernatant (S) fraction (lane 1) was taken away. Gel beads were successively washed with NaCl solutions of increasing concentrations to remove non‐specifically bound proteins. After the final 500 mM NaCl wash (W, lane 2), bound proteins were eluted with Laemmli buffer (E, lane 3). Samples were analyzed in immunoblots with monoclonal antibodies directed against GFP. Using Aut2p-GST, a clear band corresponding to Aut7p-GFP is seen in the eluate fraction (lane 3, arrowhead). As a control, GST alone was attached to the column and no band was detectable (lane 6).


( C ) As described in ( B ) , bovine tubulin binds to GST - Aut2p glutathione - Sepharose 4B ( lane 12 ) , but not to GST glutathione - Sepharose 4B ( lane 9 ) . with Coomassie is shown in lanes 1 - 6 ; in lanes 7 - 12 tubulin was detected using monoclonal antibodies ( Boehringer Mannheim ) . Further details are given in Materials methods .

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(C) As described in (B), bovine tubulin binds to GST-Aut2p glutathione-Sepharose 4B (lane 12), but not to GST glutathione-Sepharose 4B (lane 9). Protein staining with Coomassie is shown in lanes 1-6; in lanes 7-12 tubulin was detected using monoclonal antibodies (Boehringer Mannheim). Further details are given in Materials and methods.


( D ) Similar to ( C ) , bovine tubulin does not bind to GST - Aut7p glutathione - Sepharose 4B ( lane 12 ) to GST glutathione - Sepharose 4B ( lane 9 ) . Lanes 1 - 6 , with Coomassie ; lanes 7 - 12 probed with antibodies directed against tubulin .

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(D) Similar to (C), bovine tubulin does not bind to GST-Aut7p glutathione-Sepharose 4B (lane 12) and to GST glutathione-Sepharose 4B (lane 9). Lanes 1-6, protein staining with Coomassie; lanes 7-12 probed with antibodies directed against tubulin.


Benomyl sensitivity of wild ‐ type overexpressing AUT2 AUT7 , of aut2Δ aut7Δ is not altered . were grown to stationary phase dilution series with decreasing densites were dropped on CM plates without uracil containing 15 30 μg / ml benomyl . Plates were incubated at 30 ° C .

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Benomyl sensitivity of wild‐type cells overexpressing AUT2 and AUT7, and of aut2Δ and aut7Δ cells is not altered. Cells were grown to stationary phase and dilution series with decreasing cell densites were dropped on CM plates without uracil containing 15 and 30 μg/ml benomyl. Plates were incubated at 30°C.


( A ) Autophagosome ‐ like vesicles ( arrowhead ) are accumulating in cytoplasm of aut2Δ starved for 4 h on 1 % K ‐ acetate in presence of PMSF . Bar represents 100 nm .

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(A) Autophagosome‐like vesicles (arrowhead) are accumulating in the cytoplasm of aut2Δ cells starved for 4 h on 1% K‐acetate in the presence of PMSF. Bar represents 100 nm.


( B ) Electron unravels accumulation of double ‐ layered autophagosome ‐ like vesicles in cytoplasm of aut7Δ starved for 3 h in nitrogen ‐ free medium in presence of PMSF . Arrowheads indicate clearly distinguishable two layers . Bar represents 100 nm .

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(B) Electron micrographs unravels the accumulation of double membrane‐layered autophagosome‐like vesicles in the cytoplasm of aut7Δ cells starved for 3 h in nitrogen‐free medium in the presence of PMSF. Arrowheads indicate the clearly distinguishable two membrane layers. Bar represents 100 nm.


( C D ) Cytosol containing autophagosome ‐ like vesicles accumulating in cytosol of aut2Δ ( D ) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction . In wild ‐ type ( C ) , no P100 is detectable . Fatty acid synthase ( Fas ) was used as a . Further details are given in text .

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(C and D) Cytosol containing autophagosome‐like vesicles accumulating in the cytosol of aut2Δ cells (D) can be isolated with an additional 100 000 g centrifugation step yielding a P100 pellet fraction. In wild‐type cells (C), no P100 is detectable. Fatty acid synthase (Fas) was used as a cytosolic protein. Further details are given in the text.


( D , E ) HTM - mediated of MPM - 2 ( D ) H3S10P ( E ) per nucleus in U2OS treated with DMSO as a control or 50 μM P22077 ( USP7i ) for 4 hrs . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm . is shown to right of representative for each . ( * * * p < 0 . 05 ; t - ) . Grey lines indicate mean values .

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(D, E) HTM-mediated quantification of MPM-2 (D) and H3S10P (E) levels per individual nucleus in U2OS cells treated with DMSO as a control or 50 μM P22077 (USP7i) for 4 hrs. Nuclei were stained with DAPI (blue). Scale bar, 30 μm. The quantification is shown to the right of representative images for each analysis. (*** p<0.05; t-test). Grey lines indicate mean values.


( A ) Western Blot showing of USP7 , MPM - 2 , CDK1Y15P CDK1 in whole extracts of RPE treated with 25 μM P22077 for 2 - 4 hrs ( USP7i ) , an asterisk indicates a non - specific band .

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(A) Western Blot showing the levels of USP7, MPM-2, CDK1Y15P and CDK1 in whole cell extracts of RPE cells treated with 25 μM P22077 for 2-4 hrs (USP7i), an asterisk indicates a non-specific band.


( B ) Immunofluorescence of CCNB1 ( red ) CCNB1S126P ( green ) in RPE in control conditions ( Control ) after treatment with 25 μM P22077 for 2 hrs ( USP7i ) . Nuclei were stained with DAPI ( blue ) . Scale bar , 30 μm .

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(B) Immunofluorescence of CCNB1 (red) and CCNB1S126P (green) in RPE cells in control conditions (Control) and after treatment with 25 μM P22077 for 2 hrs (USP7i). Nuclei were stained with DAPI (blue). Scale bar, 30 μm.


( B ) Western blot showing of MPM - 2 , pAKT , AKT , pPRC1 , pKAP , KAP , pMAPKAPK - 2 , pSTAT3 , STAT3 β - Actin in whole extracts of RPE treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Ponceau is shown as loading control .

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(B) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pKAP, KAP, pMAPKAPK-2, pSTAT3, STAT3 and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Ponceau staining is shown as loading control.


( D ) Immunofluorescence of PP2A - A ( green ) in U2OS treated with DMSO ( CONTROL ) , 50 μM P22077 ( USP7i ) for 4 hrs or 0 . 5 μM Okadaic acid ( OA ) for 1 hr 30 min . Nuclei were stained with DAPI ( blue ) . Scale bar , 10 μm .

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(D) Immunofluorescence of PP2A-A (green) in U2OS cells treated with DMSO (CONTROL), 50 μM P22077 (USP7i) for 4 hrs or 0.5 μM Okadaic acid (OA) for 1 hr 30 min. Nuclei were stained with DAPI (blue). Scale bar, 10 μm.


( E ) Western blot showing of MPM - 2 , pAKT , AKT , pPRC1 , pMAPKAPK - 2 , p4e - BP1 , 4e - BP1 β - Actin , in whole extracts of RPE treated with DMSO ( Control ) , 25 μM P22077 ( USP7i ) 10 μM of PP2A activator DT - 061 for 2 - 4 hrs . Ponceau is shown as loading control .

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(E) Western blot showing the levels of MPM-2, pAKT, AKT, pPRC1, pMAPKAPK-2, p4e-BP1, 4e-BP1 and β-Actin, in whole cell extracts of RPE cells treated with DMSO (Control), 25 μM P22077 (USP7i) and 10 μM of the PP2A activator DT-061 for 2-4 hrs. Ponceau staining is shown as loading control.


( F ) Western blot showing of MPM - 2 , phosphor - CDK substrate motif [ ( K / H ) pSP ] β - Actin in whole extracts of RPE treated with DMSO ( Control ) , 0 . 5 μM Okadaic acid ( OA ) for 2 hrs , 10 nM Calyculin A for 2 hrs , 25 μM P22077 for 4 hrs or 25 μM BAY 11 - 7082 for 2 hrs . Calyculin A was added to this experiment to illustrate gain in CDK activity induced by an additional PP2A inhibitor . Ponceau is shown as loading control .

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(F) Western blot showing the levels of MPM-2, phosphor-CDK substrate motif [(K/H)pSP] and β-Actin in whole cell extracts of RPE cells treated with DMSO (Control), 0.5 μM Okadaic acid (OA) for 2 hrs, 10 nM Calyculin A for 2 hrs, 25 μM P22077 for 4 hrs or 25 μM BAY 11-7082 for 2 hrs. Calyculin A was added to this experiment to illustrate the gain in CDK activity induced by an additional PP2A inhibitor. Ponceau staining is shown as loading control.


( E ) Western blot showing of USP7 γH2AX in whole extracts of HCT - 116 treated with DMSO ( Control ) , 50 μM P22077 for 2 - 4 hrs ( USP7i ) , 10 μM RO3306 for 4 hrs ( CDK1i ) or 50 μM P22077 + 10 μM RO3306 for 2 - 4 hrs ( USP7i + CDK1i ) . Ponceau is shown as loading control .

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(E) Western blot showing the levels of USP7 and γH2AX in whole cell extracts of HCT-116 cells treated with DMSO (Control), 50 μM P22077 for 2-4 hrs (USP7i), 10 μM RO3306 for 4 hrs (CDK1i) or 50 μM P22077 + 10 μM RO3306 for 2-4 hrs (USP7i + CDK1i). Ponceau staining is shown as loading control.


( B ) of apoptotic induction was measured by flow cytometry in RPE after treatment with DMSO as a control , 25 μM P22077 ( USP7i ) , 10 μM RO3306 ( CDK1i ) or 10 μM DT - 061 ( PP2Aa ) , their combinations for indicated times . content was followed with Hoechst , mitochondrial potential was measured by TMRE . of apoptotic ( TMRE negative ) is indicated for each condition . This experiment was repeated 3 times . ( * p < 0 . 05 , * * p < 0 . 01 , t - ) . Bars represent mean values ± .

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(B) Quantification of apoptotic induction was measured by flow cytometry in RPE cells after treatment with DMSO as a control, 25 μM P22077 (USP7i), 10 μM RO3306 (CDK1i) or 10 μM DT-061 (PP2Aa), and their combinations for the indicated times. DNA content was followed with Hoechst, and the mitochondrial membrane potential was measured by TMRE. The percentage of apoptotic cells (TMRE negative) is indicated for each condition. This experiment was repeated 3 times. (* p<0.05, ** p<0.01, t-test). Bars represent mean values ± SEM.


( C ) GLO viability in treated with 3 independent USP7 inhibitors ( P22077 , BAY 11 - 7082 or HBX 19818 ) alone or in combination with a 24hr treatment of 10 μM RO3306 ( CDK1i ) . This experiment was repeated 3 times . Data represent mean values ± . ( * * * p < 0 . 05 , ANOVA ) .

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(C) GLO viability assay in cells treated with 3 independent USP7 inhibitors (P22077, BAY 11-7082 or HBX 19818) alone or in combination with a 24hr treatment of 10 μM RO3306 (CDK1i). This experiment was repeated 3 times. Data represent mean values ± SEM. (*** p<0.05, ANOVA).


( a ) 30 150 days post fertilization ( dpf ) zebrafish ventricles were isolated relative determined ( n = 3 ) .

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(a) 30 and 150 days post fertilization (dpf) zebrafish ventricles were isolated and relative protein levels determined (n = 3).


( c ) qPCR of mRNA of glycolytic enzyme genes in 5 dpci remote wound border cardiac tissue ( n = 2 - 3 technical replicates using pooled cDNA from 10 ventricles for each condition ( n = 2 for pkmb pdk4 , n = 3 for other genes ) ) .

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(c) qPCR analysis of mRNA levels of glycolytic enzyme genes in 5 dpci remote and wound border cardiac tissue (n = 2-3 technical replicates using pooled cDNA from 10 ventricles for each condition (n = 2 for pkmb and pdk4, and n = 3 for the other genes)).


d , Immunostaining of heart sections for PCNA MEF2 in 5 dpci treated with PBS , 2 - DG or DCA ; magnified view of in boxes shown below ; dashed lines mark wound border ; arrowheads point to PCNA + CMs ; of PCNA + CMs in border zone shown on right ( n = 4 - 5 ventricles ) .

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d, Immunostaining of heart sections for PCNA and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to PCNA+ CMs; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-5 ventricles).


e ) Immunostaining of heart sections for MEF2 N2 . 261 MEF2 in 5 dpci treated with PBS , 2 - DG or DCA ; magnified view of in boxes shown below ; dashed lines mark wound border ; arrowheads point to N2 . 261 + CMs ; of N2 . 261 + CMs in border zone shown on right ( n = 4 - 5 ventricles ) .

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e) Immunostaining of heart sections for MEF2 N2.261 and MEF2 in 5 dpci animals treated with PBS, 2-DG or DCA; magnified view of area in white boxes shown below; white dashed lines mark the wound border; arrowheads point to N2.261+ CMs; percentage of N2.261+ CMs in the border zone shown on the right (n = 4-5 ventricles).


( a Immunostaining of heart sections for PCNA MEF2 in 5 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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(a Immunostaining of heart sections for PCNA and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for N2 . 261 MEF2 in 5 dpci pkma2 + / - ; pkmb + / - pkma2 - / - ; pkmb + / - ; magnified view of in boxes shown below ; arrowheads point to N2 . 261 + CMs ; dashed lines mark wound border ; of N2 . 261 + in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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Immunostaining of heart sections for N2.261 and MEF2 in 5 dpci pkma2+/-; pkmb+/- and pkma2-/-; pkmb+/- animals; magnified view of area in white boxes shown below; arrowheads point to N2.261+ CMs; white dashed lines mark the wound border; percentage of N2.261+ in the border zone shown on the right (n = 4-6 ventricles of each genotype).


Immunostaining of heart sections for PCNA MEF2 in 5 dpci ppargc1a + / + ppargc1a - / - ; magnified view of in boxes shown below ; arrowheads point to PCNA + CMs ; dashed lines mark wound border ; of PCNA + CMs in border zone shown on right ( n = 4 - 6 ventricles of each genotype ) .

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Immunostaining of heart sections for PCNA and MEF2 in 5 dpci ppargc1a+/+ and ppargc1a-/- animals; magnified view of area in white boxes shown below; arrowheads point to PCNA+ CMs; white dashed lines mark the wound border; percentage of PCNA+ CMs in the border zone shown on the right (n = 4-6 ventricles of each genotype).