F - G : Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone ( F ) or with 300µM fn1a morpholino ( G ) . H : Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos ( 125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone ) are injected into WT , fn1a morphants ( 300µM MO ) , fn1a + / - heterozygotes and fn1a - / - mutants . Loss of a single or both copies of fn1a exacerbates reduced gna13 levels , as does knockdown of fn1a .

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

F-G: Lateral Nomarski images of 48hpf WT larval fins injected with 200µM gna13b alone (F) or with 300µM fn1a morpholino (G). H: Quantification of the proportion of larvae with fin blisters when low amounts of gna13 morpholinos (125µM each gna13a and gna13b MO combined or 200µm gna13b MO alone) are injected into WT, fn1a morphants (300µM MO), fn1a+/- heterozygotes and fn1a-/- mutants. Loss of a single or both copies of fn1a exacerbates reduced gna13 levels, as does knockdown of fn1a.


Immunoblot analyses of TDG and TDG - SUMO in whole cell extracts of mESCs and MEFs . TDG and TDG - SUMO conjugates before ( 2i , 0 h ) and during differentiation of TDG wildtype ( TDGwt ) and TDG deficient ( TDGnull ) mESC and MEFs ( 5 µM all - trans retinoic acid ( RA , 24 h , 48 h ) . Percentages of TDG - SUMO relative to total TDG signal are indicated .

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

Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs and MEFs. TDG and TDG-SUMO conjugates before (2i, 0 h) and during differentiation of TDG wildtype (TDGwt) and TDG deficient (TDGnull) mESC and MEFs (5 µM all-trans retinoic acid (RA, 24 h, 48 h). Percentages of TDG-SUMO relative to total TDG signal are indicated.


Immunoblot analyses of TDG , TDG - SUMO in whole cell extracts of mESCs TDG and TDG - SUMO before and after H2O2 treatment ( 10 mM , 15 min ) .

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

Immunoblot analyses of TDG, TDG-SUMO in whole cell extracts of mESCs TDG and TDG-SUMO before and after H2O2 treatment (10 mM, 15 min).


Immunoblot analyses of TDG and TDG - SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype ( ESC - TDGwt ) or SUMOylation - deficient TDG ( ESC - TDGsnm ) . Sol , chromatin unbound proteins ; Chr1 , chromatin associated proteins ; Chr2 , chromatin bound fraction ; Histone H2B , stably chromatin bound marker .

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

Immunoblot analyses of TDG and TDG-SUMO in whole cell extracts of mESCs Chromatin fractionation from mESCs expressing wildtype (ESC-TDGwt) or SUMOylation-deficient TDG (ESC-TDGsnm). Sol, chromatin unbound proteins; Chr1, chromatin associated proteins; Chr2, chromatin bound fraction; Histone H2B, stably chromatin bound marker.


Immunoblot analyses of XRCC1 and , XRCC1 - SUMO , in whole cell extracts of mESCs and MEFs . XRCC1 and XRCC1 - SUMO conjugates before ( 2i , 0 h ) and during RA - induced differentiation ( 24 h , 48 h ) in mESCs and MEFs . Percentages of XRCC1 - SUMO relative to total XRCC1 signal are indicated .

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

Immunoblot analyses of XRCC1 and , XRCC1-SUMO, in whole cell extracts of mESCs and MEFs. XRCC1 and XRCC1-SUMO conjugates before (2i, 0 h) and during RA-induced differentiation (24 h, 48 h) in mESCs and MEFs. Percentages of XRCC1-SUMO relative to total XRCC1 signal are indicated.


Immunoblot analyses of XRCC1 and XRCC1 - SUMO , in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease ( SENP2364 - 489 ) .

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

Immunoblot analyses of XRCC1 and XRCC1-SUMO, in whole cell extracts of mESCs XRCC1 SUMOylation in native whole cells extracts before and after incubation with purified SUMO protease (SENP2364-489).


Immunoblot analyses of , XRCC1 and XRCC1 - SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 ( 5 mM , 15 min ) .

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

Immunoblot analyses of , XRCC1 and XRCC1-SUMO in whole cell extracts of mESCs XRCC1 SUMOylation before and after exposure to H2O2 (5 mM, 15 min).


Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs . TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs .

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

Immunoblot analyses of TET1 isoforms in whole cell extracts of mESCs and MEFs. TET1 and TET1s isoform protein levels in mESCs during RA induced differentiation and in MEFs.


Fluorescence microscopy of GFP - TDGwt GFP - TDGpip in mESCs . Scale bar : 100µM . Quantitation of fluorescence signals of GFP - TDGwt and GFP - TDGpip in mESCs . median with interquartile range . > 470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs .

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

Fluorescence microscopy of GFP-TDGwt GFP-TDGpip in mESCs. Scale bar: 100µM. Quantitation of fluorescence signals of GFP-TDGwt and GFP-TDGpip in mESCs. median with interquartile range. >470 cells were analysed of each GFPTDGwt and GFPTDGpip ESCs.


Immunoblot analysis of XRCC1 and XRCC1 - SUMO in GFP - TDGwt and GFP - TDGpip mESCs .

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

Immunoblot analysis of XRCC1 and XRCC1-SUMO in GFP-TDGwt and GFP-TDGpip mESCs.


" Far Western " blot analyses of BER protein interactions . Recombinant purified human TDG ( 10 µg ) , POLß ( 1 µg ) , APE1 ( 1 µg ) , and LIGIII ( 1 µg ) probed with XRCC1 and XRCC1 - SUMO ( 20 µg ) and immune - stained for XRCC1 .

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

"Far Western" blot analyses of BER protein interactions. Recombinant purified human TDG (10 µg), POLß (1 µg), APE1 (1 µg), and LIGIII (1 µg) probed with XRCC1 and XRCC1-SUMO (20 µg) and immune-stained for XRCC1.


Immunoblot analysis of interactions of XRCC1 and XRCC1 - SUMO with endogenous TDG . Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1 - SUMO was coupled to Ni - NTA agarose and incubated with mESC extracts . Input ( I ) , last wash ( LW ) and elution ( E ) fractions .

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

Immunoblot analysis of interactions of XRCC1 and XRCC1-SUMO with endogenous TDG. Purified 6hisXRCC1 or in vitro modified and purified 6hisXRCC1-SUMO was coupled to Ni-NTA agarose and incubated with mESC extracts. Input (I), last wash (LW) and elution (E) fractions.


0 . 5 µM TDGwt , were incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) and equimolar amounts of XRCC1 , POLβ and LIGIII proteins .

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

0.5 µM TDGwt, were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) and equimolar amounts of XRCC1, POLβ and LIGIII proteins.


Schematic of biotin - 5caC DNA substrate pulldown experiment . 0 . 5 µM TDGwt or TDGcat ( TDG ( N140A ) ) incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) as indicated and equimolar amounts of XRCC1 - SUMO ( > 50 % SUMOylated ) , POLβ and LIGIII .

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

Schematic of biotin-5caC DNA substrate pulldown experiment. 0.5 µM TDGwt or TDGcat (TDG(N140A)) incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1-SUMO (>50% SUMOylated), POLβ and LIGIII.


1 µM biotinylated 5caC substrate ( G • 5caC DNA ) and 10 µM homoduplex competitor DNA ( G • C DNA ) were incubated with XRCC1 - SUMO ( 50 % SUMOylated ) and BER proteins as indicated .

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

1 µM biotinylated 5caC substrate (G•5caC DNA) and 10 µM homoduplex competitor DNA (G•C DNA) were incubated with XRCC1-SUMO (50% SUMOylated) and BER proteins as indicated.


0 . 5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate ( G • 5caC DNA ) , 0 . 5 µM SUMO and 10 µM homoduplex competitor DNA ( G • C DNA ) as indicated . Shown is an immunoblot analysis of TDG and free SUMO .

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

0.5 µM human TDGwt protein were incubated with 1 µM biotinylated 5caC substrate (G•5caC DNA), 0.5 µM SUMO and 10 µM homoduplex competitor DNA (G•C DNA) as indicated. Shown is an immunoblot analysis of TDG and free SUMO.


" Far Western " blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1 - SUMO . TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1 - SUMO ( probe : XRCC1 or XRCC1SUMO ) where indicated . TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated .

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

"Far Western" blot analyses of TDGwt and TDGE310Q with 6hisXRCC1 and 6hisXRCC1-SUMO. TDGwt or TDGE310Q was dot blotted on nitrocellulose membranes and probed with XRCC1 or XRCC1-SUMO (probe:XRCC1 or XRCC1SUMO) where indicated. TDG and XRCC1 probes were detected by immunoblotting with anti TDG and anti XRCC1 antibodies as indicated.


Schematic of biotin - 5caC DNA substrate pulldown experiment . Immunoblot analysis of purified TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) , 6hisXRCC1 / XRCC1 - SUMO and 6his BER proteins bound to Streptavidin ( St ) Biotin - 5caC DNA substrate . 0 . 5 µM TDGwt or TDG SUMO - binding mutant ( TDGE310Q ) were incubated with biotinylated 5caC substrate ( G • 5caC DNA , 1 µM ) and homoduplex competitor DNA ( G • C DNA , 10 µM ) as indicated and equimolar amounts of XRCC1 , POLβ , APE1 and LIGIII proteins . Grey ovals , BERosome .

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

Schematic of biotin-5caC DNA substrate pulldown experiment. Immunoblot analysis of purified TDGwt or TDG SUMO-binding mutant (TDGE310Q), 6hisXRCC1/XRCC1-SUMO and 6his BER proteins bound to Streptavidin (St) Biotin-5caC DNA substrate. 0.5 µM TDGwt or TDG SUMO-binding mutant (TDGE310Q) were incubated with biotinylated 5caC substrate (G•5caC DNA, 1 µM) and homoduplex competitor DNA (G•C DNA, 10 µM) as indicated and equimolar amounts of XRCC1, POLβ, APE1 and LIGIII proteins. Grey ovals, BERosome.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA .

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

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA . SUMOylation assay as in ( A ) with SUMO provided as XRCC1 - SUMO instead of free SUMO .

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

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMOylation assay as in (A) with SUMO provided as XRCC1-SUMO instead of free SUMO.


Reconstitution SUMO conjugation / de - conjugation reaction with purified SAE1 / 2 , UBC9 , SUMO protease SENP2364 - 489 ) , free SUMO in equimolar amounts and 20 - fold molar excess over TDG . TDG SUMOylation was performed in the presence or absence of AP - site containing synthetic DNA double - strands as ( as indicated ( G • AP ) . TDG and TDG - SUMO were detected by immunoblotting . SUMOylation of DNA ( G • AP ) bound TDG is strongly reduced but a 20 - fold molar excess of free SUMO compensates the inhibitory effect of DNA . SUMO transfer as depicted in ( B ) with additional APE1 , POLß , LIGIII , and TDG .

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

Reconstitution SUMO conjugation/de-conjugation reaction with purified SAE1/2, UBC9, SUMO protease SENP2364-489), free SUMO in equimolar amounts and 20-fold molar excess over TDG. TDG SUMOylation was performed in the presence or absence of AP-site containing synthetic DNA double-strands as (as indicated (G•AP). TDG and TDG-SUMO were detected by immunoblotting. SUMOylation of DNA (G•AP) bound TDG is strongly reduced but a 20-fold molar excess of free SUMO compensates the inhibitory effect of DNA. SUMO transfer as depicted in (B) with additional APE1, POLß, LIGIII, and TDG.


Immunoblot analysis of XRCC1 , TDG , TDG - SUMO in TDGwt ( wt ) , TDGsnm ( snm ) and XRCC1null mESCs ( null ) .

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

Immunoblot analysis of XRCC1, TDG, TDG-SUMO in TDGwt (wt), TDGsnm (snm) and XRCC1null mESCs (null).


Immunoblot analysis of TDG and TDG - SUMO in TDGwt ( wt ) and XRCC1null mESCs ( null ) exposed to H2O2 as indicated .

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

Immunoblot analysis of TDG and TDG-SUMO in TDGwt (wt) and XRCC1null mESCs (null) exposed to H2O2 as indicated.


Top : Schematic of the SUMO - mediated TDG - BERosome ( grey oval ) formation and DNA processing . Bottom : Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double - strands in presence of dynamic SUMO - conjugation / de - conjugation . Left panels lanes 3 - 14 : BER reconstitution with XRCC1 or XRCC1 - SUMO as indicated . Right panels lanes 15 - 21 : BER reconstitution with free SUMO or XRCC1 - SUMO as SUMO donor as indicated . ds59merDNA substrates : lane 1 - 2 CG / CG HpaII methylation sensitive restriction site , cleavable by HpaII ; lane 3 - 6 , 8 - 14 , 15 - 17 , 21 HpaII methylation sensitive restriction site restriction site with a caC ( 5caCG / CG ) , not cleavable by HpaII ; lane 7 and 20 repaired Product ( CC / CG ) that is cleavable by HpaII after excision / repair of caC / CG to CG / CG by BER . Percentage of total signal of fully repaired product is indicated . ss59merDNA , single stranded DNA .

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

Top: Schematic of the SUMO-mediated TDG-BERosome (grey oval) formation and DNA processing. Bottom: Reconstitution of active DNA demethylation on a 5caC containing synthetic DNA double-strands in presence of dynamic SUMO-conjugation/de-conjugation. Left panels lanes 3-14: BER reconstitution with XRCC1 or XRCC1-SUMO as indicated. Right panels lanes 15-21: BER reconstitution with free SUMO or XRCC1-SUMO as SUMO donor as indicated. ds59merDNA substrates: lane 1-2 CG/CG HpaII methylation sensitive restriction site, cleavable by HpaII; lane 3-6, 8-14, 15-17, 21 HpaII methylation sensitive restriction site restriction site with a caC (5caCG/CG), not cleavable by HpaII; lane 7 and 20 repaired Product (CC/CG) that is cleavable by HpaII after excision/repair of caC/CG to CG/CG by BER. Percentage of total signal of fully repaired product is indicated. ss59merDNA, single stranded DNA.


Bright field captures of TDGwt , TDGcat and TDGsnm mESCs at indicated stages of differentiation . DAPI , Alexa Fluor ® 680 phalloidin actin staining at the bottom . Scale bars 25µM , 50µM , 500µM as indicated . Quantification of neural cells with neurites of total differentiating TDGwt , TDGcat and TDGsnm mESCs . n ≥ 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney test .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

Bright field captures of TDGwt, TDGcat and TDGsnm mESCs at indicated stages of differentiation. DAPI, Alexa Fluor® 680 phalloidin actin staining at the bottom. Scale bars 25µM, 50µM, 500µM as indicated. Quantification of neural cells with neurites of total differentiating TDGwt, TDGcat and TDGsnm mESCs. n ≥ 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


Relative changes of lineage marker expression between NPCs and undifferentiated mESCs ( ESCM LIF , 0 h ) , quantified with Gapdh as normalizer . n ≥ 3 . Means with standard deviation ( SD ) ) . * : P < 0 . 05 ; * * : P < 0 . 01 by non parametric Mann - Whitney test .

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Relative changes of lineage marker expression between NPCs and undifferentiated mESCs (ESCM LIF, 0 h), quantified with Gapdh as normalizer. n ≥ 3. Means with standard deviation (SD)). *: P<0.05; **: P<0.01 by non parametric Mann-Whitney test.


LC - MS / MS analysis of global cytosine - modification levels in DNA from ground - state mESCs ( 2i ) and EBs . Means with standard deviation ( SD ) of 5mC , 5hmC , 5fC and 5caC per 106 bases ) . n ≥ 3 . * : P ≤ 0 . 05 , * * : P < 0 . 01 , * * * : P < 0 . 0001 by one - way ANOVA , Dunnett ' s multiple comparisons test .

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LC-MS/MS analysis of global cytosine-modification levels in DNA from ground-state mESCs (2i) and EBs. Means with standard deviation (SD) of 5mC, 5hmC, 5fC and 5caC per 106 bases). n ≥ 3. *: P≤0.05, **: P<0.01, ***: P<0.0001 by one-way ANOVA, Dunnett's multiple comparisons test.


Alkaline comet assay with pluripotent ( 0 h ) and differentiating ( 24 h 5 µM RA ) mESC as indicated . Olive Tail Moments and % DNA in tail . n ≥ 3 . Medians with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P ≤ 0 . 01 , * * * : P ≤ 0 . 0001 by one - way ANOVA , Sidak ' s multiple comparisons test .

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Alkaline comet assay with pluripotent (0 h) and differentiating (24 h 5 µM RA) mESC as indicated. Olive Tail Moments and % DNA in tail. n ≥ 3. Medians with standard deviation (SD). *: P≤0.05; **: P≤0.01, ***: P≤0.0001 by one-way ANOVA, Sidak's multiple comparisons test.


Cell proliferation assay . Relative viability of Talazoparib treated to untreated cells . n = 3 . Means with standard deviation ( SD ) , P ≤ 0 . 05 , * * : P < 0 . 01 , by one - way ANOVA , Dunnett ' s multiple comparisons test .

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Cell proliferation assay. Relative viability of Talazoparib treated to untreated cells. n = 3. Means with standard deviation (SD), P≤0.05, **: P<0.01, by one-way ANOVA, Dunnett's multiple comparisons test.


ChIP - qPCR analysis of TET1 , TDG and XRCC1 at gene promoters in mESCs ( 24 h 5 µM RA ) . Relative enrichments to an unbound control region ( Chr2neg ) . n ≥ 3 . Means with standard deviation ( SD ) . * : P ≤ 0 . 05 ; * * : P < 0 . 001 ; * * * : P ≤ 0 . 0001 , by two - tailed Mann - Whitney test .

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ChIP-qPCR analysis of TET1, TDG and XRCC1 at gene promoters in mESCs (24 h 5 µM RA). Relative enrichments to an unbound control region (Chr2neg). n ≥ 3. Means with standard deviation (SD). *: P≤0.05; **: P<0.001; ***: P≤0.0001, by two-tailed Mann-Whitney test.


Representative images of mESC neuronal differentiation as in Fig 4B . Shown are XRCC1wt and XRCC1null ( XRCC1null1 , XRCC1null2 ) mESCs in 2i medium supplemented with LIF ( 2i LIF ) and in ESC medium supplemented with LIF ( ESCM LIF ) , EBs in ESCM LIF and 5µM RA ( EB 4d ESCM RA ) and dissociated neuron - like cells in B27 ( B27 ) . Scale bars 25µM , 50µM , 500µM as indicated . Neurite grids were observed for XRCC1wt ; heterogeneous cell populations formed with the XRCC1null ( XRCC1null1 , null2 ) clones . Quantification ( below ) of neural cells with neurites of total differentiating XRCC1wt , XRCC1null mESCs . n = 5 . Means with standard deviation ( SD ) . * * : P < 0 . 01 . Non parametric , Mann - Whitney test .

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Representative images of mESC neuronal differentiation as in Fig 4B. Shown are XRCC1wt and XRCC1null (XRCC1null1, XRCC1null2) mESCs in 2i medium supplemented with LIF (2i LIF) and in ESC medium supplemented with LIF (ESCM LIF), EBs in ESCM LIF and 5µM RA (EB 4d ESCM RA) and dissociated neuron-like cells in B27 (B27). Scale bars 25µM, 50µM, 500µM as indicated. Neurite grids were observed for XRCC1wt; heterogeneous cell populations formed with the XRCC1null (XRCC1null1, null2) clones. Quantification (below) of neural cells with neurites of total differentiating XRCC1wt, XRCC1null mESCs. n = 5. Means with standard deviation (SD). **: P<0.01. Non parametric, Mann-Whitney test.


Quantification of mRNA expression of pluripotency and ectodermal / neurogenesis marker genes by RT - qPCR . n ≥ 3 . Mean values and standard deviations ( SD ) of EBs relative to their mESC ( ESCM LIF , 16h ) , normalised to Gapdh .

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Quantification of mRNA expression of pluripotency and ectodermal/neurogenesis marker genes by RT-qPCR. n ≥ 3. Mean values and standard deviations (SD) of EBs relative to their mESC (ESCM LIF, 16h), normalised to Gapdh.


Growth phenotypes of the wild - type ( WT , Col - 0 ) , bri1 - null mutant , and two independent ubp12i / ubp13 double mutant lines grown on 1 / 2MS medium supplemented with 10 μM DEX ( DEX medium ) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution ( 0 . 1 % [ v / v ] EtOH ) for additionally 8 days . Scale bars : 10 mm .

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Growth phenotypes of the wild-type (WT, Col-0), bri1-null mutant, and two independent ubp12i/ubp13 double mutant lines grown on 1/2MS medium supplemented with 10 μM DEX (DEX medium) for 10 days and transferred to DEX medium containing 1 μM BL or mock solution (0.1% [v/v] EtOH) for additionally 8 days. Scale bars: 10 mm.


Phenotype of the WT and ubp12i / ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days . Scale bars : 10 mm . Hypocotyl length relative to the mock control of seedlings ( B ) . Experiments were done in triplicate ( n > 15 seedlings for each line ) .

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Phenotype of the WT and ubp12i/ubp13 double mutant grown on DEX medium in the presence of increasing concentrations of BL in the light for 6 days. Scale bars: 10 mm.   Hypocotyl length relative to the mock control of seedlings (B). Experiments were done in triplicate (n >15 seedlings for each line).


Western blot ( WB ) analysis of BES1 dephosphorylation after BL treatment with the α - BES1 antibody . Total proteins were isolated from 6 - day - old seedlings grown on DEX medium in the light , either in the presence of 100 nM BL or mock . PEPC was used as a loading control . Percentage of dephosphorylated BES1 relative to the total BES1 from ( D ) ( n = 6 biological replicates ) .

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Western blot (WB) analysis of BES1 dephosphorylation after BL treatment with the α-BES1 antibody. Total proteins were isolated from 6-day-old seedlings grown on DEX medium in the light, either in the presence of 100 nM BL or mock. PEPC was used as a loading control.   Percentage of dephosphorylated BES1 relative to the total BES1 from (D) (n = 6 biological replicates).


Western blot analysis of BRI1 protein amount in ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from 18 - day - old plants grown on 1 / 2MS medium supplemented with 10 μM DEX ( + ) or not ( - ) . Western blot analysis of BRI1 protein level in concanamycin A ( ConcA ) - treated plants . Eighteen - day - old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation .

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Western blot analysis of BRI1 protein amount in ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from 18-day-old plants grown on 1/2MS medium supplemented with 10 μM DEX (+) or not (-).   Western blot analysis of BRI1 protein level in concanamycin A (ConcA)-treated plants. Eighteen-day-old plants were grown on DEX medium and exposed to 1 μM ConcA for 4 h before total protein isolation.


Deubiquitinating activity of UBP13 against K48 - and K63 - linked ubiquitination . Diubiquitins linked through K48 or K63 ( K48 Di - Ub or K63 Di - Ub ) were incubated alone or with GST - UBP13 or GST - UBP13C207S for 18 h . Deubiquitination of the diubiquitins was analyzed by western blot with the α - ubiquitin ( α - Ub , P4D1 ) antibody . The presence of GST - UBP13 / UBP13C207S recombinant proteins was confirmed by the α - GST antibody .

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Deubiquitinating activity of UBP13 against K48- and K63-linked ubiquitination. Diubiquitins linked through K48 or K63 (K48 Di-Ub or K63 Di-Ub) were incubated alone or with GST-UBP13 or GST-UBP13C207S for 18 h. Deubiquitination of the diubiquitins was analyzed by western blot with the α-ubiquitin (α-Ub, P4D1) antibody. The presence of GST-UBP13/UBP13C207S recombinant proteins was confirmed by the α-GST antibody.


UBP13 deubiquitination of K63 - ubiquitinated BRI1 in vitro . Polyubiquitinated MBP - BRI1CD was generated by incubation with GST - PUB13 ( Ub reaction ) and then with GST - UBP13 or GST - UBP13C207S ( DUB reaction - MBP pull - down , input ) . After in vitro deubiquitination , MBP - BRI1CD was purified by an MBP pull - down to analyze the BRI1CD ubiquitination ( DUB reaction - MBP pull - down , SE ) . The ubiquitinated proteins were detected by western blot with an α - ubiquitin antibody ( FK2 ) . Ubiquitin chain specificity was detected with an α - K63Ub antibody ( Apu3 ) . The presence of recombinant proteins was confirmed by the α - GST and α - MBP antibodies .

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

UBP13 deubiquitination of K63-ubiquitinated BRI1 in vitro. Polyubiquitinated MBP-BRI1CD was generated by incubation with GST-PUB13 (Ub reaction) and then with GST-UBP13 or GST-UBP13C207S (DUB reaction-MBP pull-down, input). After in vitro deubiquitination, MBP-BRI1CD was purified by an MBP pull-down to analyze the BRI1CD ubiquitination (DUB reaction-MBP pull-down, SE). The ubiquitinated proteins were detected by western blot with an α-ubiquitin antibody (FK2). Ubiquitin chain specificity was detected with an α-K63Ub antibody (Apu3). The presence of recombinant proteins was confirmed by the α-GST and α-MBP antibodies.


Growth phenotype comparison of BRI1 - mCit / bri1 / ubp12i / ubp13 and BRI125KR - mCit / bri1 / ubp12i / ubp13 . The indicated transgenic lines were grown on DEX medium for 18 days . Scale bars : 10 mm .

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Growth phenotype comparison of BRI1-mCit/bri1/ubp12i/ubp13 and BRI125KR-mCit/bri1/ubp12i/ubp13. The indicated transgenic lines were grown on DEX medium for 18 days. Scale bars: 10 mm.


Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR - mCit / bri1 / ubp12i / ubp13 plants with the α - BRI1 antibody . Total proteins were isolated from the roots of the lines indicated ( A ) . PEPC was used as a loading control .

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Western blot analysis monitoring the BRI1 protein accumulation in BRI125KR-mCit/bri1/ubp12i/ubp13 plants with the α-BRI1 antibody. Total proteins were isolated from the roots of the lines indicated (A). PEPC was used as a loading control.


A - D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 ( A ) or BRI125KR ( C ) tagged with mCitrine ( mCit ) in either bri1 / UBP12 / UBP13 or bri1 / ubp12i / ubp13 background . Six - day - old seedlings were grown on DEX medium , and kept in the dark for 2 h in the presence of 50 μM CHX before imaging . Confocal images of epidermal cells from root meristem are shown . Scale bars : 10 μm . Measurements of the relative PM / intracellular BRI1 - mCit fluorescence intensity . For each line , at least 55 and 25 cells from over 25 and 15 seedlings were measured in ( B ) and ( D ) , respectively .

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A-D Analysis of the BRI1 vacuolar targeting on Arabidopsis seedlings expressing BRI1 (A) or BRI125KR (C) tagged with mCitrine (mCit) in either bri1/UBP12/UBP13 or bri1/ubp12i/ubp13 background. Six-day-old seedlings were grown on DEX medium, and kept in the dark for 2 h in the presence of 50 μM CHX before imaging. Confocal images of epidermal cells from root meristem are shown. Scale bars: 10 μm. Measurements of the relative PM/intracellular BRI1-mCit fluorescence intensity. For each line, at least 55 and 25 cells from over 25 and 15 seedlings were measured in (B) and (D), respectively.


( A ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in wild - type or SAS6 monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(A) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in wild-type or SAS6 monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( D ) Top hits that emerged from the screens ranked by MaGeCK FDR value . Blue hits are genes required for centriole duplication or stability . Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes .

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(D) Top hits that emerged from the screens ranked by MaGeCK FDR value. Blue hits are genes required for centriole duplication or stability. Green hits are genes predicted to be required to arrest the growth of cells with extra centrosomes.


( G ) Top : Graph showing the efficiency of frameshifting INDELs measured using TIDE . N . D . = Not determined . Data shown is from n = 1 biological replicate . Bottom : Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(G) Top: Graph showing the efficiency of frameshifting INDELs measured using TIDE. N.D. = Not determined. Data shown is from n = 1 biological replicate. Bottom: Graph showing the relative growth of doxycycline treated PLK4Dox cells expressing an sgRNA targeting the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( H ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in polyclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(H) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in polyclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( A ) Top , widefield image showing the localization of CEP164 and PIDD1 at the mature - mother centriole . Bottom : Representative 3D STORM image of the same centriole showing PIDD1 localization . STORM image colors correspond to the Z - depth with red being the closest to the coverslip and blue being the most distant .

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(A) Top, widefield image showing the localization of CEP164 and PIDD1 at the mature-mother centriole. Bottom: Representative 3D STORM image of the same centriole showing PIDD1 localization. STORM image colors correspond to the Z-depth with red being the closest to the coverslip and blue being the most distant.


( B ) 3D STORM image of PIDD1 at the mature mother centriole . The overlaid mask represents the inner and outer diameters of the PIDD1 signal .

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(B) 3D STORM image of PIDD1 at the mature mother centriole. The overlaid mask represents the inner and outer diameters of the PIDD1 signal.


( C ) Inner and outer diameter measurements for distal appendage proteins and PIDD1 . Measurements in the shaded region were previously reported in ( Bowler et al . , 2019 ) . Data displayed as box and whisker plots , where the box represents the upper and lower quartile and the whiskers represent the s . d . Scale bars : 1 μm for all wide - field images of centrioles and 500 nm for STORM images . Data acquired across n ≥ 6 cells .

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(C) Inner and outer diameter measurements for distal appendage proteins and PIDD1. Measurements in the shaded region were previously reported in (Bowler et al., 2019). Data displayed as box and whisker plots, where the box represents the upper and lower quartile and the whiskers represent the s.d. Scale bars: 1 μm for all wide-field images of centrioles and 500 nm for STORM images. Data acquired across n ≥6 cells.


( D ) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes . A dot displays measurements from each experiment . Experiments were performed in PLK4Dox polyclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with PIDD1 localized to the mature mother centriole in PLK4Dox cells expressing an sgRNA targeting the indicated genes. A dot displays measurements from each experiment. Experiments were performed in PLK4Dox polyclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes . Each dot displays measurements from a single experiment . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n ≥ 3 biological replicates . Mean ± s . e . m .

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(F) Graph showing the relative growth of doxycycline treated PLK4Dox cells that were knocked out for the indicated genes. Each dot displays measurements from a single experiment. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n ≥ 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(G) Quantification of the fraction of cells with PIDD1 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( A ) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

(A) Quantification of the fraction of cells with ANKRD26 localized at the mature mother centriole. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( B - D ) Representative images of WT or ANKRD26 - / - PLK4Dox cells immunostained with the indicated antibodies . Scale bar = 5 µm .

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(B-D) Representative images of WT or ANKRD26-/- PLK4Dox cells immunostained with the indicated antibodies. Scale bar = 5 µm.


( E ) Quantification of the fraction of WT or ANKRD26 - / - hTERT RPE1 cells with cilia . Each dot displays measurements from a single experiment . Data acquired across n = 5 biological replicates . Mean ± s . e . m .

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(E) Quantification of the fraction of WT or ANKRD26-/- hTERT RPE1 cells with cilia. Each dot displays measurements from a single experiment. Data acquired across n = 5 biological replicates. Mean ± s.e.m.


( F ) Growth assay of the indicated cells with and without doxycycline - inducible overexpression of PLK4 . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(F) Growth assay of the indicated cells with and without doxycycline-inducible overexpression of PLK4. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( G ) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes . Experiments were performed in PLK4Dox monoclonal knockout cells . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(G) Quantification of centrosome number in PLK4Dox cells expressing an sgRNA targeting the indicated genes. Experiments were performed in PLK4Dox monoclonal knockout cells. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( H ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . ( I ) Quantification of pro - CASP2 levels following treatment with dox for the specified number of days . Experiments were performed in PLK4Dox monoclonal knockout cells . Each dot displays measurements from a single experiment . Data acquired across n = 6 biological replicates . Mean ± s . e . m .

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(H) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. (I) Quantification of pro-CASP2 levels following treatment with dox for the specified number of days. Experiments were performed in PLK4Dox monoclonal knockout cells. Each dot displays measurements from a single experiment. Data acquired across n = 6 biological replicates. Mean ± s.e.m.


( A ) Top : schematic representation of PIDD1 and its various cleavage products . Bottom : HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

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(A) Top: schematic representation of PIDD1 and its various cleavage products. Bottom: HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( B ) HEK293FT cells were transfected with the indicated constructs , subjected to co - immunoprecipitation and immunoblotted with the indicated antibodies .

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(B) HEK293FT cells were transfected with the indicated constructs, subjected to co- immunoprecipitation and immunoblotted with the indicated antibodies.


( C ) Western blot showing the expression of ANKRD26 . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes .

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(C) Western blot showing the expression of ANKRD26. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes.


( D ) Quantification of the fraction of cells with ANKRD26 ( left ) and PIDD1 ( right ) localized to the mature mother centriole in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(D) Quantification of the fraction of cells with ANKRD26 (left) and PIDD1 (right) localized to the mature mother centriole in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( E ) Graph showing the relative growth of doxycycline - treated , monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated ANKRD26 transgenes . Each dot displays measurements from a single experiment . Data acquired across n = 3 biological replicates . Mean ± s . e . m .

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(E) Graph showing the relative growth of doxycycline-treated, monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated ANKRD26 transgenes. Each dot displays measurements from a single experiment. Data acquired across n = 3 biological replicates. Mean ± s.e.m.


( F ) Western blot showing expression of pro - CASP2 and P21 following treatment with dox for 4 days . Experiments were performed in monoclonal ANKRD26 - / - PLK4Dox cells expressing the indicated transgenes .

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(F) Western blot showing expression of pro-CASP2 and P21 following treatment with dox for 4 days. Experiments were performed in monoclonal ANKRD26-/- PLK4Dox cells expressing the indicated transgenes.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . A ) Transcription in fft2Δ relative to WT over chromosome 1 . The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands , respectively . Coordinates are shown in the middle panel ; genes more than 2 - fold upregulated are marked : Tf2 retrotransposons ( black ) ; antisense to Tf2 elements ( gray ) ; coding genes ( green ) ; noncoding RNAs ( blue )

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). A) Transcription in fft2Δ relative to WT over chromosome 1. The top and bottom panels represent fold change in tiling array signal from the forward and reverse strands, respectively. Coordinates are shown in the middle panel; genes more than 2-fold upregulated are marked: Tf2 retrotransposons (black); antisense to Tf2 elements (gray); coding genes (green); noncoding RNAs (blue)


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . B ) Northern blot for the Tf2 ORF ; rRNA staining of the same membrane by methylene blue is shown below . Two different exposures of the molecular size marker are shown ( left ) .

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). B) Northern blot for the Tf2 ORF; rRNA staining of the same membrane by methylene blue is shown below. Two different exposures of the molecular size marker are shown (left).


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . C ) qPCR scheme . Amplicon 1 spans the end of the LTR and into the ORF . Amplicon 2 does not include the LTR but covers the translation start site . Amplicon 3 is entirely within the ORF . D ) Bar graph representing RNA levels for the amplicons depicted in ( C ) , relative to a control locus ( SPAC1F8 . 07c ) and to WT levels , as measured by qPCR . Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates .

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Retrotransposon transcription in WT (Hu303), fft2Δ(Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). C) qPCR scheme. Amplicon 1 spans the end of the LTR and into the ORF. Amplicon 2 does not include the LTR but covers the translation start site. Amplicon 3 is entirely within the ORF. D) Bar graph representing RNA levels for the amplicons depicted in (C), relative to a control locus (SPAC1F8.07c) and to WT levels, as measured by qPCR. Error bars represent standard deviation of duplicate reverse transcriptions of biological triplicates.


Retrotransposon transcription in WT ( Hu303 ) , fft2Δ ( Hu1955 ) , fft3Δ ( Hu1867 ) , and fft2Δfft3Δ ( Hu2000 ) . E ) 5 ' RACE products run on a 1 . 5 % agarose gel .

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Retrotransposon transcription in WT (Hu303), fft2Δ (Hu1955), fft3Δ (Hu1867), and fft2Δfft3Δ (Hu2000). E) 5'RACE products run on a 1.5% agarose gel.


Top panel : Y - axis shows the CAGE signal intensity ( tag - per - million normalized number of mapped CAGE tags on plus strand ) . X axis shows LTR and 5 ' of Tf2 ORF . Three biological replicates are shown for each strain : fft2Δfft3 and WT . Bottom : LTR and tf2 ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer and primer binding sequence ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of all Tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above the sequence elements . The black square indicates the 5 ' end of the Tf2 mRNA in WT and the green square indicates the 5 ' end in fft2Δ fft3Δ detected by 5 ' RACE .

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Top panel: Y-axis shows the CAGE signal intensity (tag-per-million normalized number of mapped CAGE tags on plus strand). X axis shows LTR and 5' of Tf2 ORF. Three biological replicates are shown for each strain: fft2Δfft3 and WT. Bottom: LTR and tf2 ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all Tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements. The black square indicates the 5' end of the Tf2 mRNA in WT and the green square indicates the 5' end in fft2Δ fft3Δ detected by 5'RACE.


A ) Fft2 and Fft3 are both enriched at LTRs , while Fft2 is also enriched at the tf2 coding region . Fft2 LTR enrichment increases in fft3Δ cells , while Fft3 LTR enrichment decreases slightly in fft2Δ cells . Top : Fft2 - myc enrichment in WT ( solid line ) and in fft3Δ ( dashed line ) . Bottom : Fft3 - myc enrichment in WT ( solid line ) and in fft2Δ ( dashed line ) . ChIP - chip signal is normalized against no - epitope control arrays .

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A) Fft2 and Fft3 are both enriched at LTRs, while Fft2 is also enriched at the tf2 coding region. Fft2 LTR enrichment increases in fft3Δ cells, while Fft3 LTR enrichment decreases slightly in fft2Δ cells. Top: Fft2 -myc enrichment in WT (solid line) and in fft3Δ (dashed line). Bottom: Fft3 -myc enrichment in WT (solid line) and in fft2Δ (dashed line). ChIP-chip signal is normalized against no-epitope control arrays.


B ) Nucleosome occupancy over the LTR U3 is significantly reduced ( average reduction ~ 50 % ; average p - values at each position < 0 . 001 , Poisson test ) in fft2Δ fft3Δ cells . MNase - seq data aligned at the start of tf25 ' LTRs is shown for WT ( black line ) , and fft2Δ fft3Δ ( dark gray line ) . Light gray line indicates significance of difference between WT and mutant signals : amplitude reflects significance ( p - value ) and sign reflects the sign of occupancy change in mutant vs . WT cells . Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements . LTR and tf2ORF are colored : U3 ( green ) , R ( red ) , U5 ( blue ) , self primer and primer binding sequence ( PBS ; gray ) , beginning of ORF ( gray striped ) . TSS of all tf2 transcripts from ( Rhind et al , 2011 ) are shown as gray dots above the sequence elements , while the green dot indicates the TSS in fft2Δ fft3Δ .

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B) Nucleosome occupancy over the LTR U3 is significantly reduced (average reduction ~50%; average p-values at each position <0.001, Poisson test) in fft2Δ fft3Δ cells. MNase-seq data aligned at the start of tf25'LTRs is shown for WT (black line), and fft2Δ fft3Δ (dark gray line). Light gray line indicates significance of difference between WT and mutant signals: amplitude reflects significance (p-value) and sign reflects the sign of occupancy change in mutant vs. WT cells. Error bars show standard deviation of nucleosome occupancy averaged over 13 tf2 elements. LTR and tf2ORF are colored: U3 (green), R (red), U5 (blue), self primer and primer binding sequence (PBS; gray), beginning of ORF (gray striped). TSS of all tf2 transcripts from (Rhind et al, 2011) are shown as gray dots above the sequence elements, while the green dot indicates the TSS in fft2Δ fft3Δ.


A ) Mean nucleosome occupancy over solo LTRs ( n = 236 ) and tf2 - associated LTRs ( n = 25 ) , in WT and in fft2Δ fft3Δ .

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A) Mean nucleosome occupancy over solo LTRs (n=236) and tf2-associated LTRs (n=25), in WT and in fft2Δ fft3Δ.


B ) Mean ratio of expression ( fft2Δ fft3Δ / WT ) for each of the following categories : non - coding with LTR ( n = 69 ) / without LTR ( n = 1665 ) ( p = 0 . 008 ) ; protein coding with LTR ( n = 246 ) / without LTR ( n = 4887 ) ( p < 0 . 001 ) ; Tf2s ( n = 13 ) . A and B ) Data shown as box plots , significance of difference between categories was assessed by Wilcoxon - Mann - Whitney test ( * * p < 0 . 001 ) .

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B) Mean ratio of expression (fft2Δ fft3Δ /WT) for each of the following categories: non-coding with LTR (n=69) / without LTR (n=1665) (p=0.008); protein coding with LTR (n=246) / without LTR (n=4887) (p<0.001); Tf2s (n=13). A and B) Data shown as box plots, significance of difference between categories was assessed by Wilcoxon-Mann-Whitney test (** p<0.001).


A ) Heat and oxidative stress cause a Tf2 TSS shift in WT cells , resulting in the production of reverse transcription competent Tf2 transcripts . Transcription fold change as in Figure 1D , but relative to act1 transcription and to transcription in the same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological duplicates .

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A) Heat and oxidative stress cause a Tf2 TSS shift in WT cells, resulting in the production of reverse transcription competent Tf2 transcripts. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates.


B ) Heat shock - associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy . The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells , where nucleosome occupancy is already very low ( Fig 2B , Fig S3 ) . LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF + 1 nucleosome , measured by MNase - qPCR .

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B) Heat shock-associated TSS shift is accompanied by a reduction in LTR nucleosome occupancy. The degree of nucleosome loss is greater in WT cells than in fft2Δ fft3Δ cells, where nucleosome occupancy is already very low (Fig 2B, Fig S3). LTR nucleosome occupancy shown relative to occupancy of the Tf2 ORF +1 nucleosome, measured by MNase-qPCR.


C ) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells . Transcription fold change as in Figure 1D , but relative to act1 transcription and to transcription in the same culture immediately prior to stress , as measured by qPCR . Error bars represent standard deviation of reverse transcriptions of biological

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C) Exposure to stress conditions does not increase Tf2 transcript levels further in fft2Δ fft3Δ cells. Transcription fold change as in Figure 1D, but relative to act1 transcription and to transcription in the same culture immediately prior to stress, as measured by qPCR. Error bars represent standard deviation of reverse transcriptions of biological duplicates


B ) Bar graph representing RNA levels as in Fig . 1D , relative to a control locus ( act1 ) and to WT levels , as measured by qPCR . Error bars represent standard deviation of nucleosome occupancy of biological duplicates .

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B) Bar graph representing RNA levels as in Fig. 1D, relative to a control locus (act1) and to WT levels, as measured by qPCR. Error bars represent standard deviation of nucleosome occupancy of biological duplicates.


C ) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy , measured by MNase - qPCR as in Fig . 5B .

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C) TSS shift in Fft deletion and catalytic mutants is accompanied by reduced LTR nucleosome occupancy, measured by MNase-qPCR as in Fig. 5B.


A ) Fluorescencein situ hybridization with a probe against tf2 elements ( green ) , and DAPI staining ( blue ) in WT and fft2Δ fft3Δ cells . B ) The number of tf2 element signal clusters is significantly elevated ( p = 9 . 8x10 - 18 , χ2 - test ) in fft2Δ fft3Δ cells , indicating a loss of retrotransposon clustering . Tf2 clustering was counted in wt cells ( n = 663 ) , fft2 ∆ ( n = 556 ) , fft3 ∆ ( n = 493 ) , fft2 ∆ fft3 ∆ ( n = 636 ) . Significance of difference between strains was assessed by Chi - square test .

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A) Fluorescencein situ hybridization with a probe against tf2 elements (green), and DAPI staining (blue) in WT and fft2Δ fft3Δ cells. B) The number of tf2 element signal clusters is significantly elevated (p=9.8x10-18, χ2-test) in fft2Δ fft3Δ cells, indicating a loss of retrotransposon clustering. Tf2 clustering was counted in wt cells (n=663), fft2∆ (n=556), fft3∆ (n=493), fft2∆ fft3∆ (n=636). Significance of difference between strains was assessed by Chi-square test.


A ) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D ( PP3 ) and fft2Dfft3D ( PP4 ) mutant strains with new integrations of Tf2 - 12 - neoAI . After 3 days at 32o C .

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A) Pictures of YES plates with G418 showing growth of G418 resistant subclones in fft3D (PP3) and fft2Dfft3D (PP4) mutant strains with new integrations of Tf2-12-neoAI. After 3 days at 32o C.


C ) Southern blot probed for neoA fft3D ( PP3 ) and fft2Dfft3D mutant strains ( PP4 ) and derived G418 resistant subclones .

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C) Southern blot probed for neoA fft3D (PP3) and fft2Dfft3D mutant strains (PP4) and derived G418 resistant subclones.


Western blot analysis of nephrin expression in immortalized podocytes cultured in normal ( 5 mM ) or high glucose ( HG ; 30 mM ) or high mannitol ( Mann ; 5 mM glucose plus 25 mM mannitol ) for 24 , 48 and 72 hours . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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Western blot analysis of nephrin expression in immortalized podocytes cultured in normal (5 mM) or high glucose (HG; 30 mM) or high mannitol (Mann; 5 mM glucose plus 25 mM mannitol) for 24, 48 and 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal , high glucose or high mannitol at the indicated time points . U , unmethylated - specific primers ; M , methylated - specific primers .

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B Analysis of the methylation status at nephrin gene promoter in podocytes cultured in normal, high glucose or high mannitol at the indicated time points. U, unmethylated-specific primers; M, methylated-specific primers.


C Effect of pargyline hydrochloride , a pan - KDM inhibitor , on high glucose - mediated nephrin expression . Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride ( 1 μM ) for 48 hours . Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated cells ( Wilcoxon two - sample test ; n = 3 ) .

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C Effect of pargyline hydrochloride, a pan-KDM inhibitor, on high glucose-mediated nephrin expression. Immortalized mouse podocytes were cultured in the indicated cultured media in the presence or absence of pargyline hydrochloride (1 μM) for 48 hours. Protein lysates from the treated podocytes were analyzed for nephrin expression by immunoblotting. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Wilcoxon two-sample test; n = 3).


D Effect of pargyline hydrochloride on high glucose - mediated DNA methylation at nephrin gene promoter . Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation - specific PCR .

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D Effect of pargyline hydrochloride on high glucose-mediated DNA methylation at nephrin gene promoter. Genomic DNA samples from the treated podocytes were analyzed for methylation status of nephrin gene promoter by methylation-specific PCR.


E Quantitative RT - PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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E Quantitative RT-PCR analyses of different KDMs in immortalized podocytes exposed to high glucose or high mannitol for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


F Western blot analysis of KDM6A expression in podocytes cultured in normal , high glucose or high mannitol conditions for 24 , 48 ad 72 hours . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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F Western blot analysis of KDM6A expression in podocytes cultured in normal, high glucose or high mannitol conditions for 24, 48 ad 72 hours. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


G Western blot analysis of H3K27me1 , H3K27me2 , H3K27me3 and pan - methyl H3K9 in podocytes cultured in normal , high glucose or high mannitol conditions for 9 and 24 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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G Western blot analysis of H3K27me1, H3K27me2, H3K27me3 and pan-methyl H3K9 in podocytes cultured in normal, high glucose or high mannitol conditions for 9 and 24 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


A Effect of KDM6A knockdown on the expression of nephrin and WT - 1 in immortalized mouse podocytes cultured under high glucose conditions . Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A - specific siRNAs , and cultured in high glucose ( HG ) or high mannitol ( Mann ) for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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A Effect of KDM6A knockdown on the expression of nephrin and WT-1 in immortalized mouse podocytes cultured under high glucose conditions. Western blot analyses were performed using protein lysates of podocytes that were transfected with scrambled siRNAs or KDM6A-specific siRNAs, and cultured in high glucose (HG) or high mannitol (Mann) for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


B Effect of KDM6A overexpression on expression levels of nephrin and WT - 1 . Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours , and the expression levels of KDM6A , nephrin and WT - 1 in these transfected podoytes were analyzed by immunoblotting . * P < 0 . 05 versus vector controls ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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B Effect of KDM6A overexpression on expression levels of nephrin and WT-1. Immortalized podocytes were transfected with the KDM6A expression plasmid or the empty vector control for 48 hours, and the expression levels of KDM6A, nephrin and WT-1 in these transfected podoytes were analyzed by immunoblotting. *P < 0.05 versus vector controls (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


C Changes in H3K27me3 levels in podocytes after treatment with high glucose , the combination of high glucose and KDM6A knockdown , or KDM6A overexpression for 48 hours . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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C Changes in H3K27me3 levels in podocytes after treatment with high glucose, the combination of high glucose and KDM6A knockdown, or KDM6A overexpression for 48 hours. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


D Changes in DNA methylation at nephrin gene promoter by high glucose , by the combination of high glucose plus KDM6A knockdown , or by KDM6A overexpression in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers . Experiments were repeated three times and a representative gel from one experiment is shown .

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D Changes in DNA methylation at nephrin gene promoter by high glucose, by the combination of high glucose plus KDM6A knockdown, or by KDM6A overexpression in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers. Experiments were repeated three times and a representative gel from one experiment is shown.


E Effect of GSK - J4 ( 40 μM ) , a specific KDM6A inhibitor , on high glucose - mediated changes in levels of nephrin , WT - 1 and H3K27me3 . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated HG - incubated cells ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Effect of GSK-J4 (40 μM), a specific KDM6A inhibitor, on high glucose-mediated changes in levels of nephrin, WT-1 and H3K27me3. *P < 0.05 versus normal controls, #P < 0.05 versus untreated HG-incubated cells (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Changes in DNA methylation at nephrin gene promoter by GSK - J4 ( 40 μM ) in immortalized podocytes . U , unmethylated - specific primers ; M , methylated - specific primers .

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F Changes in DNA methylation at nephrin gene promoter by GSK-J4 (40 μM) in immortalized podocytes. U, unmethylated-specific primers; M, methylated-specific primers.


Relative mRNA levels of KDM6A , nephrin and WT - 1 , normalized to β - actin , expressed in kidney glomeruli of normal and diabetic mice at 4 , 8 and 12 weeks after diabetic induction . * Significant differences ( P < 0 . 05 ) compared with normal controls ( Wilcoxon two - sample test ; n = 8 each ) .

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Relative mRNA levels of KDM6A, nephrin and WT-1, normalized to β-actin, expressed in kidney glomeruli of normal and diabetic mice at 4, 8 and 12 weeks after diabetic induction. *Significant differences (P < 0.05) compared with normal controls (Wilcoxon two-sample test; n =8 each).


B Western blot analysis of nephrin , KDM6A and H3K27me3 in kidney glomeruli of normal and diabetic mice at different time points . * P < 0 . 05 versus normal controls for the indicated time points ( Wilcoxon two - sample test ; n = 3 ) .

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B Western blot analysis of nephrin, KDM6A and H3K27me3 in kidney glomeruli of normal and diabetic mice at different time points. *P < 0.05 versus normal controls for the indicated time points (Wilcoxon two-sample test; n = 3).


C Changes in body weights and levels of blood glucose in normal , diabetic and GSK - J4 - treated diabetic mice . As noted , GSK - J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks ( Wilcoxon two - sample test ; n = 8 ) .

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C Changes in body weights and levels of blood glucose in normal, diabetic and GSK-J4-treated diabetic mice. As noted, GSK-J4 treatment did not significantly affect body weights or blood glucose levels in diabetic mice during the experimental period of 12 weeks (Wilcoxon two-sample test; n = 8).


D Levels of urinary protein excretion , weights of kidney , and levels of HbA1c in normal , diabetic and GSK - J4 - treated diabetic mice . Urinary total protein excretion , kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction . The mean relative kidney weight ( % ) shown in the study is determined as the percent of kidneys out of total body weight , and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin ( % HbA1c ; DCCT unit ) . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 8 ) .

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D Levels of urinary protein excretion, weights of kidney, and levels of HbA1c in normal, diabetic and GSK-J4-treated diabetic mice. Urinary total protein excretion, kidney weight and levels of HbA1c were measured at 12 weeks after diabetic induction. The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).


E Western blot analysis of KDM6A , nephrin , WT - 1 and H3K27me3 expressed in kidney glomeruli of normal , diabetic and GSK - J4 - treated diabetic mice at 12 weeks after diabetic induction . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Western blot analysis of KDM6A, nephrin, WT-1 and H3K27me3 expressed in kidney glomeruli of normal, diabetic and GSK-J4-treated diabetic mice at 12 weeks after diabetic induction. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence analysis of KDM6A , nephrin and WT - 1 in kidney sections from normal , diabetic , and GSK - J4 - treated diabetic mice . Green : nephrin or WT - 1 ; Red : KDM6A ; Blue : DAPI . Scale bars , 20 μm . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus untreated diabetic mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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F Immunofluorescence analysis of KDM6A, nephrin and WT-1 in kidney sections from normal, diabetic, and GSK-J4-treated diabetic mice. Green: nephrin or WT-1; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. *P < 0.05 versus normal controls, #P < 0.05 versus untreated diabetic mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Immunofluorescence staining of F - actin and KDM6A in primary podocytes isolated from normal , diabetic , and GST - J4 - treated diabetic mice . Green : F - actin ; Red : KDM6A ; Blue : DAPI . Scale bars , 20 μm . Presented experiments were performed at least three times independently .

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G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from normal, diabetic, and GST-J4-treated diabetic mice. Green: F-actin; Red: KDM6A; Blue: DAPI. Scale bars, 20 μm. Presented experiments were performed at least three times independently.


A Quantitative RT - PCR evaluation of KDM6A mRNA expression in kidney glomeruli isolated from wild - type and KDM6A - knockout ( KO ) mice . * P < 0 . 05 , significant difference versus wild - type controls ( Wilcoxon two - sample test ; n = 8 ) .

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A Quantitative RT-PCR evaluation of KDM6A mRNA expression in kidney glomeruli isolated from wild-type and KDM6A-knockout (KO) mice. *P < 0.05, significant difference versus wild-type controls (Wilcoxon two-sample test; n = 8).


B Western blot analysis of KDM6A expression in glomeruli and podocytes isolated from wild - type and KDM6A - KO mice . Presented experiments were performed at least three times independently .

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B Western blot analysis of KDM6A expression in glomeruli and podocytes isolated from wild-type and KDM6A-KO mice. Presented experiments were performed at least three times independently.


C Changes in body weights and levels of blood glucose in wild - type or KDM6A - KO mice with or without STZ treatment . No significant differences in body weights or blood glucose levels between wild - type and KDM6A - KO mice with diabetes were observed during the 8 - week experimental period ( Wilcoxon two - sample test ; n = 8 ) .

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C Changes in body weights and levels of blood glucose in wild-type or KDM6A-KO mice with or without STZ treatment. No significant differences in body weights or blood glucose levels between wild-type and KDM6A-KO mice with diabetes were observed during the 8-week experimental period (Wilcoxon two-sample test; n = 8).


D Levels of urinary protein excretion , weights of kidney and levels of HbA1c in wild - type and KDM6A - KO mice with or without STZ treatment ( at 8 weeks after the onset of diabetes ) . The mean relative kidney weight ( % ) shown in the study is determined as the percent of kidneys out of total body weight , and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin ( % HbA1c ; DCCT unit ) . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 8 ) .

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D Levels of urinary protein excretion, weights of kidney and levels of HbA1c in wild-type and KDM6A-KO mice with or without STZ treatment (at 8 weeks after the onset of diabetes). The mean relative kidney weight (%) shown in the study is determined as the percent of kidneys out of total body weight, and the HbA1c level is defined as the ratio of HbA1c to the total hemoglobin (% HbA1c; DCCT unit). *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 8).


E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild - type ( WT - NC ) , KDM6A - KO ( KO - NC ) , STZ - treated wild - type ( WT - DM ) , or STZ - treated KDM6A - KO ( KO - DM ) mice . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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E Immunofluorescence images of kidney sections stained with KDM6A and nephrin in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild - type ( WT - NC ) , KDM6A - KO ( KO - NC ) , STZ - treated wild - type ( WT - DM ) , or STZ - treated KDM6A - KO ( KO - DM ) mice . Scale bars , 20 μm . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

F Immunofluorescence images of kidney sections stained with KDM6A and H3K27me3 in wild-type (WT-NC), KDM6A-KO (KO-NC), STZ-treated wild-type (WT-DM), or STZ-treated KDM6A-KO (KO-DM) mice. Scale bars, 20 μm. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


G Immunofluorescence staining of F - actin and KDM6A in primary podocytes isolated from the above treated mice . Presented experiments were performed at least three times independently . Scale bars , 20 μm .

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G Immunofluorescence staining of F-actin and KDM6A in primary podocytes isolated from the above treated mice. Presented experiments were performed at least three times independently. Scale bars, 20 μm.


H Western blot analysis of nephrin , KDM6A and WT - 1 expressed in primary podocytes isolated from the above treated mice . * P < 0 . 05 versus untreated wild - type controls , # P < 0 . 05 versus STZ - treated wild - type mice ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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H Western blot analysis of nephrin, KDM6A and WT-1 expressed in primary podocytes isolated from the above treated mice. *P < 0.05 versus untreated wild-type controls, #P < 0.05 versus STZ-treated wild-type mice (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


I Effect of high glucose on the expression of podocyte - related markers in primary cultured podocytes isolated from wild - type or KDM6A - KO mice . The primary podocytes isolated from wild - type or KDM6A - KO mice were cultured in normal or high glucose ( 30 mM ) for 48 hours . Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies . * P < 0 . 05 versus wild - type podocytes in normal glucose , # P < 0 . 05 versus wild - type podocytes in high glucose ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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I Effect of high glucose on the expression of podocyte-related markers in primary cultured podocytes isolated from wild-type or KDM6A-KO mice. The primary podocytes isolated from wild-type or KDM6A-KO mice were cultured in normal or high glucose (30 mM) for 48 hours. Protein lysates from the cultured podocytes were subjected to Western blot analysis with the indicated antibodies. *P < 0.05 versus wild-type podocytes in normal glucose, #P < 0.05 versus wild-type podocytes in high glucose (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


A Screening of the potential KDM6A - regulated transcriptional factors involved in repression of nephrin expression . After transduction with a control lentiviral vector or a lentiviral vector expressing KDM6A into primary podocytes for 48 hours , intracellular RNAs were isolated and used for RNA sequencing ( RNA - Seq ) analysis . The transcript expression patterns of 86 selected transcriptional factors from three independent RNA - Seq experiments are presented in a heat map . Notably , among these 86 selected genes , KLF10 is the most upregulated gene .

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A Screening of the potential KDM6A-regulated transcriptional factors involved in repression of nephrin expression. After transduction with a control lentiviral vector or a lentiviral vector expressing KDM6A into primary podocytes for 48 hours, intracellular RNAs were isolated and used for RNA sequencing (RNA-Seq) analysis. The transcript expression patterns of 86 selected transcriptional factors from three independent RNA-Seq experiments are presented in a heat map. Notably, among these 86 selected genes, KLF10 is the most upregulated gene.


B Validation of increased KDM6A and KLF10 expression in primary podocytes that were infected with an empty lentiviral vector or KDM6A - expressing lentiviral vector for 48 hours . * P < 0 . 05 versus the empty vector control ( Wilcoxon two - sample test ; n = 3 ) .

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B Validation of increased KDM6A and KLF10 expression in primary podocytes that were infected with an empty lentiviral vector or KDM6A-expressing lentiviral vector for 48 hours. *P < 0.05 versus the empty vector control (Wilcoxon two-sample test; n = 3).


C Increased expression of KDM6A and KLF10 in primary podocytes cultured in high glucose for 48 hours . * P < 0 . 05 versus normal controls ( Wilcoxon two - sample test ; n = 3 ) .

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C Increased expression of KDM6A and KLF10 in primary podocytes cultured in high glucose for 48 hours. *P < 0.05 versus normal controls (Wilcoxon two-sample test; n = 3).


D Effect of KLF10 knockdown on high glucose - mediated reduction of nephrin in primary podocytes . As noted , knockdown of KLF10 prevented downregulation of nephrin and upregulation of KDM6A in high glucose - treated podocytes . * P < 0 . 05 versus normal controls , # P < 0 . 05 versus control siRNA with HG incubation ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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D Effect of KLF10 knockdown on high glucose-mediated reduction of nephrin in primary podocytes. As noted, knockdown of KLF10 prevented downregulation of nephrin and upregulation of KDM6A in high glucose-treated podocytes. *P < 0.05 versus normal controls, #P < 0.05 versus control siRNA with HG incubation (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


E Immunofluorescence analysis of KLF10 and nephrin in renal sections of normal , diabetic , and GSK - J4 - treated diabetic mice . Scale bars , 20 μm . * P < 0 . 05 versus the normal control group , # P < 0 . 05 versus the untreated diabetic group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

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

E Immunofluorescence analysis of KLF10 and nephrin in renal sections of normal, diabetic, and GSK-J4-treated diabetic mice. Scale bars, 20 μm. *P < 0.05 versus the normal control group, #P < 0.05 versus the untreated diabetic group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).


F Immunofluorescence images of KLF10 and nephrin in renal sections of wild - type or KDM6A - KO mice with or without STZ treatment . Scale bars , 20 μm . * P < 0 . 05 versus the untreated wild - type group , # P < 0 . 05 versus the STZ - treated wild - type group ( Parametric ANOVA and a Bonferroni post hoc test ; n = 3 ) .

O O O O B-MEASURED_VAR O B-MEASURED_VAR 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 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

F Immunofluorescence images of KLF10 and nephrin in renal sections of wild-type or KDM6A-KO mice with or without STZ treatment. Scale bars, 20 μm. *P < 0.05 versus the untreated wild-type group, #P < 0.05 versus the STZ-treated wild-type group (Parametric ANOVA and a Bonferroni post hoc test; n = 3).