Figure 3 ( A - J ) confocal images of the NMJ on larval muscles 6 and 7 in segment A3 , stained with anti - HRP ( magenta ) to label the nerve and anti - Dlg ( green ) as a postsynaptic marker of synaptic boutons . ( A ) w1118 control ; ( B ) btz2 / + ; ( C ) btz2 / Df ( 3R ) BSC497 ; ( D ) UAS - btz / + ; da - GAL4 , btz2 / Df ( 3R ) BSC497 ; ( E ) BG380 - GAL4 / + ; UAS - btz / + ; btz2 / Df ( 3R ) BSC497 ; ( F ) 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( G ) BG380 - GAL4 / + ; 24B - GAL4 / UAS - btz ; btz2 / Df ( 3R ) BSC497 ; ( H ) BG380 - GAL4 / + ; UAS - btz RNAi / + ; ( I ) mhc - GAL4 / UAS - btz RNAi ; ( J ) repo - GAL4 / UAS - btz RNAi . Scale bar , 30 μm . Insets in ( A , C ) show enlargements of the boxed regions , illustrating the closer spacing of boutons in btz mutants . ( K , L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 ; * * , p < 0 . 01 ; ns , not significant by unpaired t - test with Welch ' s correction . n = 23 ( w ; da - GAL4 , btz / Df ) , n = 6 ( btz / + ) , n = 10 ( Df / + , UAS - BtzGFP / + ; btz / Df ) , n = 8 ( btz / Df ) , n = 22 ( da > btz - GFP ; btz / Df ) , n = 13 ( BG380 - GAL4 ; btz / Df ; BG380 + 24B > Btz - GFP ; btz / Df ; da - GAL4 / + ) , n = 5 ( BG380 > Btz - GFP ; btz / Df ) , n = 11 ( 24B - GAL4 ; btz / Df ; da > btz RNAi ) , n = 12 ( 24B > Btz - GFP ; btz / Df ; btz RNAi / + ) , n = 9 ( BG380 + 24B - GAL4 ; btz / Df ) , n = 15 ( BG380 - GAL4 / + ) , n = 14 ( BG380 > btz RNAi ; repo > btz RNAi ) , n = 16 ( mhc - GAL4 / + ) , n = 28 ( mhc > btz RNAi ) , or n = 21 ( repo - GAL4 / + ) . Error bars show mean ± SEM . NMJ size is reduced in btz mutants and rescued by expressing UAS - btz with da - GAL4 or with the combination of the BG380 - GAL4 and 24B - GAL4 drivers , but not with any single driver . Expressing UAS - btz RNAi with da - GAL4 or in motor neurons with BG380 - GAL4 , muscles with mhc - GAL4 or glia with repo - GAL4 also reduces NMJ size .

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

Figure 3(A-J) confocal images of the NMJ on larval muscles 6 and 7 in segment A3, stained with anti-HRP (magenta) to label the nerve and anti-Dlg (green) as a postsynaptic marker of synaptic boutons. (A) w1118 control; (B) btz2/+; (C) btz2/Df(3R)BSC497; (D) UAS-btz/+; da-GAL4, btz2/Df(3R)BSC497; (E) BG380-GAL4/+; UAS-btz/+; btz2/Df(3R)BSC497; (F) 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (G) BG380-GAL4/+; 24B-GAL4/UAS-btz; btz2/Df(3R)BSC497; (H) BG380-GAL4/+; UAS-btz RNAi/+; (I) mhc-GAL4/UAS-btz RNAi; (J) repo-GAL4/UAS-btz RNAi. Scale bar, 30 μm. Insets in (A, C) show enlargements of the boxed regions, illustrating the closer spacing of boutons in btz mutants.(K, L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001; **, p<0.01; ns, not significant by unpaired t-test with Welch's correction. n=23 (w; da-GAL4, btz/Df), n=6 (btz/+), n=10 (Df/+, UAS-BtzGFP/+; btz/Df), n=8 (btz/Df), n=22 (da>btz-GFP; btz/Df), n=13 (BG380-GAL4; btz/Df; BG380+24B>Btz-GFP; btz/Df; da-GAL4/+), n=5 (BG380>Btz-GFP; btz/Df), n=11 (24B-GAL4; btz/Df; da>btz RNAi), n=12 (24B>Btz-GFP; btz/Df; btz RNAi/+), n=9 (BG380+24B-GAL4; btz/Df), n=15 (BG380-GAL4/+), n=14 (BG380>btz RNAi; repo>btz RNAi), n=16 (mhc-GAL4/+), n=28 (mhc>btz RNAi), or n=21 (repo-GAL4/+). Error bars show mean ± SEM. NMJ size is reduced in btz mutants and rescued by expressing UAS-btz with da-GAL4 or with the combination of the BG380-GAL4 and 24B-GAL4 drivers, but not with any single driver. Expressing UAS-btz RNAi with da-GAL4 or in motor neurons with BG380-GAL4, muscles with mhc-GAL4 or glia with repo-GAL4 also reduces NMJ size.


Figure 4 ( I ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; * * * , p < 0 . 001 by unpaired t - test or Mann - Whitney test . n = 15 ( BG380 - GAL4 ) , n = 12 ( BG380 > btz RNAi ; BG380 > mago RNAi ; BG380 > eIF4AIII RNAi ) , n = 16 ( btz / + ) , n = 13 ( btz / Df ) , or n = 10 ( Btz - WT ; btz / Df ; Btz - HD ; btz / Df ) . Error bars show mean ± SEM . NMJ size is reduced by knocking down btz in motor neurons , but slightly increased by knocking down mago or eIF4AIII . The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction - defective btz transgenes .

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Figure 4(I) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ***, p<0.001 by unpaired t-test or Mann-Whitney test. n=15 (BG380-GAL4), n=12 (BG380>btz RNAi; BG380>mago RNAi; BG380>eIF4AIII RNAi), n=16 (btz/+), n=13 (btz/Df), or n=10 (Btz-WT; btz/Df; Btz-HD; btz/Df). Error bars show mean ± SEM. NMJ size is reduced by knocking down btz in motor neurons, but slightly increased by knocking down mago or eIF4AIII. The size deficit in btz mutants is equally well rescued by the wild type or EJC interaction-defective btz transgenes.


Figure 5 ( L ) quantifications of the number of boutons normalized to muscle surface area ( x1000 ) in the indicated genotypes . * * * * , p < 0 . 0001 ; ns , not significant by unpaired t - test or Mann - Whitney test . n = 19 ( UAS - daw RNAi / + ) , n = 9 ( BG380 > daw RNAi ; btz / Df ; tub > daw ) , n = 23 ( mhc > daw RNAi ) , n = 10 ( repo > daw RNAi ; tub > daw ) , n = 12 ( btz / Df ; UAS - daw ) , or n = 14 ( btz / Df ; tub > btz ) . Error bars show mean ± SEM . Knocking down daw in motor neurons , muscles or glia reduces NMJ size . Expressing UAS - daw with tub - GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS - btz , but has no significant effect on NMJ size in a wild - type background .

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Figure 5(L) quantifications of the number of boutons normalized to muscle surface area (x1000) in the indicated genotypes. ****, p<0.0001; ns, not significant by unpaired t-test or Mann-Whitney test. n=19 (UAS-daw RNAi/+), n=9 (BG380>daw RNAi; btz/Df; tub>daw), n=23 (mhc>daw RNAi), n=10 (repo>daw RNAi; tub>daw), n=12 (btz/Df; UAS-daw), or n=14 (btz/Df; tub>btz). Error bars show mean ± SEM. Knocking down daw in motor neurons, muscles or glia reduces NMJ size. Expressing UAS-daw with tub-GAL4 rescues NMJ size in btz mutants as efficiently as expressing UAS-btz, but has no significant effect on NMJ size in a wild-type background.


Figure 1Immunofluorescence labeling of QSOX1 and a Golgi marker ( GM130 ) in epithelial cells isolated from murine colon . QSOX1 is green , GM130 is red , and DAPI staining of nuclei is blue in the merged image . Scale bar is 10 µm . Top , average colon length of WT and QSOX1 KO mice following indicated treatments . For untreated mice , colon lengths were calculated for 3 WT and 3 KO . For treated mice , lengths were calculated from 5 mice except for the DSS - treated WT , for which 3 mice were analyzed . Error bars are standard deviation . Statistical analysis was performed using Tukey multiple comparison of means ( * * p < 0 . 01 ) . Administration of QSOX1 inhibitory antibody MAb316 . 1 ( αQSOX1 ) , did not enhance the sensitivity of WT colons to DSS ( p > 0 . 4 for MAb316 . 1 - treated WT vs . WT control antibody ( IgG ) - treated ; p > 0 . 5 for MAb316 . 1 - treated WT vs . WT without antibody treatment ) . Bottom , representative images of colons following DSS treatment . Scale bar is 1 cm .

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Figure 1Immunofluorescence labeling of QSOX1 and a Golgi marker (GM130) in epithelial cells isolated from murine colon. QSOX1 is green, GM130 is red, and DAPI staining of nuclei is blue in the merged image. Scale bar is 10 µm.Top, average colon length of WT and QSOX1 KO mice following indicated treatments. For untreated mice, colon lengths were calculated for 3 WT and 3 KO. For treated mice, lengths were calculated from 5 mice except for the DSS-treated WT, for which 3 mice were analyzed. Error bars are standard deviation. Statistical analysis was performed using Tukey multiple comparison of means (** p<0.01). Administration of QSOX1 inhibitory antibody MAb316.1 (αQSOX1), did not enhance the sensitivity of WT colons to DSS (p>0.4 for MAb316.1-treated WT vs. WT control antibody (IgG)-treated; p>0.5 for MAb316.1-treated WT vs. WT without antibody treatment). Bottom, representative images of colons following DSS treatment. Scale bar is 1 cm.


Figure 3Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well - packed goblet cell granules in WT and QSOX1 KO colons . Scale bar is 5 µm . Alcian blue and PAS staining of reduced guanidine - insoluble mucins from WT and QSOX1 KO mice , separated on a 6 % polyacrylamide gel . Data information : each lane corresponds to a sample from one mouse . Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels . Data information : each lane corresponds to a sample from one mouse . Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels . Data information : each lane corresponds to a sample from one mouse . QSOX1 immunofluorescence ( green ) in control ( siControl ) and QSOX1 knockdown ( siQSOX1 ) MDA - MB - 231 cells . Blue is DAPI staining of nuclei . Scale bar is 20 µm . Data information : The results described in are representative of 3 experiments that were performed . Western blot analysis of the MUC2 N - terminal region and indicated cysteine mutants in supernatants of transfected cell cultures . No differences in disulfide - mediated dimerization were observed for any MUC2 variant between siC ( siControl ) and siQ ( siQSOX1 ) . Data information : The results described in G are representative of 3 experiments that were performed .

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

Figure 3Representative TEM micrographs of about a hundred collected from 3 mice of each genotype show large and well-packed goblet cell granules in WT and QSOX1 KO colons. Scale bar is 5 µm.Alcian blue and PAS staining of reduced guanidine-insoluble mucins from WT and QSOX1 KO mice, separated on a 6% polyacrylamide gel. Data information: each lane corresponds to a sample from one mouse.Muc5b fluorescent immunoblot of WT and QSOX1 KO lung lavage samples separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.Western blot analysis of blood Vwf from WT and QSOX1 KO mice separated on agarose gels. Data information: each lane corresponds to a sample from one mouse.QSOX1 immunofluorescence (green) in control (siControl) and QSOX1 knockdown (siQSOX1) MDA-MB-231 cells. Blue is DAPI staining of nuclei. Scale bar is 20 µm. Data information: The results described in are representative of 3 experiments that were performed.Western blot analysis of the MUC2 N-terminal region and indicated cysteine mutants in supernatants of transfected cell cultures. No differences in disulfide-mediated dimerization were observed for any MUC2 variant between siC (siControl) and siQ (siQSOX1). Data information: The results described in G are representative of 3 experiments that were performed.


Figure 4Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice . Lysates were treated with PEG - mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1 . As for panel A but using antibodies to St3gal1 . As for panel A but using antibodies to B3galt5 . Data information : For the experiments in panels A - C , equal amounts of total protein were applied to each gel lane , and no reducing agent was added . Experiments were repeated 3 times with fresh new lysates , and representative blots are shown . QSOX1 oxidizes St6gal1 in vitro . MAb492 . 1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG - mal additions ( 2PEG ) , whereas protein with two pairs of free cysteines acquires four PEG - mal additions ( 4PEG ) . The change in migration per two PEG - mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs . protein QSOX1 oxidation of St6gal1 mutants . The C350 - C361 disulfide ( the redox - active disulfide present in the C139A - C403A mutant ) is oxidized rapidly , while the C142 - C406 disulfide ( present in the C350A - C361A mutant ) is oxidized slowly . Data information : Experiments were repeated 3 times with fresh new lysates , and representative blots are shown .

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

Figure 4Western blot analysis of colon epithelial cell lysates from WT and QSOX1 KO mice. Lysates were treated with PEG-mal 2 kDa or NEM as indicated above each blot and probed using antibodies to St6gal1. As for panel A but using antibodies to St3gal1. As for panel A but using antibodies to B3galt5. Data information: For the experiments in panels A-C, equal amounts of total protein were applied to each gel lane, and no reducing agent was added. Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.QSOX1 oxidizes St6gal1 in vitro. MAb492.1 is a monoclonal antibody that inhibits human QSOX1 St6gal1 with one pair of free cysteines is modified by two PEG-mal additions (2PEG), whereas protein with two pairs of free cysteines acquires four PEG-mal additions (4PEG). The change in migration per two PEG-mal modifications appears greater than 4 kD as expected due to the differences in hydrodynamic properties and SDS binding of PEG vs. protein QSOX1 oxidation of St6gal1 mutants. The C350-C361 disulfide (the redox-active disulfide present in the C139A-C403A mutant) is oxidized rapidly, while the C142-C406 disulfide (present in the C350A-C361A mutant) is oxidized slowly. Data information: Experiments were repeated 3 times with fresh new lysates, and representative blots are shown.


Figure 1 ( A ) Kinetics of cell numbers in Lineage - EPCR + CD150 + CD48 - ( L - ESLAM ) HSCs , Lineage - EPCR + CD150 + CD48 + ( CD150 + CD48 + ) progenitor cells , and Lineage - EPCR + CD150 - CD48 + ( CD150 - CD48 + ) progenitor cells after the administration of 5 - FU ( 250 mg / kg , i . v . ) . The small graph is an enlargement of 0 to 8 days after the administration of 5 - FU . ( * or * * ) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days , respectively .

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Figure 1(A) Kinetics of cell numbers in Lineage-EPCR+CD150+CD48- (L-ESLAM) HSCs, Lineage-EPCR+CD150+CD48+ (CD150+CD48+) progenitor cells, and Lineage-EPCR+CD150-CD48+ (CD150-CD48+) progenitor cells after the administration of 5-FU (250 mg/kg, i.v.). The small graph is an enlargement of 0 to 8 days after the administration of 5-FU. (* or **) in the small graph represents the statistical analysis of HSC between 3 and 4 or 7 days, respectively.


Figure 3 ( A ) A principal component analysis ( PCA ) using the ATAC - seq data of HSCs in the steady state ( untreated ) , early phase ( 5 - FU 6d ) , or late phase ( 5 - FU 10d ) .

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Figure 3(A) A principal component analysis (PCA) using the ATAC-seq data of HSCs in the steady state (untreated), early phase (5-FU 6d), or late phase (5-FU 10d).


Figure 4mRNA ( C ) in Acly in HSCs after the administration of 5 - FU . protein ( D ) , and phosphorylation levels of Ser455 ( E ) in Acly in HSCs after the administration of 5 - FU . The levels of intracellular Glu ( G ) in HSCs obtained from untreated or 5 - FU - treated mice . total acetylated lysine ( I ) in HSCs obtained from untreated or 5 - FU - treated mice .

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Figure 4mRNA (C) in Acly in HSCs after the administration of 5-FU.protein (D), and phosphorylation levels of Ser455 (E) in Acly in HSCs after the administration of 5-FU.The levels of intracellular Glu (G) in HSCs obtained from untreated or 5-FU-treated mice.total acetylated lysine (I) in HSCs obtained from untreated or 5-FU-treated mice.


Figure 5 ( B - C ) The effects of CTPI - 2 ( B ) or BMS303141 ( BMS ) ( C ) on H3K27ac in HSCs in the early phase after the administration of 5 - FU . ( D - E ) The effects of CTPI - 2 ( D ) , BMS303141 ( BMS ) ( E ) , A - 485 ( D ) , and TV - 3664 ( E ) on HSC expansion in the early phase after the administration of 5 - FU . Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells .

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Figure 5(B-C) The effects of CTPI-2 (B) or BMS303141 (BMS) (C) on H3K27ac in HSCs in the early phase after the administration of 5-FU.(D-E) The effects of CTPI-2 (D), BMS303141 (BMS) (E), A-485 (D), and TV-3664 (E) on HSC expansion in the early phase after the administration of 5-FU. Numbers in the dot plots represent the frequencies of the indicated fractions within total BM cells.


Figure 7Global H3K27ac ( A ) , Acly levels ( B ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU . acetylated lysine ( C ) , and ΔΨm ( D ) in CD48 - HSCs and CD48 + progenitor cells ( HPCs ) in the late phase after the administration of 5 - FU .

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Figure 7Global H3K27ac (A), Acly levels (B) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.acetylated lysine (C), and ΔΨm (D) in CD48- HSCs and CD48+ progenitor cells (HPCs)in the late phase after the administration of 5-FU.


Figure 8 ( C ) The frequencies of the CD150 + CD48 - and CD48 + fractions within the lineage - EPCR + fraction after a 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ; 100 μM ) , SB204990 ( SB ; 100 μM ) , an alternative Acly inhibitor , or CTPI - 2 ( 50 μM ) . ( D and E ) Total cell number ( D ) or CD48 + cell number ( E ) after the 4 - day culture of HSCs in the late phase with or without BMS303141 ( BMS ) , SB204990 ( SB ) , or CTPI - 2 . Numbers within the graph represent the inhibitory effects of each treatment ( fold decrease by an inhibitor treatment ) .

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Figure 8(C) The frequencies of the CD150+CD48- and CD48+ fractions within the lineage-EPCR+ fraction after a 4-day culture of HSCs in the late phase with or without BMS303141 (BMS; 100 μM), SB204990 (SB; 100 μM), an alternative Acly inhibitor, or CTPI-2 (50 μM). (D and E) Total cell number (D) or CD48+ cell number (E) after the 4-day culture of HSCs in the late phase with or without BMS303141 (BMS), SB204990 (SB), or CTPI-2. Numbers within the graph represent the inhibitory effects of each treatment (fold decrease by an inhibitor treatment).


Figure 1B , C . Cryosections of samples from patients with prostate carcinoma and SCC of the lung . Samples were processed as described in the methods section . Ve = vessel , Tu = tumor . Scale 50µm . See also Fig 1D for comparative semi - quantitative analysis of a panel of samples from patients with prostate carcinoma , lung SCC and non - lung SCC . D . Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities . For definition of the H - score reflecting binding intensity , refer to the methods section . Statistical analysis was performed using the paired , non - parametric Wilcoxon test . Sample size ( n ) and the corresponding P - values are indicated in the graphs .

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Figure 1B, C. Cryosections of samples from patients with prostate carcinoma and SCC of the lung. Samples were processed as described in the methods section. Ve=vessel, Tu=tumor. Scale 50µm. See also Fig 1D for comparative semi-quantitative analysis of a panel of samples from patients with prostate carcinoma, lung SCC and non-lung SCC.D. Semi quantitative analysis of binding of the PSMA antibodies 10B3 and J591 to cryosections from different tumor entities. For definition of the H-score reflecting binding intensity, refer to the methods section. Statistical analysis was performed using the paired, non-parametric Wilcoxon test. Sample size (n) and the corresponding P-values are indicated in the graphs.


Figure 2E , F . Binding of the Fabsc - and IgGsc - molecules to PSMA - expressing LNCaP ( E ) or 22Rv1low ( F ) cells was assessed by flow cytometry , as described in the methods section . The EC50 values were calculated using GraphPad Prism software and are presented below the figure . G - I . Binding of the Fabsc - and IgGsc - molecules to CD3 - expressing Jurkat - cells ( G ) and to CD4 T cells ( H ) and CD8 T cells ( I ) in PBMC preparations was assessed by flow cytometry . The results represent the average of three independent experiments performed with different PBMC donors . EC50 values are presented in the table below the figure .

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Figure 2E, F. Binding of the Fabsc- and IgGsc-molecules to PSMA-expressing LNCaP (E) or 22Rv1low (F) cells was assessed by flow cytometry, as described in the methods section. The EC50 values were calculated using GraphPad Prism software and are presented below the figure.G-I. Binding of the Fabsc- and IgGsc-molecules to CD3-expressing Jurkat-cells (G) and to CD4 T cells (H) and CD8 T cells (I) in PBMC preparations was assessed by flow cytometry. The results represent the average of three independent experiments performed with different PBMC donors. EC50 values are presented in the table below the figure.


Figure 3BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP - expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements are indicated . BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low - cells expressing high and low amounts of PSMA , respectively . After three days , cytokine release were determined by flow cytometry as described in the methods section . Mean values and standard deviations of triplicate measurements are indicated .

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Figure 3BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with LNCaP- expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.BsAb were incubated at the indicated concentrations with PBMC of healthy donors together with 22Rv1low-cells expressing high and low amounts of PSMA, respectively. After three days, cytokine release were determined by flow cytometry as described in the methods section. Mean values and standard deviations of triplicate measurements are indicated.


Figure 4B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs , and after three days cytokine release ( B ) were measured using a 3H - thymidine uptake - and a Legendplex assay , respectively . Mean values and standard deviations from triplicate samples .

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Figure 4B PBMC of a healthy donor were incubated with the indicated concentrations of bsAbs, and after three days cytokine release (B) were measured using a 3H-thymidine uptake- and a Legendplex assay, respectively. Mean values and standard deviations from triplicate samples.


Figure 5C - F . 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors ( 5 animals per group ) , and uptake at the indicated locations was determined by PET quantification over time ( C , D ) and at termination of the experiment after 48 hours ( E , F ) as described in the methods section . Error bars represent standard deviation of the mean value .

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Figure 5C-F. 64Cu labeled bsAbs were injected into NSG mice carrying 22Rv1high tumors (5 animals per group), and uptake at the indicated locations was determined by PET quantification over time (C, D) and at termination of the experiment after 48 hours (E, F) as described in the methods section. Error bars represent standard deviation of the mean value.


Figure 6B , C . 106 LNCaP cells were injected s . c . into the right flank of NSG mice ( 5 per group ) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm . BsAbs ( 2µg per dose ) were applied at day 1 , 8 and 15 ( IgGsc ) and daily at day 1 - 3 , day 8 - 10 and day 15 - 17 ( Fabsc ) , PBMC were applied at day 1 , 8 and 15 . Mean values and standard deviations of five mice per group are indicated .

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Figure 6B, C. 106 LNCaP cells were injected s.c. into the right flank of NSG mice (5 per group) and treatment with PBMC and bsAbs was started when tumors had reached a diameter of 5mm. BsAbs (2µg per dose) were applied at day 1, 8 and 15 (IgGsc) and daily at day 1-3, day 8-10 and day 15-17 (Fabsc), PBMC were applied at day 1,8 and 15. Mean values and standard deviations of five mice per group are indicated.


Figure 7The IgGsc - molecule was applied in escalating doses , with a starting dose of 20µg ( patient 1 ) or 40µg ( patients 2 and 3 ) to a maximum of approx . 2 . 6mg , as indicated . PSA values were monitored and T - cell activation was assessed by flow cytometry .

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Figure 7The IgGsc-molecule was applied in escalating doses, with a starting dose of 20µg (patient 1) or 40µg (patients 2 and 3) to a maximum of approx. 2.6mg, as indicated. PSA values were monitored and T-cell activation was assessed by flow cytometry.


Figure 1 ( E ) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 ( LxxAxA ) . Binding of YFP - B56α to indicated proteins was determined and quantified by LiCor . ( G ) YFP - B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins . Representative of 4 independent experiments .

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Figure 1(E) Peptide competition assay with a WT LxxIxE peptide or a mutated variant that does not bind B56 (LxxAxA). Binding of YFP-B56α to indicated proteins was determined and quantified by LiCor.(G) YFP-B56α pull down from cells stably expressing the indicated LxxIxE binding pocket variants of B56α and subsequent immunoblotting of indicated proteins. Representative of 4 independent experiments.


Figure 2 ( B ) IP of YFP - B56α from cells stably expressing the B56α WT , R222E , and 5A followed by immunoblotting of indicated proteins . Representative blots are shown ( top ) . hSgo1 signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) . ( C ) Reciprocal IP of ( B ) . YFP - hSgo1 expression construct was transfected into cells stably expressing FLAG - B56α WT , R222E and 5A , followed by YFP IP and immunoblotting of indicated proteins . Representative blots are shown ( top ) . B56α signals were normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 ) . ( D ) Representative images of chromosome spreads from the indicated conditions . Scale bar , 5μm . ( E ) Quantification of ( D ) . The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 4 independent experiments and the mean and SD are indicated . ( F ) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere - targeting domain ( CB ) . Representative images of chromosome spreads are shown . CB targets all the rescue constructs ( green ) to the centromere . Scale bar , 5μm . ( G ) Quantification of ( F ) . The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted . The data are from 3 independent experiments and the mean and SD are indicated .

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

Figure 2(B) IP of YFP-B56α from cells stably expressing the B56α WT, R222E, and 5A followed by immunoblotting of indicated proteins. Representative blots are shown (top). hSgo1 signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).(C) Reciprocal IP of (B). YFP-hSgo1 expression construct was transfected into cells stably expressing FLAG-B56α WT, R222E and 5A, followed by YFP IP and immunoblotting of indicated proteins. Representative blots are shown (top). B56α signals were normalized to YFP and plotted (bottom). Error bars represent SD (n=4).(D) Representative images of chromosome spreads from the indicated conditions. Scale bar, 5μm. (E) Quantification of (D). The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 4 independent experiments and the mean and SD are indicated.(F) hSgo1 RNAi and rescue with the indicated B56α variants fused to YFP and the Cenp B centromere-targeting domain (CB). Representative images of chromosome spreads are shown. CB targets all the rescue constructs (green) to the centromere. Scale bar, 5μm. (G) Quantification of (F). The distance between the two peak intensities of YFP was measured for 5 kinetochore pairs and averaged for a single cell and plotted. The data are from 3 independent experiments and the mean and SD are indicated.


Figure 3 ( D ) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP - hSgo1 variants . Representative immunofluorescent images are shown . Scale bar , 5μm . ( E ) B56α signal intensity was quantified . B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP - hSgo1 signal . Each circle represents an individual cell , and the average and SD are indicated . ( F ) Mitotic U2OS LacO Haspin CM cells expressing hSgo1 - LacI - GFP variants or LacI - GFP ( control ) were stained for PP2A - C . Scale bar , 5μm . ( G ) PP2A - C signal intensity was quantified , normalized to GFP , and then plotted . Each circle represents an individual cell , and the mean fluorescent intensity is indicated . Representative of at least 3 independent experiments .

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Figure 3(D) Localization of B56α in cells depleted of hSgo1 and expressing the indicated YFP-hSgo1 variants. Representative immunofluorescent images are shown. Scale bar, 5μm. (E) B56α signal intensity was quantified. B56α signal from each cell was determined from 5 kinetochore pairs and normalized to YFP-hSgo1 signal. Each circle represents an individual cell, and the average and SD are indicated.(F) Mitotic U2OS LacO Haspin CM cells expressing hSgo1-LacI-GFP variants or LacI-GFP (control) were stained for PP2A-C. Scale bar, 5μm. (G) PP2A-C signal intensity was quantified, normalized to GFP, and then plotted. Each circle represents an individual cell, and the mean fluorescent intensity is indicated. Representative of at least 3 independent experiments.


Figure 4 ( A ) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP - hSgo1 variants . All the YFP - hSgo1 rescue constructs ( green ) localize to the centromeres ( CREST , red ) . Scale bar , 5μm . ( B ) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in ( A ) and averaged for a single cell . The data are from 3 independent experiments and the mean and SD are indicated . ( C ) IP of YFP - hSgo1 from cells stably expressing hSgo1 WT , 3A , and 4A followed by immunoblotting of cohesin components , Smc1 and Smc3 . Representative blots are shown ( top ) . Signals were quantification by LiCor , normalized to YFP and plotted ( bottom ) . Error bars represent SD ( n = 4 for Smc1 and n = 3 for Smc3 ) . ( F ) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi - resistant constructs was performed . Representative still images captured during the live cell imaging showing DIC and YFP - hSgo1 WT , 3A , and 4A localization during mitosis . Time ( min ) from nuclear envelop breakdown ( NEBD ) is indicated . Scale bar , 15μm .

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Figure 4(A) Representative images of chromosome spreads from hSgo1 RNAi treated cells stably expressing the indicated YFP-hSgo1 variants. All the YFP-hSgo1 rescue constructs (green) localize to the centromeres (CREST, red). Scale bar, 5μm. (B) The distance between the two peak intensities of CREST was measured for 5 kinetochore pairs from the chromosome spreads in (A) and averaged for a single cell. The data are from 3 independent experiments and the mean and SD are indicated.(C) IP of YFP-hSgo1 from cells stably expressing hSgo1 WT, 3A, and 4A followed by immunoblotting of cohesin components, Smc1 and Smc3. Representative blots are shown (top). Signals were quantification by LiCor, normalized to YFP and plotted (bottom). Error bars represent SD (n=4 for Smc1 and n=3 for Smc3).(F) hSgo1 RNAi and rescue with the indicated hSgo1 RNAi-resistant constructs was performed. Representative still images captured during the live cell imaging showing DIC and YFP-hSgo1 WT, 3A, and 4A localization during mitosis. Time (min) from nuclear envelop breakdown (NEBD) is indicated. Scale bar, 15μm.


Figure 1 ( A ) LC - MS / MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA . Signal intensities of the modifications are scaled to that for m1A , the ribonucleoside with the highest MS

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Figure 1(A) LC-MS/MS extracted ion chromatograms of 28 modified ribonucleosides analyzed in parasite tRNA. Signal intensities of the modifications are scaled to that for m1A, the ribonucleoside with the highest MS signal


Figure 2 ( A ) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC - MS / MS in total tRNA extracted from parasites across the IDC time course . The average fold - change values ( relative to an arbitrary average control ) were subjected to hierarchical clustering analysis ( log2 - transformed data ) . The scale of the heat map has saturated the fold - change values of some modifications . Refer to Appendix Table S6 . ( B ) Three groups of modifications show differential regulation during the course of IDC . The majority ( 22 ) increase synchronously across the IDC ( blue ) , while 4 show asynchronous or irregular behavior ( purple ) and 2 ( orange ) are uniquely up - regulated in the ring stage . Log2Fold - change values plotted in the three graphs represent mean ± SEM ( N = 3 ) . Differences between the highest value ( denoted as peak ) and the lowest value ( denoted as trough ) were subjected to a two - tailed Student ' s t - test : NS , not

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Figure 2(A) Changes in the relative quantities of individual modified ribonucleosides were quantified by LC-MS/MS in total tRNA extracted from parasites across the IDC time course. The average fold-change values (relative to an arbitrary average control) were subjected to hierarchical clustering analysis (log2-transformed data). The scale of the heat map has saturated the fold-change values of some modifications. Refer to Appendix Table S6. (B) Three groups of modifications show differential regulation during the course of IDC. The majority (22) increase synchronously across the IDC (blue), while 4 show asynchronous or irregular behavior (purple) and 2 (orange) are uniquely up-regulated in the ring stage. Log2Fold-change values plotted in the three graphs represent mean ± SEM (N = 3). Differences between the highest value (denoted as peak) and the lowest value (denoted as trough) were subjected to a two-tailed Student's t-test: NS, not significant


Figure 3iTRAQ proteomics analysis ( Appendix Fig . S2 ) resulted in 2 , 100 proteins consistently quantified at every point across the IDC . The heat map shows hierarchical clustering analysis of fold - changes ( log2 - transformed ) in the levels of the proteins relative to an arbitrary average across the IDC . The 100 most up - regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P . falciparum 3D7 . The significantly ( P < 0 . 05 ) enriched pathways among the up - regulated proteins at each time point are summarized in tables for each time

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Figure 3iTRAQ proteomics analysis (Appendix Fig. S2) resulted in 2,100 proteins consistently quantified at every point across the IDC. The heat map shows hierarchical clustering analysis of fold-changes (log2-transformed) in the levels of the proteins relative to an arbitrary average across the IDC. The 100 most up-regulated proteins at each time point were subjected to pathway enrichment analysis based on GO and MPM pathways annotated for P. falciparum 3D7. The significantly (P < 0.05) enriched pathways among the up-regulated proteins at each time point are summarized in tables for each time point


Figure 5LC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of the unmodified ( 3055 . 425 Da ) and modified ( 3143 . 43 Da ) oligos for Glu - tRNAGAA ( BCollision - induced dissociation of the parent molecular ions for m / z 1046 . 477 - 3 from Glu - tRNAGA produced expected - c , - w and - y ions for the sequences CU [ mcm5s2U ] UCACCCLC - MS / MS analysis was performed on T1 - digested tRNA and deconvolution yielded precise masses of th unmodified ( 1835 . 20 Da ) and modified ( 1923 . 262 Da ) oligos for Gln - tRNACAA ( E ) Collision - induced dissociation of the parent molecular ions fo m / z 1923 . 262 from Gln - tRNACAA produced expected - c , - w and - y ions for the sequence CC [ mcm5s2U ] CCAARatios of modified oligonucleotides from the anticodon stem - loop o ( H ) GluGA isoacceptors expressed as percentages of their sum totalRatios of modified oligonucleotides from the anticodon stem - loop o ( G ) GlnCARatios of modified oligonucleotides from the anticodon stem - loop o ( I ) GlyGGA isoacceptors expressed as percentages of their sum

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Figure 5LC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of the unmodified (3055.425 Da) and modified (3143.43 Da) oligos for Glu-tRNAGAA (BCollision-induced dissociation of the parent molecular ions for m/z 1046.477-3 from Glu-tRNAGA produced expected -c, -w and -y ions for the sequences CU[mcm5s2U]UCACCCLC-MS/MS analysis was performed on T1-digested tRNA and deconvolution yielded precise masses of th unmodified (1835.20 Da) and modified (1923.262 Da) oligos for Gln-tRNACAA (E)Collision-induced dissociation of the parent molecular ions fo m/z 1923.262 from Gln-tRNACAA produced expected -c, -w and -y ions for the sequence CC[mcm5s2U]CCAARatios of modified oligonucleotides from the anticodon stem-loop o (H)GluGA isoacceptors expressed as percentages of their sum totalRatios of modified oligonucleotides from the anticodon stem-loop o (G)GlnCARatios of modified oligonucleotides from the anticodon stem-loop o (I)GlyGGA isoacceptors expressed as percentages of their sum total


Figure 6 ( A , B ) Comparison of arbitrary translation efficiency ( TE ) values of ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) proteins across all experimental time points . The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system . The top 5 % up - regulated proteins ( n = 100 ) at ( A ) late trophozoite stage ( TP 40 ) and ( B ) schizont stage ( TP 46 ) were used for this analysis . Differences among all groups were first tested by one - way ANOVA analysis , followed by Dunnett ' s test as multiple comparison post - test to find significant differences between pairs of ( A ) TP 40 or ( B ) TP 46 and other time points . P < 0 . 01 is denoted as * * . P < 0 . 0001 is denoted as * * * * . Scatterplots of mRNA and protein fold - changes ( log - transformed ) at time points ( C ) 40 h and ( D ) 46 h in the erythrocytic cycle . Quadrants are designated with roman numerals . Data points falling into quadrant I are designated as high translationally efficient ( HTE ) proteins while those falling into quadrant IV are designated low translationally efficient ( LTE )

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Figure 6(A, B) Comparison of arbitrary translation efficiency (TE) values of (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) proteins across all experimental time points. The arbitrary TE is defined here as the normalized protein abundance per mRNA abundance to mimic the translational output per mRNA copy in a biological system. The top 5% up-regulated proteins (n = 100) at (A) late trophozoite stage (TP 40) and (B) schizont stage (TP 46) were used for this analysis. Differences among all groups were first tested by one-way ANOVA analysis, followed by Dunnett's test as multiple comparison post-test to find significant differences between pairs of (A) TP 40 or (B) TP 46 and other time points. P<0.01 is denoted as **. P<0.0001 is denoted as ****. Scatterplots of mRNA and protein fold-changes (log-transformed) at time points (C) 40 h and (D) 46 h in the erythrocytic cycle. Quadrants are designated with roman numerals. Data points falling into quadrant I are designated as high translationally efficient (HTE) proteins while those falling into quadrant IV are designated low translationally efficient (LTE) proteins


Figure 1Chromatin - enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2 / 3 antibody . except that cells were preextracted in 0 . 2 % Triton X - 100 , fixed and immunostained with SUMO2 / 3 antibody . Representative images are shown . Scale bar , 5 μm . Relative DPC levels in cells treated were quantified using a KCl / SDS precipitation assay ( mean ± SD ; n = 3 independent experiments ) . HeLa cells were treated with formaldehyde for 1 h , and where indicated propagated for an additional h in the absence of formaldehyde ( recovery ) . Cells were then fractionated into soluble and chromatin - enriched fractions and immunoblotted with SUMO2 / 3 and SUMO1 antibodies . using HeLa cells transfected with non - targeting control ( CTRL ) or SPRTN siRNAs . Mass spectrometry - based analysis of formaldehyde - induced SUMOylation changes . His10 - SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to formaldehyde were purified on Ni - NTA under stringent conditions and analyzed by mass spectrometry . All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database , at the default interaction confidence setting of 0 . 4 . Proteins not connected to the network were omitted . SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies .

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Figure 1Chromatin-enriched fractions of HeLa cells exposed to the indicated genotoxic agents for 1 h were subjected to immunoblot analysis using SUMO2/3 antibody.except that cells were preextracted in 0.2% Triton X-100, fixed and immunostained with SUMO2/3 antibody. Representative images are shown. Scale bar, 5 μm.Relative DPC levels in cells treated were quantified using a KCl/SDS precipitation assay (mean±SD; n=3 independent experiments).HeLa cells were treated with formaldehyde for 1 h, and where indicated propagated for an additional h in the absence of formaldehyde (recovery). Cells were then fractionated into soluble and chromatin-enriched fractions and immunoblotted with SUMO2/3 and SUMO1 antibodies.using HeLa cells transfected with non-targeting control (CTRL) or SPRTN siRNAs.Mass spectrometry-based analysis of formaldehyde-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. All proteins displaying significant upregulation of SUMOylation in response to formaldehyde treatment were subjected to network analysis using the STRING database, at the default interaction confidence setting of 0.4. Proteins not connected to the network were omitted.SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to formaldehyde or heat stress for 1 h were purified and immunoblotted with indicated antibodies.


Figure 2HeLa cells were transfected with non - targeting control ( CTRL ) or DNMT1 siRNAs , exposed or not to 5 - azadC and collected 2 h later . Chromatin - enriched fractions were immunoblotted with antibodies to SUMO2 / 3 , DNMT1 and MCM6 ( loading control ) . Representative images of HeLa cells that were treated and co - immunostained with antibodies to DNMT1 , SUMO2 / 3 and PCNA . Scale bar , 10 μm . Proliferative capacity of HeLa / GFP - DNMT1 cells transfected with indicated siRNAs and exposed to the indicated 5 - azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) . Extracts of HeLa / GFP - DNMT1 cells treated with 5 - azadC for the indicated times were subjected to GFP immunoprecipitation ( IP ) under denaturing conditions followed by immunoblotting with SUMO2 / 3 and GFP antibodies . HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive ( CI ) GFP - DNMT1 . Cells were then left untreated or incubated with 5 - azadC for 2 h , and SUMOylation of GFP - DNMT1 was analyzed Asterisk denotes unmodified GFP - DNMT1 recognized by the SUMO2 / 3 antibody due to weak cross - reactivity . Mass spectrometry analysis of 5 - azadC - induced SUMOylation changes . His10 - SUMO2 conjugates from HeLa / His10 - SUMO2 cells subjected or not to 5 - azadC were purified on Ni - NTA under stringent conditions and analyzed by mass spectrometry . Proteins displaying significantly altered SUMOylation in response to 5 - azadC treatment relative to the control condition were visualized through two - sample t - testing using permutation - based FDR to achieve q - values of < 0 . 05SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown . Both control ( CTRL ) and DNMT1 siRNA - transfected cells were treated with 5 - azadC .

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Figure 2HeLa cells were transfected with non-targeting control (CTRL) or DNMT1 siRNAs, exposed or not to 5-azadC and collected 2 h later. Chromatin-enriched fractions were immunoblotted with antibodies to SUMO2/3, DNMT1 and MCM6 (loading control).Representative images of HeLa cells that were treated and co-immunostained with antibodies to DNMT1, SUMO2/3 and PCNA. Scale bar, 10 μm.Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with indicated siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).Extracts of HeLa/GFP-DNMT1 cells treated with 5-azadC for the indicated times were subjected to GFP immunoprecipitation (IP) under denaturing conditions followed by immunoblotting with SUMO2/3 and GFP antibodies.HeLa cells were subjected to consecutive rounds of transfection with DNMT1 siRNA targeting the UTR and expression plasmid encoding WT or catalytically inactive (CI) GFP-DNMT1. Cells were then left untreated or incubated with 5-azadC for 2 h, and SUMOylation of GFP-DNMT1 was analyzed Asterisk denotes unmodified GFP-DNMT1 recognized by the SUMO2/3 antibody due to weak cross-reactivity.Mass spectrometry analysis of 5-azadC-induced SUMOylation changes. His10-SUMO2 conjugates from HeLa/His10-SUMO2 cells subjected or not to 5-azadC were purified on Ni-NTA under stringent conditions and analyzed by mass spectrometry. Proteins displaying significantly altered SUMOylation in response to 5-azadC treatment relative to the control condition were visualized through two-sample t-testing using permutation-based FDR to achieve q-values of <0.05SUMO target proteins displaying altered SUMOylation upon DNMT1 knockdown are shown. Both control (CTRL) and DNMT1 siRNA-transfected cells were treated with 5-azadC.


Figure 3Chromatin - enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5 - azadC for 2 h were analyzed by immunoblotting with SUMO2 / 3 and Actin antibodies . HeLa cells stably expressing GFP - DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5 - azadC for 2 h . Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal . Mean detergent - resistant GFP signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown . Proliferative capacity of HeLa / GFP - DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5 - azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay ( mean ± SEM ; n = 3 independent experiments ; * * * p < 0 . 001 , Student ' s t - test ) . untransfected HeLa cells were exposed or not to 5 - azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor ( SUMO - E1i ) . HeLa cells treated with 5 - azadC for 2 h in the presence or absence of SUMO - E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5 - azadC removal , and immunostained with DNMT1 antibody . Mean detergent - resistant DNMT1 signal was determined by quantitative image analysis ( > 6000 cells analyzed per condition ) . Data from a representative experiment are shown . Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5 - azadC doses for 2 h was assayediPOND analysis of 5 - azadC - induced DNMT1 trapping and SUMOylation . HeLa cells were mock - treated or pre - incubated with 5 - azadC for 5 min before addition of EdU for 10 min . Cells were then washed and incubated with thymidine for 0 ( pulse ) or 60 min ( chase ) before performing iPOND . HeLa cells stably expressing GFP - DNMT1 were pre - incubated or not with SUMO - E1i for 30 min , and where indicated 5 - azadC was added to the medium for an additional 60 min . Cells were then processed for GFP immunoprecipitation ( IP ) followed by immunoblotting for the indicated proteins . Immunoblot analysis of chromatin - enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block , pulse - labeled with 5 - azadC for 30 min and grown in the presence or absence of proteasome inhibitor ( MG132 ) for the indicated times . cells were treated or not with aphidicolin following pulse - labeling with 5 - azadC .

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Figure 3Chromatin-enriched fractions of HeLa cells transfected with indicated siRNAs and subsequently exposed or not to 5-azadC for 2 h were analyzed by immunoblotting with SUMO2/3 and Actin antibodies.HeLa cells stably expressing GFP-DNMT1 and transfected with indicated siRNAs were left untreated or exposed to 5-azadC for 2 h. Cells were then preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal. Mean detergent-resistant GFP signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.Proliferative capacity of HeLa/GFP-DNMT1 cells transfected with control or UBC9 siRNAs and exposed to the indicated 5-azadC doses for 2 h was assayed by measuring cell proliferation with the SRB assay (mean±SEM; n=3 independent experiments; ***p<0.001, Student's t-test).untransfected HeLa cells were exposed or not to 5-azadC in the presence or absence of a small molecule SUMO E1 enzyme inhibitor (SUMO-E1i).HeLa cells treated with 5-azadC for 2 h in the presence or absence of SUMO-E1i were preextracted in stringent preextraction buffer and fixed at the indicated time points after 5-azadC removal, and immunostained with DNMT1 antibody. Mean detergent-resistant DNMT1 signal was determined by quantitative image analysis (>6000 cells analyzed per condition). Data from a representative experiment are shown.Proliferative capacity of HeLa cells transfected with SPRTN or ZNF451 siRNAs and exposed to indicated 5-azadC doses for 2 h was assayediPOND analysis of 5-azadC-induced DNMT1 trapping and SUMOylation. HeLa cells were mock-treated or pre-incubated with 5-azadC for 5 min before addition of EdU for 10 min. Cells were then washed and incubated with thymidine for 0 (pulse) or 60 min (chase) before performing iPOND.HeLa cells stably expressing GFP-DNMT1 were pre-incubated or not with SUMO-E1i for 30 min, and where indicated 5-azadC was added to the medium for an additional 60 min. Cells were then processed for GFP immunoprecipitation (IP) followed by immunoblotting for the indicated proteins.Immunoblot analysis of chromatin-enriched fractions of HeLa cells that were synchronized in early S phase by double thymidine block, pulse-labeled with 5-azadC for 30 min and grown in the presence or absence of proteasome inhibitor (MG132) for the indicated times.cells were treated or not with aphidicolin following pulse-labeling with 5-azadC.


Figure 4Representative images of U2OS cells stably expressing GFP - SPRTN or GFP - ACRC that were transfected with control ( CTRL ) or UBC9 siRNAs , exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme ( UBA1 ) inhibitor TAK - 243 ( Ub - E1i ) as indicated and fixed one h later . Scale bar , 10 μm . Quantification of data showing proportion of U2OS / GFP - SPRTN cells displaying nuclear GFP - SPRTN foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) . As in ( C ) , but showing proportion of U2OS / GFP - ACRC cells displaying nuclear GFP - ACRC foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) . U2OS cells expressing GFP - ACRC were exposed to the indicated genotoxic agents ( formaldehyde : 600 μM , 1 h ; UV : 20 J / m2 , 6 h recovery ; hydroxyurea ( HU ) : 2mM , 24 h ; mitomycin C ( MMC ) : 40 ng / mL , 24 h ) , preextracted and fixed , and analyzed by microscopy . Representative images are shown . Scale bar , 10 μm . HeLa cells transfected with plasmids encoding GFP alone or indicated GFP - ACRC alleles were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO22 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies . Representative images of U2OS cells expressing indicated GFP - ACRC alleles that were treated with 5 - azadC in the presence or absence of SUMO inhibitor ( SUMO - E1i ) , fixed 2 h later and immunostained with DNMT1 antibody . Scale bar , 10 μm . Quantification of data in ( G ) , showing proportion of cells displaying GFP - ACRC co - localization with DNMT1 foci ( mean ± SEM ; at least 100 cells quantified per condition per experiment ; n = 3 independent experiments ) .

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Figure 4Representative images of U2OS cells stably expressing GFP-SPRTN or GFP-ACRC that were transfected with control (CTRL) or UBC9 siRNAs, exposed to formaldehyde in the presence or absence of ubiquitin E1 enzyme (UBA1) inhibitor TAK-243 (Ub-E1i) as indicated and fixed one h later. Scale bar, 10 μm.Quantification of data showing proportion of U2OS/GFP-SPRTN cells displaying nuclear GFP-SPRTN foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments). As in (C), but showing proportion of U2OS/GFP-ACRC cells displaying nuclear GFP-ACRC foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).U2OS cells expressing GFP-ACRC were exposed to the indicated genotoxic agents (formaldehyde: 600 μM, 1 h; UV: 20 J/m2, 6 h recovery; hydroxyurea (HU): 2mM, 24 h; mitomycin C (MMC): 40 ng/mL, 24 h), preextracted and fixed, and analyzed by microscopy. Representative images are shown. Scale bar, 10 μm.HeLa cells transfected with plasmids encoding GFP alone or indicated GFP-ACRC alleles were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO22-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.Representative images of U2OS cells expressing indicated GFP-ACRC alleles that were treated with 5-azadC in the presence or absence of SUMO inhibitor (SUMO-E1i), fixed 2 h later and immunostained with DNMT1 antibody. Scale bar, 10 μm.   Quantification of data in (G), showing proportion of cells displaying GFP-ACRC co-localization with DNMT1 foci (mean±SEM; at least 100 cells quantified per condition per experiment; n=3 independent experiments).


Figure 5HeLa cells transfected with plasmids encoding GFP alone , GFP - ACRC or GFP - tagged C . elegans GCNA - 1 were subjected to GFP IP under denaturing conditions . Beads were incubated with recombinant polySUMO23 - 8 chains , washed extensively and processed for immunoblotting with SUMO2 / 3 and GFP antibodies . GFP expression driven by the gcna - 1 promoter was observed in germ cells , proliferating embryos and young larvae but not in post - mitotic tissues in the head Scale bars , 50 µm . Formaldehyde survival of wild type ( wt ) , dvc - 1 , gcna - 1 loss - of - function ( lof ) and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 4 independent experiments ) . Cisplatin survival of wild type , dvc - 1 , gcna - 1 and gcna - 1 ; dvc - 1 double mutant C . elegans ( mean ± SEM ; n = 3 independent experiments ) . Formaldehyde survival of gcna - 1 deletion ( del ) and E364Q mutant C . elegans ( mean ± SEM ; n = 2 independent experiments ) . Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) . Formaldehyde survival of C . elegans grown on L4440 control ( CTRL ) or gei - 17 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) . Formaldehyde survival of wild type and gcna - 1 deletion ( del ) mutant C . elegans grown on L4440 control ( CTRL ) or smo - 1 RNAi bacteria ( mean ± SEM ; n = 2 independent experiments ) .

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Figure 5HeLa cells transfected with plasmids encoding GFP alone, GFP-ACRC or GFP-tagged C. elegans GCNA-1 were subjected to GFP IP under denaturing conditions. Beads were incubated with recombinant polySUMO23-8 chains, washed extensively and processed for immunoblotting with SUMO2/3 and GFP antibodies.GFP expression driven by the gcna-1 promoter was observed in germ cells, proliferating embryos and young larvae but not in post-mitotic tissues in the head Scale bars, 50 µm.Formaldehyde survival of wild type (wt), dvc-1, gcna-1 loss-of-function (lof) and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=4 independent experiments).Cisplatin survival of wild type, dvc-1, gcna-1 and gcna-1; dvc-1 double mutant C. elegans (mean±SEM; n=3 independent experiments).Formaldehyde survival of gcna-1 deletion (del) and E364Q mutant C. elegans (mean±SEM; n=2 independent experiments).Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).Formaldehyde survival of C. elegans grown on L4440 control (CTRL) or gei-17 RNAi bacteria (mean±SEM; n=2 independent experiments).Formaldehyde survival of wild type and gcna-1 deletion (del) mutant C. elegans grown on L4440 control (CTRL) or smo-1 RNAi bacteria (mean±SEM; n=2 independent experiments).


Figure 1A Detection of the RBPJ / L3MBTL3 interaction using the yeast two - hybrid ( Y2H ) assay . In this Y2H experiment , RBPJ is fused to the GAL4 DNA - binding ( DB ) domain and L3MBTL3 is fused to the GAL4 activation domain ( AD ) . The DB - RBPJ and AD - L3MBTL3 fusion proteins interact with each other , leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine . The six Y2H controls were previously described ( Dreze et al , 2010 ) . The experiment was independently replicated thrice . B Endogenous L3MBTL3 co - purifies specifically with HA - RBPJ but not with HA - EGFP , HA - TBL1X or HA - HEY2 . Immuno - precipitation ( IP ) of HA - tagged RBPJ , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or L3MBTL3 antibody . The experiment was independently replicated twice . C Endogenous RBPJ co - purifies specifically with HA - L3MBTL3 but not with HA - EGFP , HA - TBL1X or HA - HEY2 . IPs of HA - tagged L3MBTL3 , EGFP , TBL1X or HEY2 in U87 - MG cells followed by Western blot analyses using HA or RBPJ antibody . The experiment was independently replicated twice . EV : Empty Vector control ; WB : Western blot ; IP : immuno - precipitation .

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Figure 1A Detection of the RBPJ/L3MBTL3 interaction using the yeast two-hybrid (Y2H) assay. In this Y2H experiment, RBPJ is fused to the GAL4 DNA-binding (DB) domain and L3MBTL3 is fused to the GAL4 activation domain (AD). The DB-RBPJ and AD-L3MBTL3 fusion proteins interact with each other, leading to the activation of the ADE2 and HIS3 reporter genes and allowing yeast cells to grow on selective media lacking adenine or histidine. The six Y2H controls were previously described (Dreze et al, 2010). The experiment was independently replicated thrice.B Endogenous L3MBTL3 co-purifies specifically with HA-RBPJ but not with HA-EGFP, HA-TBL1X or HA-HEY2. Immuno-precipitation (IP) of HA-tagged RBPJ, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or L3MBTL3 antibody. The experiment was independently replicated twice. C Endogenous RBPJ co-purifies specifically with HA-L3MBTL3 but not with HA-EGFP, HA-TBL1X or HA-HEY2. IPs of HA-tagged L3MBTL3, EGFP, TBL1X or HEY2 in U87-MG cells followed by Western blot analyses using HA or RBPJ antibody. The experiment was independently replicated twice. EV: Empty Vector control; WB: Western blot; IP: immuno-precipitation.


Figure 2B L3MBTL3 - Δ ( 1 - 64 ) does not interact with RBPJ . IP of HA - FLAG - tagged RBPJ in the presence of FLAG - tagged L3MBTL3 ( WT or deletion mutants ) in HEK293T cells followed by Western blotting using FLAG antibody . The experiment was independently replicated twice . D Deletion of the BTD domain impairs the RBPJ / L3MBTL3 interaction . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and deletion mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . The experiment was independently replicated twice . E RBPJF261R point mutant does not interact with L3MBTL3 . IP of HA - tagged L3MBTL3 in the presence of FLAG - tagged RBPJ ( WT and point mutants ) in HEK293T cells followed by Western blotting using HA or FLAG antibody . RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3 . The experiment was independently replicated twice . WB : Western blot ; IP : immuno - precipitation .

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Figure 2B L3MBTL3-Δ(1-64) does not interact with RBPJ. IP of HA-FLAG-tagged RBPJ in the presence of FLAG-tagged L3MBTL3 (WT or deletion mutants) in HEK293T cells followed by Western blotting using FLAG antibody. The experiment was independently replicated twice.D Deletion of the BTD domain impairs the RBPJ/L3MBTL3 interaction. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and deletion mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. The experiment was independently replicated twice. E RBPJF261R point mutant does not interact with L3MBTL3. IP of HA-tagged L3MBTL3 in the presence of FLAG-tagged RBPJ (WT and point mutants) in HEK293T cells followed by Western blotting using HA or FLAG antibody. RBPJV263R and RBPJA284R also show a reduced ability to interact with L3MBTL3. The experiment was independently replicated twice. WB: Western blot; IP: immuno-precipitation.


Figure 3A Thermodynamic characterization of the RBPJ / L3MBTL3 interaction . Representative thermograms ( raw heat signal and nonlinear least squares fit to the integrated data ) for L3MBTL3 - ( 31 - 70 ) binding to RBPJ - ( 53 - 474 ) . B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( B ) SBP - FLAG - RBPJ and HA - L3MBTL3 - Δ ( SAM ) in the presence of an increasing amount of HA - NOTCH1 ICD . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation . B / C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose - dependent manner . IPs were performed in CRISPR / Cas9 - mediated L3MBTL3 knockout ( KO ) HEK293T cells . ( C ) SBP - FLAG - RBPJ and HA - NOTCH1 ICD in the presence of an increasing amount of HA - L3MBTL3 - Δ ( SAM ) . The L3MBTL3 - Δ ( SAM ) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to express HA - L3MBTL3 - Δ ( SAM ) . The experiment was independently replicated thrice . WB : Western blot ; IP : immuno - precipitation .

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

Figure 3A Thermodynamic characterization of the RBPJ/L3MBTL3 interaction. Representative thermograms (raw heat signal and nonlinear least squares fit to the integrated data) for L3MBTL3-(31-70) binding to RBPJ-(53-474).B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (B) SBP-FLAG-RBPJ and HA-L3MBTL3-Δ(SAM) in the presence of an increasing amount of HA-NOTCH1 ICD. The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.B/C NOTCH1 ICD outcompetes L3MBTL3 for binding to RBPJ in a dose-dependent manner. IPs were performed in CRISPR/Cas9-mediated L3MBTL3 knockout (KO) HEK293T cells. (C) SBP-FLAG-RBPJ and HA-NOTCH1 ICD in the presence of an increasing amount of HA-L3MBTL3-Δ(SAM). The L3MBTL3-Δ(SAM) mutant construct was used instead of the L3MBTL3 WT construct in order to allow the analysis of both NOTCH1 ICD and L3MBTL3 proteins in the same Western blot. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to express HA-L3MBTL3-Δ(SAM). The experiment was independently replicated thrice. WB: Western blot; IP: immuno-precipitation.


Figure 4A De - repression of Notch target genes upon RBPJ knockdown . Shown are means ± s . d . of quadruplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data . Inset : Western blot analysis validates the shRNA - mediated depletion of RBPJ . B De - repression of Notch target genes in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of quadruplicate experiments . [ * * ] P < 0 . 01 , NS : Not Significant ; two - sample T - test on log - transformed data . Inset : Western blot analysis validates the CRISPR / Cas9 - mediated KO of L3MBTL3 . C RBPJ and L3MBTL3 co - localize at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . D L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes decreases upon RBPJ knockdown . Shown are means ± s . d . of triplicate ChIP experiments . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . E The repressive activity of L3MBTL3 at Notch target genes is RBPJ - dependent . Expression analysis of Notch target genes upon RBPJ knockdown and / or overexpression of L3MBTL3 . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA model on log - transformed data where the difference of differences was tested , which is equivalent to testing the interaction in a two - way ANOVA model . Western blot analysis validates the overexpression of L3MBTL3 and the shRNA - mediated depletion of RBPJ ( Appendix Fig S3E ) . Gene expression analyses of OCT4 was performed as control ( Appendix Fig S3F ) . F L3MBTL3 occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on its RBPJ interaction domain . ChIP analyses of HA - L3MBTL3 WT and HA - L3MBTL3 - Δ ( 1 - 64 ) occupancy at the proximal Notch - responsive elements of Notch target genes . Shown are means ± s . d . of duplicate experiments measured twice each . In panels C , D and F : distance in base pairs ( bp ) relative to the transcriptional start site ( TSS ) is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . G The L3MBTL3 - ( 1 - 64 ) domain is required for the downregulation of HES1 and HEY2 in U87 - MG cells . Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT , L3MBTL3 - Δ ( 1 - 64 ) or LacZ control ( Control ) . Shown are means ± s . d . of triplicate experiments . [ * ] P < 0 . 05 , [ * * ] P < 0 . 01 , NS : Not Significant ; one - way ANOVA model on log - transformed data .

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

Figure 4A De-repression of Notch target genes upon RBPJ knockdown. Shown are means ±s.d. of quadruplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data. Inset: Western blot analysis validates the shRNA-mediated depletion of RBPJ.B De-repression of Notch target genes in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of quadruplicate experiments. [**] P<0.01, NS: Not Significant; two-sample T-test on log-transformed data. Inset: Western blot analysis validates the CRISPR/Cas9-mediated KO of L3MBTL3.C RBPJ and L3MBTL3 co-localize at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes decreases upon RBPJ knockdown. Shown are means ±s.d. of triplicate ChIP experiments.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).E The repressive activity of L3MBTL3 at Notch target genes is RBPJ-dependent. Expression analysis of Notch target genes upon RBPJ knockdown and/or overexpression of L3MBTL3. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA model on log-transformed data where the difference of differences was tested, which is equivalent to testing the interaction in a two-way ANOVA model. Western blot analysis validates the overexpression of L3MBTL3 and the shRNA-mediated depletion of RBPJ (Appendix Fig S3E). Gene expression analyses of OCT4 was performed as control (Appendix Fig S3F).F L3MBTL3 occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on its RBPJ interaction domain. ChIP analyses of HA-L3MBTL3 WT and HA-L3MBTL3-Δ(1-64) occupancy at the proximal Notch-responsive elements of Notch target genes. Shown are means ±s.d. of duplicate experiments measured twice each.In panels C, D and F: distance in base pairs (bp) relative to the transcriptional start site (TSS) is indicated below the gene names. Chrom8 was used as negative control (NEG).G The L3MBTL3-(1-64) domain is required for the downregulation of HES1 and HEY2 in U87-MG cells. Expression analysis of Notch target genes upon overexpression of L3MBTL3 WT, L3MBTL3-Δ(1-64) or LacZ control (Control). Shown are means ±s.d. of triplicate experiments. [*] P<0.05, [**] P<0.01, NS: Not Significant; one-way ANOVA model on log-transformed data.


Figure 5A The RBPJ / KDM1A interaction is indirect and occurs via L3MBTL3 . IP of HA - KDM1A in the presence of overexpressed V5 - L3MBTL3 or V5 - L3MBTL3 - Δ ( 1 - 64 ) in L3MBTL3 KO U87 - MG cells . CRISPR / Cas9 sg - L3MBTL3 - resistant plasmids were used to overexpress the L3MBTL3 proteins . The experiment was independently replicated twice . B KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is L3MBTL3 - dependent . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . C KDM1A occupancy at the proximal Notch - responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains . ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87 - MG cells upon overexpression of L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) . Control : empty vector . Shown are means ± s . d . of duplicate experiments measured twice each . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . D L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , leads to decreasing H3K4me2 at the proximal Notch - responsive element of HES1 . ChIP analysis of H3K4me2 at the proximal Notch - responsive element of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of duplicate experiments measured twice each . P values were estimated via a one - way ANOVA on log - transformed data . Panels B , C and D : distance in bp relative to the TSS is indicated below the gene names . Chrom8 was used as negative control ( NEG ) . E L3MBTL3 , but neither L3MBTL3 - Δ ( 1 - 64 ) nor L3MBTL3 - Δ ( SAM ) , represses HES1 . Expression analysis of HES1 upon overexpression of LacZ control ( Control ) , L3MBTL3 , L3MBTL3 - Δ ( 1 - 64 ) or L3MBTL3 - Δ ( SAM ) mutants in L3MBTL3 KO U87 - MG cells . Shown are means ± s . d . of triplicate experiments . P values were estimated via a one - way ANOVA on log - transformed data . NS : Not Significant . WB : Western blot ; IP : immuno - precipitation . We note that in the context of this experiment , i . e . , in the absence of endogenous L3MBTL3 , the overexpression of L3MBTL3 - Δ ( 1 - 64 ) does not result in the increased expression of HES1 , contrasting with the result obtained in Fig 4G , i . e . , in the presence of endogenous L3MBTL3 . Indeed , as expected , the dominant negative effect of L3MBTL3 - ( 1 - 64 ) on endogenous WT L3MBTL3 ' s ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed .

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

Figure 5A The RBPJ/KDM1A interaction is indirect and occurs via L3MBTL3. IP of HA-KDM1A in the presence of overexpressed V5-L3MBTL3 or V5-L3MBTL3-Δ(1-64) in L3MBTL3 KO U87-MG cells. CRISPR/Cas9 sg-L3MBTL3-resistant plasmids were used to overexpress the L3MBTL3 proteins. The experiment was independently replicated twice.B KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is L3MBTL3-dependent. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).C KDM1A occupancy at the proximal Notch-responsive elements of Notch target genes is dependent on L3MBTL3 and both its RBPJ interaction and KDM1A interaction domains. ChIP analysis of endogenous KDM1A in L3MBTL3 KO U87-MG cells upon overexpression of L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM). Control: empty vector. Shown are means ±s.d. of duplicate experiments measured twice each.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).D L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), leads to decreasing H3K4me2 at the proximal Notch-responsive element of HES1. ChIP analysis of H3K4me2 at the proximal Notch-responsive element of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of duplicate experiments measured twice each. P values were estimated via a one-way ANOVA on log-transformed data.Panels B, C and D: distance in bp relative to the TSS is indicated below the gene names. Chrom8 was used as negative control (NEG).E L3MBTL3, but neither L3MBTL3-Δ(1-64) nor L3MBTL3-Δ(SAM), represses HES1. Expression analysis of HES1 upon overexpression of LacZ control (Control), L3MBTL3, L3MBTL3-Δ(1-64) or L3MBTL3-Δ(SAM) mutants in L3MBTL3 KO U87-MG cells. Shown are means ±s.d. of triplicate experiments. P values were estimated via a one-way ANOVA on log-transformed data. NS: Not Significant. WB: Western blot; IP: immuno-precipitation. We note that in the context of this experiment, i.e., in the absence of endogenous L3MBTL3, the overexpression of L3MBTL3-Δ(1-64) does not result in the increased expression of HES1, contrasting with the result obtained in Fig 4G, i.e., in the presence of endogenous L3MBTL3. Indeed, as expected, the dominant negative effect of L3MBTL3-(1-64) on endogenous WT L3MBTL3's ability to repress the expression of Notch target genes can only be observed when WT L3MBTL3 is expressed.