Fig 1: Trim33KOEBs are morphologically distinct when compared to control and SB431542-treated EBs. A, representative immuno blot (left) of control and mutant EBs during the first three days of differentiation analyzed for Trim33 (4-OHT, 1 μg/mL, was administered at day 0 of differentiation). The bar graph (right) shows relative quantification of Trim33 in control (blue bars) and mutant (red bars) (normalized to β-actin; n = 3). Error bars, S.E; * ≤ 0.05. B-G, Representative images of EBs at day 5 (B-D) and 9 (E-G) in differentiation. 4-OHT (C, F) or SB431542 (D, G) were added at the initiation of EB cultures (day 0). B, E; untreated cultures. (H) mRNA expression for Trim33, pluripotency markers (Pou5f1 and Nanog), mesendoderm markers (Gsc and Mixl1) in untreated EB cultures (blue bars), 4-OHT-treated EB cultures (red bars) and SB431542-treated EB cultures (green bars). Scale bars in B-D, 500 µm; E-G, 1 mm; *, p ≤ 0.05; **, p ≤ 0.01.
Fig 2: Several pluripotency and Stat3 target genes are upregulated while Rac1, Nanog and mesoderm markers are downregulated in Trim33 mutant EBs. A, Dot plots showing gene expression in normalized counts of the differentially expressed genes shown in C, control; M, mutant. Horizontal lines present the median of 3 independent findings; * ≤ 0.05; ** ≤ 0.01. B, representative immuno blot (left) of control and mutant EBs during the first three days of differentiation analyzed for the presence of Tfcp2l1 and Rac1 (4-OHT, 1 μg/mL, was administered at day 0 of differentiation). The bar graph (right) shows relative quantification of Tfcp2l1 and Rac1 in control (blue columns) and mutant (red columns) (normalized to β-actin; n = 3). Error bars, S.E; *, p ≤ 0.05; **, p ≤ 0.01.
Fig 3: Characterization of control andTrim33-deficient ESCs cultured under non-differentiation conditions. A, a schematic presentation of the targetedTrim33 locus. Arrows a, c, and d depict the primers used in the genotyping assay (B). B, Trim33FF:UbcCreERT2, 4-OHT- (1) and Trim33FF:UbcCreERT2, 4-OHT+ (2). Lanes 3–6 are known controls: #3, Trim33FKO; #4, Trim33FF; #5, Trim33KOKO; #6, Trim33WTKO. C-D, Phase contrast (top left) and immunofluorescence images for SSEA-1 (red) and Pou5f1 (green) of control (C, 4-OHT-) and Trim33KO (D, 4-OHT+) cells showing characteristic ESC colony morphology. Cultured in the presence of LIF and 2i on gelatin-coated tissue culture dishes; top right, merge, counterstaining with DAPI (blue); bottom raw: Pou5f1 (green) and SSEA-1 (red). E-F, Phase contrast (top left) and immunofluorescence images for SSEA-1 (red) and Trim33 (green) of control (E, 4-OHT-) and Trim33KO (F, 4-OHT+) cells showing characteristic ESC colony morphology. Cultured in the presence of LIF and 2i on gelatin-coated tissue culture dishes; top right, merge, counterstaining with DAPI (blue); bottom raw: Pou5f1 (green) and SSEA-1 (red). G-H, Phase contrast (left) and immunofluorescence images for Tfcp2l1 (right, green) of control (G, 4-OHT-) and Trim33KO (H, 4-OHT+) cells showing characteristic ESC colony morphology. Cultured in the presence of LIF and 2i on gelatin-coated tissue culture dishes. I-J, Phase contrast and immunofluorescence images (right) for SSEA-1 (red) and Pou5f1 (green) of control (E, 4-OHT-) and Trim33KO (F, 4-OHT+) cells cultured for 7 days without LIF and 2i on gelatin-coated tissue culture dishes. Scale bars in C-J, 100 µm. K, Proliferation curves of Trim33FF:UbcCreERT2 cells cultured in KSR with 2i+LIF for 24, 48 and 72 h; control cells (blue line), mutant cells (treated with 4-OHT for 24 h before plating (red line). L, 4-OHT-treatment of wildtype cells (Trim33WTWT:UbcCreERT2) did not have a similar effect suggesting that reduced proliferation in Trim33FF:UbcCreERT2 cultures was not caused by 4-OHT treatment (culture conditions as in K).
Fig 4: E2f4 promotes Recql binding to chromatin in Trim33-KO cells.a Immunoprecipitation from p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql; n = 3. b Representative genome browser tracks of Cut&Run analysis of HA-Recql binding in p19/Nras Trim33-WT and Trim33-KO cells expressing HA-tagged Recql. Lower two tracks—E2f4 ChIP-seq. c Sequencing tag coverage for Recql Cut&Run signal from the experiment shown in b) at all TSS and at E2f4 sites. d Immunoprecipitation analysis with Recql antibodies from formaldehyde-crosslinked p19/Nras Trim33-WT and Trim33-KO cells, transfected with E2f4-targeting or a control siRNA. e Cut&Run assays with Recql antibodies in p19/Nras Trim33-WT and Trim33-KO cells followed by qPCR analysis at the TSS sites of indicated genes. Mean values of percent input for three technical replicates and standard deviation are plotted. f Representative genome browser tracks of the Cut&Run assays with Recql antibodies in Trim33-KO p19/Nras cells expressing the indicated E2f4 variants. g Cut&Run sequencing tag coverage at all TSS for the experiment shown in f). h DNA fiber assays in Trim33-KO cells, expressing siRNA against Recql or control siRNA, untreated or released from a 4 h HU treatment; n = 2. 100 fibers were counted per sample and the data were analyzed using Kruskal–Wallis test followed by Dunn’s multiple comparison. Boxplots represent median±quartiles with whiskers ranging up to 1.5-fold of the interquartile range. i Immunoblotting analysis of Recql expression in Trim33-KO cells, used in h). Source data are provided as a Source Data file.
Fig 5: Trim33 loss exacerbates DNA damage following release from replicative stress.a Immunofluorescence analysis with pH2AX and 53bp1 antibodies in p19/Nras Trim33-WT and Trim33-KO cells cultured in the presence of 0.1 mM HU. Scale bar = 5 µm. Graphs show pH2AX signal intensity and 53bp1 nuclear bodies (n.b.) per nucleus for n = 231,228 cells (Trim33-WT,KO). Significance was determined by a two-tailed Mann–Whitney test. A representative of two independent experiments is shown. b Distribution of pH2AX signal intensity in p19/Nras Trim33-WT and Trim33-KO cells, upon 24 h exposure to HU (HU) or after a 24-h release from HU treatment (HU-rel). c Immunoblots of p19/Nras Trim33-KO cells transfected with the indicated siRNAs; n = 3. d Immunofluorescence staining with pH2AX antibody in p19/Nras Trim33-WT and Trim33-KO cells expressing the indicated siRNAs 24 h after release from HU. From left, n = 161,219,97,139 cells; significance was determined using Kruskal–Wallis test with Dunn’s multiple comparisons; scale bar = 20 µm. e Neutral comet assays in p19/Nras Trim33-WT and Trim33-KO cells transfected with the indicated siRNAs, untreated or released from HU treatment as in d). Scale bar = 20 µm. From left, n = 59,34,64,60 cells. Significance was determined by one-way ANOVA with Tukey’s multiple comparisons. f Representative genome browser tracks of DSB capture and sequencing signal in untreated (Ctrl), HU-treated (HU), or HU-treated and released (HU-rel) Trim33-WT and Trim33-KO cells. g DSB capture tag density at all TSS for HU-treated (24 h) or HU-treated and released for 24 h p19/Nras Trim33-WT and Trim33-KO cells. h Linear regression analysis of Recql recruitment (normalized HA-Recql Cut&Run tags for HU-treated cells) vs. DSB capture tags at 24 h after HU release for 3000 sites with strongest Recql binding, sorted by the number of Recql tags and binned at 60 sites/bin. a, d, e Boxplots represent median ± quartiles with whiskers ranging up to 1.5-fold of the interquartile range. Source data are provided as a Source Data file.
Supplier Page from MilliporeSigma for Anti-TRIM33 antibody produced in rabbit