Journal: Nature Communications

Article Title: PBAF/cBAF reorganization on H3.3 chromatin regulates BMAL1 activity in the absence of circadian negative feedback

doi: 10.1038/s41467-025-64045-2

Figure Lengend Snippet: a Native HA-tag IP on soluble FH-H3.3A chromatin at circadian times (CT) CT8 and CT20 from wild-type and FH-H3.3A;PerKO mouse livers. Wild-type chromatin was used as a negative control (blue box). Nuclear extracts (0.6%) were used as Input (lanes 2-5). Immunoblots of HA (FH-H3.3A), CLOCK, BMAL1, H3.1/2 and H2A.Z. M, protein ladder. Results represent n = 3 independent biological replicates, see Source data. b Native HA-tag IP on soluble FH-H3.3A chromatin from CT0-20 (lanes 9–15), with wild-type chromatin used as a negative control (blue box). Nuclear extracts (0.6%) were loaded as Input (lanes 2–8). Immunoblots of HA (FH-H3.3A), PBRM1, ARID2, BRG1, BMAL1 and H3.1/2. Results represent n = 3 independent biological replicates. c Native PBRM1 IP on soluble wild-type liver chromatin over CT (lanes 10-15); IgG antibody was used as a negative control (lanes 17-22). Nuclear extracts (0.6%) were loaded on a separate gel as Input (lanes 2–7). Immunoblots of PBRM1, ARID2, BRD7, PHF10, BRG1 and BMAL1. Results represent n = 3 independent biological replicates. d Native FH-H3.3A circadian ChIP-seq. Input signal was plotted along with the normalized ChIP signal on the same graph. Y-axis corresponds to the mean of normalized scores per genomic region. X-axis represents the distance from the TSS or the center of the given site/region ( ± 1 kb). Each color represents a given CT, with yellow/orange for the day- and blue colors for the night-time points. Results represent n = 3 independent biological replicates, see Supplementary Data . e Mean H3.3A and H3.1/2 occupancy at day- vs. night-time points at TSS of all genes. ChIP-seq graph axes are as in (d). Results represent n = 3 independent biological replicates. f Real-time bioluminescence recordings of synchronized wild-type (blue) and H3.3 double knock-out (red) mouse embryonic fibroblasts expressing Bmal1:Luciferase reporter. Detrended analysis over 7 d of measurements is shown. Results represent n = 5 (wild-type) and n = 6 (knock-out) replicates; the bar graph shows the calculated period length represented as mean ± s.e.m. with individual points indicated for each replicate. P -value between wild-type and H3.3DKO was calculated with paired, one-tailed t-test; *** P = 1.12542E-05. Knock-out efficiency was confirmed by western-blotting. Source Data are provided as a Source Data file.

Article Snippet: Following primary antibodies were used for western-blotting in this study, dilutions were prepared in 3% BSA- PBS 1X as indicated: HA-Tag (C29F4) Rabbit mAb (Cell Signaling, #3724), 1:1000; PBRM1/BAF180 (E9X2Z) Rabbit mAb (Cell Signaling, #89123), 1:1000; ARID2 (GT7311) Mouse mAb (Sigma-Aldrich, SAB2702340), 1:500; ARID2 (GT7311) Mouse mAb (GENETEX, GTX632011), 1:500; BRG1/SMARCA4 Rabbit pAb (Bethyl Laboratories, A300-813A), 1:500; BRD7 Rabbit pAb (Proteintech, 51009-2-AP), 1:500; PHF10 Mouse mAb (Proteintech, 66341-1-Ig), 1:500; PHF10 Rabbit pAb (GENETEX, GTX116314), 1:500; BMAL1 Rabbit pAb (Bethyl Laboratories, A302-616A), 1:1000; CLOCK Mouse mAb (MBL, D334-3), 1:500; Histone H3.1/3.2 (1D4F2) Mouse mAb (Active Motif, 61629), 1:500; Tri-Methyl-Histone H3 (Lys4) Rabbit pAb (Cell Signaling, #9727), 1:500; Acetyl-Histone H3 (Lys115) Rabbit pAb (PTM Bio, PTM-170), 1:1000; Histone H3 (acetyl K122) Rabbit pAb (Abcam, ab33309), 1:500; Histone H2A.Z Rabbit pAb (Active Motif, 39113), 1:1000; Homemade H2A.Z Rabbit pAb (kind gift from Stefan Dimitrov), 1:500; ARID1B Rabbit pAb (GENETEX, GTX130708), 1:500; SMARCA2 (HL1115) Rabbit mAb (GENETEX, GTX636330), 1:500; Histone H3 Rabbit pAb (Proteintech, 17168-1-AP), 1:2000 in 1% milk- PBS 1X; Histone H4 Rabbit pAb (Proteintech, 16047-1-AP), 1:2000 in 1% milk- PBS 1X; U2AF65 Rabbit mAb (Abcam, ab197031), 1:500; HIRA (clone WC119) Mouse mAb (Sigma-Aldrich, 04-1488), 1:500; DAXX anti-Mouse (Precision antibody), 1:500.

Techniques: Negative Control, Western Blot, ChIP-sequencing, Knock-Out, Expressing, Luciferase, One-tailed Test

Journal: Nature Communications

Article Title: PBAF/cBAF reorganization on H3.3 chromatin regulates BMAL1 activity in the absence of circadian negative feedback

doi: 10.1038/s41467-025-64045-2

Figure Lengend Snippet: a Native HA-tag IP at day vs. night time-points in FH-H3.3A wild-type and PerKO livers (lanes 8–11), with wild-type chromatin used as a negative control (blue box). 0.6% of the nuclear extracts were loaded as Input (lanes 2–5). Immunoblots of HA epitope (FH-H3.3A), PBRM1, ARID2, BRD7, PHF10, BRG1 and BMAL1. M, protein ladder. Results are representative of n = 3 independent biological replicates, see Source data. b Native PBRM1 IP at day vs. night time-points in FH-H3.3A wild-type and PerKO livers (lanes 7–10); IgG antibody was used as a negative control (lanes 12–15). 0.6% of the nuclear extracts were loaded as Input (lanes 2–5). Immunoblots of PBRM1, ARID2, BRD7, PHF10, BRG1 and BMAL1. Results are representative of n = 4 independent biological replicates. c mRNA relative expression levels of specific PBAF components and shared ATPase subunit Brg1 normalized to Rps9 ; Per1 was used as control. Results are represented as mean ± s.e.m. with individual points indicated for each replicate. P -values were calculated for all PBAF components between wild-type CT8 and PerKO CT8, and between wild-type CT20 and PerKO CT20 with paired, one-tailed Welch’s (unequal variance) t-test; P values were not significant, except for Arid2 CT8 time point; * P = 0.04346. Results are representative of n = 3 independent biological replicates. Source Data are provided as a Source Data file.

Article Snippet: Following primary antibodies were used for western-blotting in this study, dilutions were prepared in 3% BSA- PBS 1X as indicated: HA-Tag (C29F4) Rabbit mAb (Cell Signaling, #3724), 1:1000; PBRM1/BAF180 (E9X2Z) Rabbit mAb (Cell Signaling, #89123), 1:1000; ARID2 (GT7311) Mouse mAb (Sigma-Aldrich, SAB2702340), 1:500; ARID2 (GT7311) Mouse mAb (GENETEX, GTX632011), 1:500; BRG1/SMARCA4 Rabbit pAb (Bethyl Laboratories, A300-813A), 1:500; BRD7 Rabbit pAb (Proteintech, 51009-2-AP), 1:500; PHF10 Mouse mAb (Proteintech, 66341-1-Ig), 1:500; PHF10 Rabbit pAb (GENETEX, GTX116314), 1:500; BMAL1 Rabbit pAb (Bethyl Laboratories, A302-616A), 1:1000; CLOCK Mouse mAb (MBL, D334-3), 1:500; Histone H3.1/3.2 (1D4F2) Mouse mAb (Active Motif, 61629), 1:500; Tri-Methyl-Histone H3 (Lys4) Rabbit pAb (Cell Signaling, #9727), 1:500; Acetyl-Histone H3 (Lys115) Rabbit pAb (PTM Bio, PTM-170), 1:1000; Histone H3 (acetyl K122) Rabbit pAb (Abcam, ab33309), 1:500; Histone H2A.Z Rabbit pAb (Active Motif, 39113), 1:1000; Homemade H2A.Z Rabbit pAb (kind gift from Stefan Dimitrov), 1:500; ARID1B Rabbit pAb (GENETEX, GTX130708), 1:500; SMARCA2 (HL1115) Rabbit mAb (GENETEX, GTX636330), 1:500; Histone H3 Rabbit pAb (Proteintech, 17168-1-AP), 1:2000 in 1% milk- PBS 1X; Histone H4 Rabbit pAb (Proteintech, 16047-1-AP), 1:2000 in 1% milk- PBS 1X; U2AF65 Rabbit mAb (Abcam, ab197031), 1:500; HIRA (clone WC119) Mouse mAb (Sigma-Aldrich, 04-1488), 1:500; DAXX anti-Mouse (Precision antibody), 1:500.

Techniques: Negative Control, Western Blot, Expressing, Control, One-tailed Test

Journal: Science Advances

Article Title: ARID1A loss in neuroblastoma promotes the adrenergic-to-mesenchymal transition by regulating enhancer-mediated gene expression

doi: 10.1126/sciadv.aaz3440

Figure Lengend Snippet: ( A ) Western blot analysis of ARID1A protein expression in control (Ctrl), ARID1A +/− , ARID1A −/− #1, and ARID1A −/− #2 NGP cells. α-Tubulin was used as a loading control. ( B ) Cell growth was measured at days 1, 3, 5, and 7. Values are means ± SEM of triplicate experiments. RLU, relative light unit. ( C and D ) Transwell invasion and migration assay of the indicated cells. The fold change of the number of migrated cells passing through the membrane per field was relative to the mean for control NGP cells and was compared with the two-tailed unpaired t test (C). ** P < 0.01; *** P < 0.001. Cells were stained with 0.1% crystal violet (D). Scale bars, 100 μm. ( E ) Immunoprecipitation (IP) of the components of the BAF complexes using an anti-ARID1B antibody. PHF10 is present exclusively in the PBAF complexes and served as a negative control in this experiment; β-actin was used as a loading control. ( F ) Control luciferase shRNA (shLuc) or each of the ARID1B -targeting shRNAs (shARID1B #1 and #2) was induced by doxycycline (1.5 μg/ml) for 6 days in the indicated cells. The expression of ARID1B was detected by western blot analysis. α-Tubulin was used as a loading control. ( G ) Cell growth was measured at days 0, 3, 5, 7, and 9 after pretreatment with doxycycline (1.5 μg/ml) for 3 days to induce the expression of shLuc, shARID1B #1, or shARID1B #2. Values are means ± SEM of triplicate experiments.

Article Snippet: Proteins were detected by the anti-ARID1A rabbit polyclonal antibody (Novus Biologicals, #NB100-55334), anti-ARID1B mouse monoclonal antibody (Abcam, #ab57461), anti-BRG1 mouse monoclonal (EPNCIR111A) antibody (Abcam, #ab110641), anti-BAF155 rabbit monoclonal (D7F8S) antibody (Cell Signaling Technology, #11956), anti-SS18 rabbit monoclonal (D6I4Z) antibody (Cell Signaling Technology, #21792), anti-BAF47 mouse monoclonal (F-4) antibody (Santa Cruz Biotechnology, #sc-166164), anti-plant homeodomain finger protein 10 (PHF10) rabbit polyclonal antibody (GeneTex, #GTX116314), and anti–β-actin rabbit polyclonal antibody (Abcam, #ab8227).

Techniques: Western Blot, Expressing, Control, Migration, Membrane, Two Tailed Test, Staining, Immunoprecipitation, Negative Control, Luciferase, shRNA