Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Integrative copy number analysis of uveal melanoma reveals novel candidate genes involved in tumorigenesis including a tumor suppressor role for PHF10/ BAF45a

doi: 10.1158/1078-0432.CCR-18-3052

Figure Lengend Snippet: A. Enrichment analysis between copy number and expression at the cytogenetic band level. Results are presented for TCGA and CC cohorts where correlation coefficients were > 0.5. For each cytogenetic band the total number of genes analysed, and the frequency of the significantly correlated genes is shown, along with the P values and FDR P values. K is the number of genes in the set from MsigDB and k is the number of genes in the intersection of the query set with a set from MsigDB. The ratio of k/K in each cohort (CC and TCGA) is also provided.

Article Snippet: Established cell lines derived from primary uveal melanomas (Mel202 ( 22 ), 92–1 ( 23 ) and Mel290 ( 24 ) (described in Supplementary Table 1 ) were subjected to PHF10 knockdown with siRNA (Santa-cruz, sc-95343) or a siRNA control that consisted of scrambled sequence/non-silencing siRNA (Santa-cruz, sc-37007) (SiCtrl).

Techniques: Expressing, Amplification

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Integrative copy number analysis of uveal melanoma reveals novel candidate genes involved in tumorigenesis including a tumor suppressor role for PHF10/ BAF45a

doi: 10.1158/1078-0432.CCR-18-3052

Figure Lengend Snippet: The region of overlap that harbors PHF10, WDR27 and c6orf120 is shown. The location of the frameshift mutation identified in MM133 (a class 2 tumor) is also shown. The CC sample with the PHF10 HD was GSM1082739. The WU cohort sample with a PHF10 HD was MM016, a class 2 tumor in a female who was diagnosed with UM in her 20s, did not harbor a detectable mutation in BAP1, SF3B1 or EIF1AX and who died from metastasis.

Article Snippet: Established cell lines derived from primary uveal melanomas (Mel202 ( 22 ), 92–1 ( 23 ) and Mel290 ( 24 ) (described in Supplementary Table 1 ) were subjected to PHF10 knockdown with siRNA (Santa-cruz, sc-95343) or a siRNA control that consisted of scrambled sequence/non-silencing siRNA (Santa-cruz, sc-37007) (SiCtrl).

Techniques: Mutagenesis

Journal: Clinical cancer research : an official journal of the American Association for Cancer Research

Article Title: Integrative copy number analysis of uveal melanoma reveals novel candidate genes involved in tumorigenesis including a tumor suppressor role for PHF10/ BAF45a

doi: 10.1158/1078-0432.CCR-18-3052

Figure Lengend Snippet: A. GO analysis of differentially expressed transcripts. For RNA sequencing, three biological replicates were prepared for each condition. Transcripts with a fold change >2 and adjusted p-value < 0.5 following PHF10 kd were selected, and those shared by at least two cell lines were used for pathway analysis with g:Profiler (https://biit.cs.ut.ee/gprofiler/gost). GO redundant terms were removed with REVIGO (65); B. Venn diagram showing the number of differentially expressed transcripts unique to and shared by each cell line; C. Western blot analysis of some transcripts encoding chromatin remodelers that were differentially expressed in PHF10 mutant tumors. 30μg of whole cell lysate was loaded. Knockdown was confirmed with qRT-PCR, RNA-Seq and Knockdown was confirmed with Western blotting and an antibody to PHF10 at a concentration of 1:1000; D, E, F: Results of adhesion assays: ~105 cells (PHF10kd or siCtrl for 24h) were seeded on top of a collagen insert in serum-free media and medium containing 10% FBS and extracellular matrix proteins were added to the lower chamber. Cells were left to invade for 48h and then lysed and stained with CyQuant GR dye and fluorescence was measured at 480/520nm. The extracellular matrix proteins tested here were Col I, Col II and Col IV corresponding to Collagen 1, 2 and 4 respectively, FN (Fibronectin), LN (laminin), TN (tenascin), VN (vitronectin). Neg corresponds to the negative control. Three biological replicates were analysed and results are shown with error bars represent standard deviation of the mean * P-value <0.05. G: Result of migration assays performed as described elsewhere (27).

Article Snippet: Established cell lines derived from primary uveal melanomas (Mel202 ( 22 ), 92–1 ( 23 ) and Mel290 ( 24 ) (described in Supplementary Table 1 ) were subjected to PHF10 knockdown with siRNA (Santa-cruz, sc-95343) or a siRNA control that consisted of scrambled sequence/non-silencing siRNA (Santa-cruz, sc-37007) (SiCtrl).

Techniques: RNA Sequencing, Western Blot, Mutagenesis, Knockdown, Quantitative RT-PCR, Concentration Assay, Staining, CyQUANT Assay, Fluorescence, Negative Control, Standard Deviation, Migration

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 2 The dynamic effect of differentiation markers followed by PHF10 in transiently transfected cells. A Parietal cell marker ATP4B. B Main cell marker Pepsinogen I. C Gastric neuroendocrine cell marker GAST. D Gastric mucus cell marker TFF1. E GC stem cell marker CD44. F Gastric progenitor cell marker SOX9. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Transfection, Marker

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 3 PHF10 inhibits subcellular morphology and enhances sphere formation ability in GC cells. A SGC7901 cells exhibited numerous microvilli on the cell surface and pits on the nuclear membrane (×15,000). B SGC7901 cells manifested a significant presence of rough endoplasmic reticulum in the cytoplasm (white arrow), limited mitochondrial cristae (black arrow) (×60,000). C SGC7901-shPHF10 cells exhibited a decrease in microvilli and pits on the nuclear membrane (×10,000). D SGC7901-shPHF10 cells presented a notable quantity of lysosomes with high electron density (white arrow) (×60,000). E The cytoplasm of SGC7901-shPHF10 cell exhibited an increase in mitochondrial cristae (white arrow) (×60,000). F Cells with PHF10 knockdown displayed alterations in the Golgi complex (white arrow) (×30,000). G Cytoplasmic vacuolization was observed in SGC7901-shPHF10 cells (×30,000). H Apoptotic cells were identified in SGC7901- shPHF10 cells (×15,000). I–K The sphere formation capacity of SGC-7901, MKN28 and GES-1 cells manipulated by PHF10 was evaluated by measuring the number and diameter of primary and secondary spheres separately. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Membrane, Knockdown

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 5 PHF10 serves as a member of SWI/SNF complex in GC cells. A, B The nuclear co-localization of PHF10 and BRG1 was observed in SGC7901 cells (A) and MKN28 cells (B). C–F Co-IP assays were performed using antibodies against PHF10 (C), BRG1 (D), BAF155 (E) and SNF5 (F) in SGC7901-shPHF10 and control cells. G–J Co-IP experiments for PHF10 (G), BRG1 (H), BAF155 (I) and SNF5 (J) were conducted in MKN28- PHF10 and control cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Co-Immunoprecipitation Assay, Control

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 6 PHF10 directly targets DUSP5 through SWI/SNF complex. A–C Detection of E2F1, PHF10 and DUSP5 expression in SGC7901 cells by qRT-PCR. D–F The mRNA levels of E2F1, PHF10 and DUSP5 in MKN28 cells were also assessed. G, H WB analysis was performed to determine the levels of DUSP5, ERK1/2 and pERK1/2 levels in SGC7901 cells. I The levels of E2F1, PHF10, DUSP5, ERK1/2 and pERK1/2 levels in MKN28 cells were evaluated. J ChIP-qPCR was carried out using antibodies against PHF10, BRG1, BAF155 and SNF5 in SGC7901 cells, with IgG antibodies serving as a negative control. The purified DNA after precipitation was subsequently amplified using the same set of primers for PCR analysis. K The promoter region of DUSP5 was amplified in MKN28-PHF10 cells using antibodies against PHF10, BRG1, BAF155 and SNF5. L A dual luciferase reporter assay was conducted in both MKN28-PHF10-Vector and MKN28-PHF10 cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Expressing, Quantitative RT-PCR, ChIP-qPCR, Negative Control, Luciferase, Reporter Assay, Plasmid Preparation

Journal: Cancer gene therapy

Article Title: PHF10 inhibits gastric epithelium differentiation and induces gastric cancer carcinogenesis.

doi: 10.1038/s41417-024-00820-5

Figure Lengend Snippet: Fig. 7 PHF10 mediates differentiation disorders in GC through E2F1-PHF10-DUSP5-pERK1/2. A The relationship between E2F1 or PHF10 and DUSP5, was examined in 30 pairs of GC tissues through qRT-PCR analysis. B IHC staining was performed for E2F1, PHF10 and DUSP5 in two patient samples. Patient 1 exhibited poorly differentiated and diffuse GC, while patient 2 had well differentiated and intestinal GC. C WB analysis was conducted to measure the levels of E2F1, PHF10, DUSP5 and pERK1/2 in the aforementioned patient samples. D The expression levels of gastric epithelium differentiation markers (ATP4B, PG I, GAST, TFF1, CD44 and SOX9) were detected by qRT-PCR in SGC7901 cells. E The mRNA levels of gastric epithelium differentiation markers were assessed in MKN28 cells. F, G Sphere formation was quantified in SGC7901 cells (F) and MKN28 cells (G). H A schematic illustrating the mechanism of PHF10-mediated dysdifferentiation in GC cells.

Article Snippet: The following antibodies were used: PHF10 (Novus, USA, H00055274-D01), E2F1 (Santa Cruz, sc-193), BRG1 (Santa Cruz, sc-17796), SNF5 (Santa Cruz, sc-16189), BAF155 (Santa Cruz, sc-10756).

Techniques: Quantitative RT-PCR, Immunohistochemistry, Expressing

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