Journal: Science signaling

Article Title: ATM directs DNA damage responses and proteostasis via genetically separable pathways

doi: 10.1126/scisignal.aan5598

Figure Lengend Snippet: (A) In vitro kinase assay with immunoprecipitated CK2. HEK-293T cells stably expressing V5-tagged CK2β were treated with 10 μM ATM inhibitor (KU-55933) or an equivalent amount of DMSO for 16 hours and CK2β was immunoprecipitated with magnetic anti-V5 beads. CK2 was incubated with 1.67 mM ATP, 5 fCi ATP-γ-32P, and 1.15 μg of GST-CK2 substrate for 1 hour, and 32P-labeled substrate was analyzed by phosphorimager. (B) Normalized amount of immunoprecipitated CK2β-V5 from (A) with the levels of CK2β-V5 normalized to levels in mock-treated cells. Quantitation was performed on the Licor system using Image Studio Ver 4.0. P-values: * < 0.05 (C-E) Distribution profile of CK2 subunits CK2α (C), CK2α′ (D), and CK2β (E), in HEK-293T cells after sucrose gradient sedimentation. HEK-293T cells expressing CK2α-V5, CK2α′-V5, or CK2β-V5 were treated with 10 μM KU-55933 or an equivalent amount of DMSO for 16 hours, harvested, and lysed in the absence of detergent. 1 mg of lysate in 500 μl lysis buffer total was added to the top of a sucrose gradient made with 1 ml layers of 50% to 5% sucrose in 5% increments. After ultracentrifugation, 500 μl fractions were collected and analyzed by western blot. (F and G) the distribution profile of CK2α (F) and CK2β (G) from the AT1ABR cells expressing wild-type or CL ATM alleles, analyzed by sucrose gradient sedimentation. Lysates were analyzed as in (C-E) except with induced AT1ABR cells expressing wild-type or CL alleles of ATM as indicated. (H) Sucrose gradient sedimentation pattern of V5-tagged CK2β stably expressed in U2OS cells treated with 10 μM KU-55933 or an equivalent amount of DMSO for 16 hours, or treated with a combination of KU-55933 and 1 mM NAC, as indicated. (I) Analysis of detergent-resistant aggregates in U2OS cells treated with 10 μM KU-55933 or DMSO, or a combination of KU-55933 and 1 mM NAC, as indicated. Aggregate fractions were isolated and compared with total lysate using western blot analysis for stably expressed V5-tagged CK2β. (J) Analysis of detergent-resistant aggregates in U2OS Flp-In cells expressing wt, CL, or 2RA ATM treated with 25 μM arsenite as shown in (I). (K) Aggregate fractions were isolated from U2OS cells expressing shCTRL or ATM shRNA and probed for CK2β, ATM, and β-actin as indicated from two independent cultures. (L) Means ± S.D. of CK2β abundance in aggregates were quantified from 3 independent experiments [including the replicates shown in (K)]. ** p< 0.005.

Article Snippet: V5-tagged CK2 levels were probed using rabbit anti-V5 (Novus Biologicals, NB600-381) and Alexa Fluor 680 goat anti-rabbit (Invitrogen, {"type":"entrez-nucleotide","attrs":{"text":"A21076","term_id":"579037","term_text":"A21076"}} A21076 ) and scanned and quantitated on the Li-Cor Odyssey system.

Techniques: In Vitro, Kinase Assay, Immunoprecipitation, Stable Transfection, Expressing, Incubation, Labeling, Quantitation Assay, Sedimentation, Lysis, Western Blot, Isolation, shRNA

Journal: Nucleic Acids Research

Article Title: Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress

doi: 10.1093/nar/gkab362

Figure Lengend Snippet: HF lowers charging of tRNA Pro and induces GCN2 and translational control. ( A ) MEF cells (left panel) and HepG2 cells (right panel) were treated with HF at the designated concentrations for 6 h and levels of the indicated proteins were measured by immunoblot analyses using specific antibodies. In parallel, levels of phosphorylation of GCN2 and eIF2α were measured by immunoblot. ( B ) MEF cells were treated with 100 nM HF or 2 μM tunicamycin (TUN) for 6 h or left untreated (NT). Cells were collected, lysed, and separated by centrifugation in a sucrose gradient. Gradients were fractionated with monitored at absorbance of 254 nm. Free 40S and 60S ribosomes, 80S monosomes, and large polysomes are indicated. Relative amounts of monosomes (M) and large polysomes (P) are presented. ( C ) MEF cells were treated with 100 nM HF alone or along with 3 mM proline for 6 h, or left untreated (NT). The percentage of uncharged tRNA Pro (left panel) and relative amounts of total tRNA Pro (right panel) were measured by a RT-qPCR as described in the materials and methods. Significant differences between treatments are indicated. **** P < 0.0001; ns, not significant.

Article Snippet: Antibodies using in the immunoblot analyses include the following: P-GCN2 (Abcam, #Ab75836), total GCN2 (Abcam, #ab137543), P-eIF2α (Abcam, #ab32157), total eIF2α (Cell Signaling Technology, #5324S), ATF4 antibody was prepared against the corresponding recombinant human proteins, which were affinity purified , P-mTOR (Cell Signaling Technology, #2971S), total mTOR (Cell Signaling Technology, #2983S), P-S6K1 (Cell Signaling Technology, #9205S), total S6K1 (Cell Signaling Technology, #9202S), P -4EBP1 (Cell Signaling Technology, #2855S), total 4EBP1 (Cell Signaling Technology, #9644S), PRODH (Proteintech, # 22980-1-AP) and β-actin (Sigma, #A5441).

Techniques: Western Blot, Centrifugation, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress

doi: 10.1093/nar/gkab362

Figure Lengend Snippet: Elevated free amino acids during HF treatment is suggested to enhance mTORC1 signaling. ( A ) MEF cells were treated with 100 nM HF for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), followed by immunoblot analyses with the indicated antibodies. ( B ) The bar graph shows the changes in the levels of phosphorylated GCN2, eIF2α, S6K1, and 4EBP1. Results were derived from 3 independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: * P < 0.05; ** P < 0.005; **** P < 0.0001. ( C ) MEF cells were treated with 100 nM HF for 6 h or left untreated (NT), followed by lysis and free amino acids were measured and illustrated in the bar graph. The data was derived from 3 independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: * P < 0.05; ** P < 0.005; *** P < 0.0005. ( D ) MEF cells were treated with 2 mM histidinol for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), followed by immunoblot analyses with the indicated antibodies. ( E ) The bar graph shows the changes in the levels of phosphorylated GCN2, eIF2α, S6K1 and 4EBP1. The data was derived from three independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: * P < 0.05; ** P < 0.005; **** P < 0.0001. ( F ) WT and Gcn2 –/– MEF cells were treated with either 50 or 100 nM HF for 6 h in presence or absence of 25 nM Torin1 or left untreated (NT), as indicated. Fresh media was then applied to cells for 18 h for a total of 24 h. Cell viability was monitored by the MTT assay. The data was derived from three independent experiments, with error bars represented as the means ± SD. Significant differences between treatment groups are indicated: * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001.

Article Snippet: Antibodies using in the immunoblot analyses include the following: P-GCN2 (Abcam, #Ab75836), total GCN2 (Abcam, #ab137543), P-eIF2α (Abcam, #ab32157), total eIF2α (Cell Signaling Technology, #5324S), ATF4 antibody was prepared against the corresponding recombinant human proteins, which were affinity purified , P-mTOR (Cell Signaling Technology, #2971S), total mTOR (Cell Signaling Technology, #2983S), P-S6K1 (Cell Signaling Technology, #9205S), total S6K1 (Cell Signaling Technology, #9202S), P -4EBP1 (Cell Signaling Technology, #2855S), total 4EBP1 (Cell Signaling Technology, #9644S), PRODH (Proteintech, # 22980-1-AP) and β-actin (Sigma, #A5441).

Techniques: Western Blot, Derivative Assay, Lysis, MTT Assay

Journal: Nucleic Acids Research

Article Title: Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress

doi: 10.1093/nar/gkab362

Figure Lengend Snippet: The ISR and mTORC1 are active in livers of mice treated with HF. WT and whole body Gcn2 –/– mice were injected with HF (0.5 mg/kg) or 0.5% DMSO vehicle. Following 8 h, one group of mice were sacrificed and liver tissues were measured for ISR and mTORC1 signaling, charging of tRNA Pro , and histological analyses. Additionally, another group of mice received a second injection of HF at 24 hours and were sacrificed after 44 h for liver analyses. ( A ) The indicated proteins were measured by immunoblot analyses with specific antibodies. Bar graphs to the right of the immunoblot panels show quantification of phosphorylated S6K1 and eIF2α from three biological replicates. The bar graphs are represented as the means ± SD and * P < 0.05. ( B ) Measurements of the percent uncharged tRNA Pro (top panel) and relative total tRNA Pro (bottom panel) by RT-qPCR. Significant differences between treatments are indicated. * P < 0.05; ** P < 0.005; *** P < 0.0005; **** P < 0.0001. ( C ) Left panel: hepatic PRODH expression was measured by immunoblot analyses with indicated antibodies; right panel: quantification of hepatic PRODH expression. ( D ) The ratio of hepatic glutamate to proline amino acid was measured. ( E ) Representative images of H&E staining of hepatic tissue for lipid droplets accumulation. Images were captured using light microscopy (100× magnification) (left panel). Quantitation is featured in the accompanying bar graph (right panel). Significant differences between treatments are indicated. *** P < 0.0008; **** P < 0.0001. ( F ) Hepatic tissue levels of TG were measured in lipid extracts. ( G ) Liver ALT levels were measured in the serum samples. Bar graphs are represented as the means ± SD, and significant differences between treatments are indicated: * P < 0.05; ** P < 0.005.

Article Snippet: Antibodies using in the immunoblot analyses include the following: P-GCN2 (Abcam, #Ab75836), total GCN2 (Abcam, #ab137543), P-eIF2α (Abcam, #ab32157), total eIF2α (Cell Signaling Technology, #5324S), ATF4 antibody was prepared against the corresponding recombinant human proteins, which were affinity purified , P-mTOR (Cell Signaling Technology, #2971S), total mTOR (Cell Signaling Technology, #2983S), P-S6K1 (Cell Signaling Technology, #9205S), total S6K1 (Cell Signaling Technology, #9202S), P -4EBP1 (Cell Signaling Technology, #2855S), total 4EBP1 (Cell Signaling Technology, #9644S), PRODH (Proteintech, # 22980-1-AP) and β-actin (Sigma, #A5441).

Techniques: Injection, Western Blot, Quantitative RT-PCR, Expressing, Staining, Light Microscopy, Quantitation Assay

Journal: Nucleic Acids Research

Article Title: Discordant regulation of eIF2 kinase GCN2 and mTORC1 during nutrient stress

doi: 10.1093/nar/gkab362

Figure Lengend Snippet: Model for discordant regulation of the GCN2 and mTORC1-directed pathways in response to HF. HF treatment sharply reduces aminoacylation of tRNA Pro , which contributes to accumulation of free amino acids and to pausing of elongating ribosomes at proline codons. The HF-triggered events, including accumulating uncharged tRNA Pro activate GCN2, leading to enhanced eIF2α-P and induction of the ISR that enhances cell viability. In parallel, there are high levels of mTORC1 activity, which contributes to enhanced translation of 5′-TOP mRNAs that signal elevated protein biogenesis. Enhanced mTORC1 signaling serves to reduce cell viability in response to HF.

Article Snippet: Antibodies using in the immunoblot analyses include the following: P-GCN2 (Abcam, #Ab75836), total GCN2 (Abcam, #ab137543), P-eIF2α (Abcam, #ab32157), total eIF2α (Cell Signaling Technology, #5324S), ATF4 antibody was prepared against the corresponding recombinant human proteins, which were affinity purified , P-mTOR (Cell Signaling Technology, #2971S), total mTOR (Cell Signaling Technology, #2983S), P-S6K1 (Cell Signaling Technology, #9205S), total S6K1 (Cell Signaling Technology, #9202S), P -4EBP1 (Cell Signaling Technology, #2855S), total 4EBP1 (Cell Signaling Technology, #9644S), PRODH (Proteintech, # 22980-1-AP) and β-actin (Sigma, #A5441).

Techniques: Activity Assay

Journal: Cell death discovery

Article Title: Exploring the potential of selective FKBP51 inhibitors on melanoma: an investigation of their in vitro and in vivo effects.

doi: 10.1038/s41420-025-02430-y

Figure Lengend Snippet: Fig. 3 SAFits impair TGF-β expression and signaling. Western blot assay showing both the monomeric and dimeric form of TGF-β (a) and p- Smad2/3 (b) in A375 melanoma incubated in the absence or presence of 20 nM SAFit 1 or 30 nM SAFit 2. γ-Tubulin and total Smad were used as loading control. Full length western blots are shown as Supplemental Material. A densitometric analysis of bands was performed using ImageJ 1.42q for Macintosh. c Relative normalized expression values of TβRI mRNA levels in A375 melanoma cells incubated for 5 h in the presence or absence of 20 nM SAFit 1 or 30 nM SAFit2. Relative quantitation of the transcript was performed using co-amplified β-Actin as an internal control for normalization. Lower, Western blot of protein extracted from the same cells for TβRI assay. Full gels are shown as Supplemental Material. d Representative images (10x magnification fields containing ≥100 cells) of transwell migration and invasion assay of A375 melanoma cells cultured 48 h in the presence of 20 nM SAFit 1 or 30 nM SAFit2. Means of cell count values and crystal violet O.D. quantization obtained from different experiments (N = 3) are indicated below.

Article Snippet: Primary antibodies against the following proteins were diluted as follows: Flag (M2, mouse monoclonal, Merck) 1:5000; β-Actin, (15G5A11/E2, mouse monoclonal, Thermo Fisher Scientific, Waltham, Massachusetts, USA) 1:5000; TGF-β (V; rabbit polyclonal, Santa Cruz Biotechnology) 1:500; γ-Tubulin (GTU-88, mouse monoclonal, Merck) 1:5000; phospho-Smad2 (Ser465/467, Cell Signaling Technology, Danvers, MA, USA) 1:500; Smad 2/3 (H465, rabbit polyclonal, Santa Cruz Fig. 5 SAFit-induced changes of TME composition in TAMs. a Representative flow cytometry gating of CD45+ cells infiltrating the tumors. b, c Graphic representation of cell count values from TME immunophenotyping (Tumor Infiltrating Leukocytes TILs: macrophages, B and T cells, NK) of SAFit untreated (white histograms) or treated (grey histograms) tumors. d Characterization of F4/80 macrophage component of the TME.

Techniques: Expressing, Western Blot, Incubation, Control, Quantitation Assay, Migration, Invasion Assay, Cell Culture, Cell Counting

Journal: Stem Cell Research & Therapy

Article Title: Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway

doi: 10.1186/s13287-022-02992-z

Figure Lengend Snippet: Metformin enhanced PDLSC osteogenic differentiation under high glucose via inhibition of the NPR3-mediated MAPK pathway. A Gene expression of C-type natriuretic peptide (CNP) in PDLSCs transfected with or without LV-NPR3 when they were cultured in normal, high glucose, or high glucose with metformin addition (mRNA expression levels detected by qRT-PCR). B Total CNP content in PDLSCs transfected with or without LV-NPR3 when they were cultured in normal, high glucose, or high glucose with metformin addition (total content in the cell culture supernates detected by ELISA). C , D Protein expression of MAPK pathway-related proteins (p38 MAPK, p-p38 MAPK, Erk1/2, p-Erk1/2, JNK, p-JNK) in PDLSCs transfected with or without LV-NPR3 when they were cultured in normal, high glucose, or high glucose with metformin addition (protein expression levels detected by Western blot analysis). The displayed bands were cropped from the corresponding original blots. HG, high glucose. Met, metformin. LV-NPR3, NPR3-overexpression lentivirus. Experiments for P4 cells from three different donors were repeated independently for at least 3 times, and data are presented as the means ± SD ( n = 3). p value was based on one-way analysis of variance (one-way ANOVA). * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences between the indicated columns, while NS represents no significant difference

Article Snippet: * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences between the indicated columns, while NS represents no significant difference To further validate our findings, p38 MAPK and Erk1/2 pathway were inhibited with specific inhibitors SB203580 (MedChemExpress, Monmouth Junction, NJ, USA) and U0126 (MedChemExpress) respectively.

Techniques: Inhibition, Gene Expression, Transfection, Cell Culture, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Western Blot, Over Expression

Journal: Stem Cell Research & Therapy

Article Title: Metformin combats high glucose-induced damage to the osteogenic differentiation of human periodontal ligament stem cells via inhibition of the NPR3-mediated MAPK pathway

doi: 10.1186/s13287-022-02992-z

Figure Lengend Snippet: Inhibition of the NPR3-mediated p38 MAPK or Erk1/2 pathway enhanced PDLSC osteogenic differentiation under glucose. A Expression of osteoblast differentiation-related genes ( ALP , RUNX2 , BMP2 and OCN ) in PDLSCs transfected with or without LV-NPR3 when they were cultured in high glucose with metformin addition or high glucose with metformin and SB203580 addition following a 14-day osteogenic induction (mRNA expression levels detected by qRT-PCR). B Expression of osteoblast differentiation-related genes ( ALP , RUNX2 , BMP2 and OCN ) in PDLSCs transfected with or without LV-NPR3 when they were cultured in high glucose with metformin addition or high glucose with metformin and U0126 addition following a 14-day osteogenic induction (mRNA expression levels detected by qRT-PCR). C Expression of osteoblast differentiation-related proteins (ALP, RUNX2, BMP2 and OCN) in PDLSCs transfected with or without LV-NPR3 when they were cultured in high glucose with metformin addition or high glucose with metformin and SB203580 addition following a 14-day osteogenic induction (protein expression levels detected by Western blot analysis). D Expression of osteoblast differentiation-related proteins (ALP, RUNX2, BMP2 and OCN) in PDLSCs transfected with or without LV-NPR3 when they were cultured in high glucose with metformin addition or high glucose with metformin and U0126 addition following a 14-day osteogenic induction (protein expression levels detected by Western blot analysis). The displayed bands were cropped from the corresponding original blots. HG, high glucose. Met, metformin. LV-NPR3, NPR3-overexpression lentivirus. SB203580, p38 MAPK pathway inhibitor. U0126, Erk1/2 pathway inhibitor. Experiments for P4 cells from three different donors were repeated independently for at least 3 times, and data are presented as the means ± SD ( n = 3). p value was based on one-way analysis of variance (one-way ANOVA). * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences between the indicated columns, while NS represents no significant difference

Article Snippet: * p < 0.05, ** p < 0.01, and *** p < 0.001 represent significant differences between the indicated columns, while NS represents no significant difference To further validate our findings, p38 MAPK and Erk1/2 pathway were inhibited with specific inhibitors SB203580 (MedChemExpress, Monmouth Junction, NJ, USA) and U0126 (MedChemExpress) respectively.

Techniques: Inhibition, Expressing, Transfection, Cell Culture, Quantitative RT-PCR, Western Blot, Over Expression

Journal: Pathogens and Disease

Article Title: A suitable and efficient optimization system for the culture of Chlamydia trachomatis in adult inclusion conjunctivitis

doi: 10.1093/femspd/ftae020

Figure Lengend Snippet: Growth and characterization of six epithelial cell lines for culture of C. trachomatis . (A) Morphology of MA104, MA104 N*V, HCEC, Hela, BGMK, and Vero cells under contrast phase microscope. Scale bar = 100 µm. (B) Immunofluorence staining of STAT1 and IRF3 within MA104 and MA104 N*V cells. Nuclei were counterstained with DAPI. Scale bar = 20 µm. (C) Western blotting demonstrating the decreased expression of STAT1 and IRF3 in MA104 N*V cells. GAPDH was used as a loading control. (D) RT-qPCR showing the significantly decreased expression levels of STAT1 and IRF3 in the MA104 N*V cell line (** P < .01, *** P < .001).

Article Snippet: Nuclei were counterstained with DAPI (C10005, Beyotime Biotechnology), and representative images were captured using an Olympus microscope (Olympus BX-51, Olympus, Tokyo, Japan).

Techniques: Microscopy, Staining, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: Pathogens and Disease

Article Title: A suitable and efficient optimization system for the culture of Chlamydia trachomatis in adult inclusion conjunctivitis

doi: 10.1093/femspd/ftae020

Figure Lengend Snippet: Slit-lamp images from seven confirmed AIC cases, accompanied by laboratory test in MA104 N*V cells with IF and Giemsa staining. The arrow indicates typical C. trachomatis intracytoplasmic inclusions detected under the microscope. Scale bar = 50 µm.

Article Snippet: Nuclei were counterstained with DAPI (C10005, Beyotime Biotechnology), and representative images were captured using an Olympus microscope (Olympus BX-51, Olympus, Tokyo, Japan).

Techniques: Staining, Microscopy

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1-BARD1 ubiquitinates TOP2B in a phosphorylation-dependent manner. ( a ) (i) Immunoprecipitation with control IgG against 5 mg of HeLa NE. P, pellet (bound) and S, supernatant (unbound) fraction. Pellet and supernatant were loaded at 1 : 10 and 1 : 100 inputs, respectively. H chain, IgG heavy chain. (ii) Immunoprecipitation of TOP2B antibody against HeLa NE, followed by immunoblotting. ( b ) Immunoblotting data showing that BARD1 KD decreases the level of TOP2B proteins and ubiquitination. CDK9 was used as a loading control. HeLa NE was included as a technical control. ( c ) In vitro ubiquitination assay followed by immunoblotting showing discrete bands at about 56, 68, 81 and 91 KDa for 0, 1 (U•), 2 (U••) and 3 (•••) ubiquitin proteins ligated to TOP2B 1–566 by SA BRCA1-BARD1. W, WT BRCA1; A, SA BRCA1; D, SD BRCA1. ( d ) In vitro ubiquitination assay and immunoblotting screening the E2 enzymes, UBCH1, UBCH3, UBCH5b, UBCH6 and UBCH13/MMS2 for the ubiquitination of TOP2B deubi in collaboration with SA BRCA1-BARD1. Red boxes indicate UBCH5b and UBCH13/MMS2 to collaborate with the BRCA1-BARD1 complex to ubiquitinate TOP2B.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Immunoprecipitation, Control, Western Blot, Ubiquitin Proteomics, In Vitro

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 regulates serum-induced transcriptional activation. ( a ) Schematic overview of cell cycle synchronization and serum-induced transcriptional activation in hIEGs with or without chemical kinase inhibitors. FBS, fetal bovine serum. ( b ) ChIP-qPCR showing BRCA1 (i) and pBRCA1 (ii) occupancy at FOS , MYC , JUN and EGR1 . S0, serum-starved cells at G 0 ; S15, serum-induced cells. Error bars show standard deviations (s.d., n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05. ( c ) BRCA1 and BARD1 KD using siRNA species. (i), immunoblotting results showing the protein level of BRCA1, BARD1 and α-tubulin (tubulin, loading control, 70 µg cell lysate/lane) using siRNA species targeting BRCA1 (si-BR1) or BARD1 (si-BD1). SCR, scrambled siRNA. (ii, iii), BRCA1 and BARD1 mRNA expression in SCR control versus BRCA1 and BARD1 KD cells. Error bars show s.d. ( n = 3). ** p < 0.005. ( d ) qRT-PCR data showing the effects of BRCA1 or BARD1 KD on EGR1 (i), JUN (ii) and FOS (iii) mRNA expression. β-actin was used as a reference gene. Error bars show s.d. ( n = 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01. ( e ) ChIP-qPCR showing impaired Pol II, S2 Pol II and TOP2B recruitment upon gene activation at EGR1 (i), MYC (ii), FOS (iii) and JUN (iv) in BRCA1 KD cells. Error bars show s.d. ( n ≥ 3). **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Activation Assay, ChIP-qPCR, Western Blot, Control, Expressing, Quantitative RT-PCR

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 is phosphorylated by ATM and ATR upon transcriptional activation. ( a ) ChIP-qPCR of pBRCA1 with or without KU55933 (ATMi) in the EGR1 TSS. Error bars show s.d. ( n = 2). * p < 0.05. ( b ) ChIP-qPCR of pBRCA1 with or without VE-821 (ATRi) in EGR1 TSS. Error bars show s.d. ( n = 2). * p < 0.05. ( c ) ChIP-qPCR showing that caffeine, which inhibits both ATM and ATR, alleviates the accumulation of pBRCA1 on EGR1 TSS upon transcriptional activation. Error bars show s.d. ( n = 3). * p < 0.05. ( d ) Immunoblotting showing that caffeine reduces the cellular level of pBRCA1 overall and upon transcriptional activation. α-tubulin was used as a loading control. ( e ) qRT-PCR showing the reduction of EGR1 mRNA level in caffeine-treated HEK293 cells upon transcriptional activation. β-actin was used as a reference and normalizer. Error bars show in s.d. ( n = 3). * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Activation Assay, ChIP-qPCR, Western Blot, Control, Quantitative RT-PCR

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 phosphorylation at S1524 is important for transcriptional activation. ( a ) Schematic overview of immobilized template and in vitro transcription assays. ( b ) Validation of PIC formation on the EGR1 TSS (–423 to +332) using immunoblotting. ( c ) In vitro transcription assay using recombinant WT, S1524A (SA), and S1524D (SD) BRCA1 showing that SD activates EGR1 transcription. ( d ) Quantification of the efficiency of S1524A and S1524D BRCA1 mutant proteins in stimulating transcription, relative to that of WT BRCA1. Error bars show s. d. ( n = 3). ** p < 0.002, * p < 0.02. ( e ) Immunofluorescence-confocal microscopy results showing increased levels of TOP2B and pBRCA1 in nuclei upon etoposide treatment. pBRCA1 intensity was quantified. n = 65. **** p < 0.0001. ( f ) Chromosome views of total BRCA1 (red) and TOP2B (etoposide-trapped, blue), and input control (grey) on representative hIEGs, EGR1 , JUN , MYC and FOS , illuminating genomic colocalization and functional collaboration. Black bars indicate binding sites of a given factor, identified by HOMER with a false discovery rate (FDR)-adjusted p -value of 0.001.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Activation Assay, In Vitro, Biomarker Discovery, Western Blot, Transcription Assay, Recombinant, Mutagenesis, Immunofluorescence, Confocal Microscopy, Control, Functional Assay, Binding Assay

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: TOP2B active (etoposide-captured) and BRCA1-binding sites are largely overlapped genome-wide. ( a ) Venn diagram showing the overlap of BRCA1 and TOP2B-binding sites in MCF10A cells. ( b ) Heat maps of the genes co-occupied by TOP2B and BRCA1 ( n = 6587).

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Binding Assay, Genome Wide

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: TOP2B and BARD1 bind to EGR1 TSS between –132 and –15. ( a ) DNA sequence of EGR1 TSS (–132 to +332). Orange flash signs indicate the restriction enzyme sites for NruI, SfoI and SacII used to map the factor binding region. Coloured letters indicate those that were subjected to mutations for the purposes indicated in the text. Altered sequences are presented under the original ones. TOP2B and BARD1 mutual binding site mapped in this study was boxed with light green. ( b ) Immobilized template assay combined with restriction enzyme digestion using EGR1 TSS (–423 to +332) and SacII (Sac, to digest at +92), followed by immunoblotting. SacII added immediately after PIC formation or NTP addition. Un, undigested template DNA; I.B., immunoblotting probing BARD1 and TOP2B; DNA, native PAGE detecting the released DNA fragment (241 nt) after SacII digestion; Silver, silver staining visualizing the proteins bound on the DNA. P, pellet; S, supernatant fraction. ( c ) Immobilized template assay using the EGR1 template (−132 to +332) in the presence or absence of BRCA1, combined with restriction enzyme digestion with NruI and SfoI that cut at −15 and +68, respectively. Recombinant WT BRCA1 was added at T2, immediately before the template was digested by restriction enzymes. The pellet and supernatant fractions were analysed by immunoblotting. ( d ) (i) Diagram of the four EGR1 TSS fragments. (ii) EMSA followed by silver staining showing TOP2B ubi binding to EGR1 TSS fragments with different affinities. The strongest binding was observed with EGR1 TSS no. 3 (–132 to +62). SM, size marker; P, TOP2B ubi . The second lane shows TOP2B ubi only.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Sequencing, Binding Assay, Western Blot, Clear Native PAGE, Silver Staining, Recombinant, Marker

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: BRCA1 phosphorylation controls TOP2B ubiquitination and DNA-binding affinity. ( a ) Purified TOP2B ubi (ubi) and TOP2B deubi (Deu) shown by silver staining and immunoblotting. ( b ) EMSA comparing TOP2B ubi versus TOP2B deubi for their binding affinity to EGR1 TSS no. 3 (–132 to +62). Silver-stained. SM, DNA size marker. ( c ) Ubiquitinated TOP2B binds to DNA with much higher affinity than deubiquitinated one. A plot summarizing EMSA to derive K D values. ( d ) Immobilized template assay results. Tight TOP2B association with EGR1 TSS (Pellet, –423 to +332) before NTP addition, regardless of recombinant BRCA1 species added at T1 during PIC formation. ( e ) Immobilized template assay results. Comparison of the degrees of TOP2B associated (Pellet, P) with and dissociated from EGR1 TSS (Supernatant, S) when WT, SA and SD BRCA1 were added after NTP addition (T2). ( f ) Quantification of TOP2B in pellet (DNA bound, i) and in supernatant (released, ii) in immobilized template assays. Error bars in s.d. ( n = 3). *** p < 0.005, ** p < 0.01, * p < 0.05.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, Ubiquitin Proteomics, Binding Assay, Purification, Silver Staining, Western Blot, Staining, Marker, Recombinant, Comparison

Journal: Open Biology

Article Title: BRCA1-BARD1 regulates transcription through modulating topoisomerase IIβ

doi: 10.1098/rsob.210221

Figure Lengend Snippet: Model of the BRCA1-BARD1 complex-mediated TOP2B regulation. During the resting state of transcription, Pol II is paused in the promoter-proximal site in hIEGs. The pausing is induced and stabilized by various factors including transcription factors, nucleosome modifiers and nucleic acids. The BRCA1-BARD1 complex that is engaged with hIEGs interacts with and ubiquitinates TOP2B in the EGR1 TSS. This ubiquitination (marked as ub) confers an enhanced DNA-binding affinity and stability to TOP2B (Tight TOP2B). In transcriptional activation, ATM/ATR phosphorylates its substrates including BRCA1 at S1524 (phosphorylation, marked as P in a blue circle). Phosphorylated BRCA1 alters the functional interaction between BRCA1-BARD1 and TOP2B to mitigate TOP2B ubiquitination. This event destabilizes and loosens TOP2B (Loose TOP2B) from the TSS. The interplay between BRCA1-BARD1 complex and TOP2B appears to be crucial in transcriptional regulation of hIEGs. Lightning marks are proposed catalysis sites of TOP2B.

Article Snippet: The antibodies used in immunoprecipitation were Pol II (ab817, Abcam; no. 2629, Cell signaling; A304-405A, Bethyl Laboratories), phosphorylated S2 Pol II (ab5095, Abcam), BARD1 (A300-263A, Bethyl Laboratories), TOP2B (A300-949A, Bethyl Laboratories; sc-25330, Santa Cruz Biotechnology), BRCA1 (A300-000A, Bethyl Laboratories; sc-6954, Santa Cruz Biotechnology), phosphorylated BRCA1 (S1524) (A300-001A, Bethyl Laboratories; NB100-200, Novus Biologicals) and IgG (sc-2025, Santa Cruz Biotechnology).

Techniques: Ubiquitin Proteomics, Binding Assay, Activation Assay, Phospho-proteomics, Functional Assay