Journal: iScience

Article Title: Histone H2A ubiquitination resulting from Brap loss of function connects multiple aging hallmarks and accelerates neurodegeneration.

doi: 10.1016/j.isci.2022.104519

Figure Lengend Snippet: Figure 6. Histone H2A ubiquitination accompanied by BRCA1 activation is the hallmark phenotype of BRAP LOF (A) Immunoblotting of histone extracts from NPCs as well as from embryonic, neonatal, adult cerebral cortical tissues, and quantification (Mean G SD) of increases in histone H2Aub (total H2Aub and H2AubK119, respectively) resulted from Brap LOF. n = 3–6 biological replicates. p-values calculated by Student’s t test are indicated. (B) Immunoblotting of Brca1 in various cells and tissues, showing that Brap LOF results in increased Brca1 abundance. (C) Immunoblotting of nuclear vs cytoplasmic fractions of MEFs at P1, showing increased nuclear localization of Brca1 in Brap/ cells. (D) Brca1 (red) and NeuN (green) double immunohistology images of cerebral cortical sections of BrapcKONPC and control mice at four months of age. Representative images are shown. Note the increased intensity and density of Brca1 puncta in the nuclei of BrapcKONPC cortical neurons (NeuN+). (E and F) Immunoblotting analyses of histone extracts from cerebral cortical tissues of three-month-old mice, showing increased ubiquitination of H2A variants targeted by Brca1 (E) along with total histone H2A ubiquitination (F). (G) Double immunohistology staining of cortical sections of 4-month old WT or BrapcKONPC mice with antibodies against Gfap (green) and histone H3 (red), showing reduced nuclear histones in cells surrounded by reactive astrocytes (circles) in BrapcKONPC cortical tissues. Representative images are shown. Nuclear DNA was stained with Hoechst 33342. Bars: 50 um or as indicated. See also Figure S3.

Article Snippet: Phospho-p53 (Ser15) Abcam Cat# ab1431; RRID:AB_301090 Phospho-p53 (Ser15) (D4S1H) Cell Signaling Technology Cat# 12571; RRID:AB_2714036 Phospho-p53 (Ser15) (16G8) Cell Signaling Technology Cat# 9286; RRID:AB_331741 Phospho-ATM(Ser1981) Novus Biologicals Cat# AF1655 Phospho-ATR (Ser 428) Cell Signaling Technology Cat# 2853; RRID:AB_2290281 53 BP1 Novus Biologicals Cat# NB100-304 Brca1 Santa Cruz Biotechnology Cat# sc-642; RRID:AB_630944 Brca1 Novus Biologicals Cat# MAB22101 Brca1 (clone 8F7) Novus Biologicals Cat# NBP1-41186 LC3B (G-9) Santa Cruz Biotechnology Cat# sc-376404; RRID:AB_11150489 LC3B Cell Signaling Technology Cat# 2775; RRID:AB_915950 LC3B Novus Biologicals Cat# NB100-2220 p62SQSTM1 Novus Biologicals Cat# MAB8028 p62SQSTM1 GeneTex Cat# GTX100685; RRID:AB_2038029

Techniques: Ubiquitin Proteomics, Activation Assay, Western Blot, Control, Staining

Journal: DNA repair

Article Title: DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR.

doi: 10.1016/j.dnarep.2022.103289

Figure Lengend Snippet: Fig. 6. Damage induced sumoylation of Sp1 and interaction with RNF4 are necessary for BRCA1 recruitment. (a-h) U2OS were transduced with shRNA against RNF4 or non-targeting (a), or endogenous Sp1 was depleted using CRISPR/Cas9 and Flag-tagged Sp1WT or Sp1K16R was expressed using lentiviral transduction. (a,c,e,g) Cells were pretreated with 10 μM BrdU for 3 h to label cells in S phase (blue). Cells were treated with 0.1 μM Adriamycin for 1 h and processed for immunofluo rescence using 53BP1, BRCA1, γH2Ax, or BrdU antibodies. (b,d,f,h) Cells expressing I-Ppol were synchronized in G1 or S phase followed by DSB induction with 4- OHT. DNA was isolated from cell lysates at various time points post-induction of I-PpoI, and ChIP was performed using a 53BP1 or BRCA1 antibody, followed by qPCR using primers flanking the break site.r

Article Snippet: Antibodies used were γH2Ax (Millipore Sigma 05–636), γH2Ax (Abcam ab11174), Sp1pS101 [29], 53BP1 (Millipore MAB3802), BRCA1 (Santa Cruz sc-642), BRCA1 (Novus Biologicals 6B4), and BrdU (Abcam ab6326).

Techniques: Transduction, shRNA, CRISPR, Expressing, Isolation

Journal: DNA repair

Article Title: DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR.

doi: 10.1016/j.dnarep.2022.103289

Figure Lengend Snippet: Fig. 7. Sumoylation of Sp1 and its interaction with RNF4 are necessary for HR. (a) U2OS cells expressing EJ5 or DR-GFP reporter constructs were depleted of Sp1 using CRISPR/Cas9 and were transduced with Flag-tagged Sp1WT or Sp1K16R. Knockdown of DNA-PK and BRCA1 in U2OS cells expressing EJ5 or DR-GFP reporter constructs were used as controls. Cells were infected with I-Sce1 lentivirus. 72 h post infection, DNA repair was assessed by flow cytometry of GFP positive cells. (b) In cells expressing I-Ppol, Sp1 was knocked out using sgRNA targeting Sp1 and either Flag-tagged Sp1WT, Sp1K16R, Sp1–30N was expressed by lentiviral transduction. Cells were synchronized in G1 or S phase followed by DSB induction with 4-OHT. DNA was isolated from cell lysates at various time points post-induction of I-PpoI, and ChIP was performed using a γH2Ax antibody, followed by qPCR using primers spanning the break site. (c) Sp1 was knocked out in UWB 1.289 BRCAD11q or BRCA1+/+ cells and Flag-tagged Sp1WT, Sp1–30N, Sp1K16R, or Sp1K16R with 53BP1 additionally knocked down by shRNA were expressed. (c) Cells were seeded in agar and treated with indicated doses of Olaparib. Cells were incubated for 14 days followed by staining and quantification. (d) Cells were pretreated with 10 μM BrdU for 3 h to label cells in S phase (blue). Cells were treated with 0.1 μM Adriamycin for 1 h and processed for immunofluorescence using BRCA1, γH2Ax, or BrdU antibodies. (a-c) Data represent means and SEM from 3 independent experiments assessed in triplicate. Significant differences between groups were determined by two-tailed Student’s t-test. *, ** or *** indicate p values < 0.05, 0.01 or 0.001, respectively. No * indicates p value > 0.05.

Article Snippet: Antibodies used were γH2Ax (Millipore Sigma 05–636), γH2Ax (Abcam ab11174), Sp1pS101 [29], 53BP1 (Millipore MAB3802), BRCA1 (Santa Cruz sc-642), BRCA1 (Novus Biologicals 6B4), and BrdU (Abcam ab6326).

Techniques: Expressing, Construct, CRISPR, Transduction, Knockdown, Infection, Flow Cytometry, Isolation, shRNA, Incubation, Staining, Immunofluorescence, Two Tailed Test

Journal: Cancer cell

Article Title: Therapeutic Targeting of CDK12/CDK13 in Triple-Negative Breast Cancer.

doi: 10.1016/j.ccell.2019.09.004

Figure Lengend Snippet: Figure 2. SR-4835 Downregulates DNA Damage Repair Proteins, and Provokes DNA Damage and Apoptosis in TNBC (A) Analysis of indicated gene expression by qPCR at 6 h with the indicated concentrations of SR-4835 or vehicle in MDA-MB-231 cells. Data are presented as mean values ± SD of triplicate points. *p < 0.05, **p < 0.01, ***p < 0.001 by t test. (B) Analysis of the indicated proteins at selected time points and concentrations of SR-4835 by immunoblot in MDA-MB-231 cells. GAPDH used as loading control. (C) Representative images of MDA-MB-231 cells treated with the indicated concentrations of SR-4835 or vehicle for the indicated times and then assessed for BRCA1 (green) and g-H2AX (red) foci by confocal immunofluorescence microscopy. Nuclei were stained with Hoechst (blue). Scale bar represents 10 mm. (D) Quantification of experiment presented in (C) using IN Cell Analyzer software. + g-H2AX represents cells with more than ten g-H2AX foci detected. + g-H2AX pan-nuclear are cells in which less than ten foci could be detected, but the intensity of the signal surpasses a threshold selected for each experiment based on (legend continued on next page)

Article Snippet: Cells were then incubated in primary antibodies against BRCA1 (Cell Signaling, Cat#9010S), and g-H2AX (S139, Millipore, clone JBW301).

Techniques: Gene Expression, Western Blot, Control, Microscopy, Staining, Software

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 was analyzed using immunoblotting in Cal-27 cells under cisplatin treatment. B , BRCA1 attenuated the cisplatin-induced decrease of miR-593-5p. Cal-27 cells were transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) and then treated with cisplatin for 24h. miR-593-5p were detected using qRT-PCR (upper panel), whereas BRCA1 was analyzed using immunoblotting (lower panel). *P< 0.01 versus cisplatin alone. C , ChIP-qPCR analysis of BRCA1 binding to the promoter of miR-593-5p in the BS3 region. **P< 0.001. D , ChIP-qPCR analysis of the association levels of BRCA1 with the miR-593-5p promoter in the BS3 region under cisplatin treatment. E , A luciferase assay indicated that cisplatin induced a reduction of miR-593-5p promoter activity in the BS3 region. Cal-27 cells were transfected with the wild-type promoter (wt) in the BS3 or empty vector (pGL3-Basic). F , A luciferase assay indicated that BRCA1 activated miR-593-5p promoter activity in the BS3 region. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or empty vector (Vec) were treated with the wild-type promoter (wt) or a promoter with mutations in the BS3 (mut). **P< 0.001.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Western Blot, Transfection, Expressing, Plasmid Preparation, Quantitative RT-PCR, Binding Assay, Luciferase, Activity Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , BRCA1 attenuated mitochondrial fission in Cal-27 cells under cisplatin treatment. Cal-27 cells transiently transfected with BRCA1 expressing plasmids or vector control (Vec) were treated with cisplatin for 24h. Scale bar equals 3 μm. **P < 0.001 versus cisplatin alone. B , C and D , Apoptosis was detected using TUNEL, flow cytometry, and caspase-3/7 activity assays. *P < 0.01 versus cisplatin alone; **P < 0.001 versus cisplatin alone. E , The knockdown of miR-593-5p leads to the attenuation of the BRCA1 inhibitory effect on MFF protein levels under cisplatin treatment. Cal-27 cells stably expressing BRCA1 or vector control (Vec) were transfected with miR-593-5p inhibitors or inhibitor-negative control (inhibitor-NC). MFF levels were analyzed using immunoblotting. F , Mitochondrial fission and apoptosis were detected via staining with MitoTracker Red and TUNEL.*P < 0.01.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Transfection, Expressing, Plasmid Preparation, TUNEL Assay, Flow Cytometry, Activity Assay, Stable Transfection, Negative Control, Western Blot, Staining

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A, B, C, BALB/c-nu mice bearing Cal-27 cells with the stable expression of MFF shRNA or its scramble form (sc) were treated with saline or cisplatin. ( A ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( B ) Representative photomicrographs of tumors from each group at day 35. ( C ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. D, E, F, BALB/c-nu mice bearing Cal-27 cells with the stable expression of miR-593-5p or its control (con) were treated with saline or cisplatin. ( D ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( E ) Representative photomicrographs of tumors from each group at day 35. ( F ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone. G, H, I, BALB/c-nu mice bearing Cal-27 cells with the stable expression of BRCA1 or empty vector (Vec) were treated with saline or cisplatin. ( G ) Tumor growth curves for Cal-27 tumors treated with saline or cisplatin. ( H ) Representative photomicrographs of tumors from each group at day 35. ( I ) Apoptosis was detected via TUNEL assay. n=6 for each group. For TUNEL assay, n=24 slices from 6 xenograft tumors were sampled per group. Bar=20 μm; # P < 0.05 versus cisplatin alone; *P< 0.01 versus cisplatin alone.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, shRNA, TUNEL Assay, Plasmid Preparation

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: A , MFF, miR-593-5p and BRCA1 expression and apoptosis were demonstrated in cisplatin-sensitive versus non-sensitive TSCCs. Left panel: MFF and BRCA1 expression were analyzed via immunohistochemistry; miR-593-5p expression was analyzed using in situ hybridization (×200). Apoptosis was detected using a TUNEL assay. Bar=20 μm. Right panel: Quantification of MFF, miR-593-5p and BRCA1 expression in cisplatin-sensitive versus non-sensitive TSCCs. # P < 0.05; *P < 0.01. B , Associations between MFF, miR-593-5p and BRCA1 expression in TSCCs were analyzed via Spearman order correlation. C , Kaplan-Meier survival curves for TSCCs are plotted for MFF, miR-593-5p and BRCA1 expression, and survival differences were analyzed using a log-rank test. D , Model of the BRCA1–miR-593-5p–MFF axis in regulating mitochondrial fission and cisplatin sensitivity. The dotted line indicates the commonly accepted mechanism of BRCA1 regulation of cisplatin sensitivity, whereas the solid line represents the novel mechanism of BRCA1-mediated cisplatin sensitivity identified in the present study.

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing, Immunohistochemistry, In Situ Hybridization, TUNEL Assay

Journal: Oncotarget

Article Title: Mitochondrial fission determines cisplatin sensitivity in tongue squamous cell carcinoma through the BRCA1–miR-593-5p–MFF axis

doi:

Figure Lengend Snippet: Correlation among clinicopathological status and the expression of MFF, miR-593-5p or BRCA1 in TSCC patients

Article Snippet: An MFF shRNA retrovirus vector (pSR-puro-MFF1 shRNA, 37247) [ ] and a BRCA1 (pBABE-puro HA BRCA1, 14999) [ ] retrovirus vector were obtained from Addgene (MA, USA).

Techniques: Expressing