anti brca1  (Santa Cruz Biotechnology)

Journal: Nucleic Acids Research

Article Title: USP37 counteracts HLTF to protect damaged replication forks and promote survival of BRCA1-deficient cells and PARP inhibitor resistance

doi: 10.1093/nar/gkaf544

Figure Lengend Snippet: USP37 is a novel target showing synthetic lethality with BRCA1 loss. ( A ) Schematic of the CRISPR screen to define the genetic interaction with BRCA1 loss with or without treatment with 25 nM olaparib (Ola) in HeLa BRCA1 mAID cells. Ranking of co-essential genes in HeLa BRCA1 −/− not treated with olaparib ( B ) or treated with olaparib ( C ) based on drugZ analysis of the results of CRISPR/Cas9 screen. The Z -score was used to define possible vulnerability to cell lethality with BRCA1 loss with or without treatment with olaparib. ( D ) Western blot analysis to confirm USP37 knockout (KO) in HeLa BRCA1 mAID cells. WT: wild type. ( E ) Representative crystal violet images of the clonogenic survival assay in USP37-depleted HeLa BRCA1 mAID cells treated with or without BRCA1 depletion and/or olaparib. ( F ) Quantification of the data in panel (E). Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. * P < .05, ** P < .01, *** P < .001, **** P < .0001. ( G ) Western blot analysis to confirm USP37 KO in HEK293A BRCA1 dTAG cells with or without induction of BRCA1 degradation. ( H ) Representative crystal violet images of the clonogenic survival assay in USP37-depleted HEK293A BRCA1 dTAG cells treated with the indicated doses of dTAG V -1 (50 and 100 nM) or dTAG V -1 Neg (100 nM). ( I ) CellTiter-Glo assay to examine the synthetic lethality of USP37 loss and BRCA1 loss in HEK293A BRCA1 dTAG cells with various doses of dTAG V -1 NEG or dTAG V -1 treatment.

Article Snippet: Antibodies used in the current study include BRCA1 (sc-6954, Santa Cruz Biotechnology), Vinculin (V9131, Millipore Sigma), Flag (F3165, Millipore Sigma), β-tubulin (T5168, Millipore Sigma), RAD51 (ab63801, Abcam), RPA2 (2208S, Cell Signaling Technology), USP37 (ab190184, Abcam), HLTF (43345S, Cell Signaling Technology), 53BP1 (NB100-304, Novus Biologicals), PCNA (sc-56, Santa Cruz Biotechnology), GAPDH (sc-32233, Santa Cruz Biotechnology), DNA-PKcs (ab70250, Abcam), phospho-DNA-PKcs S2056 (ab18192, Abcam), KAP1 (A300-274A, Bethyl Laboratories), phospho-KAP1 S824 (4127 S, Cell Signaling Technology), phospho-ATM S1981 (13050 S; Cell Signaling Technology), ATM (2873S; Cell Signaling Technology), ATR (2790S; Cell Signaling Technology), phospho-ATR T1989 (30632 S; Cell Signaling Technology), phospho-CHK2 Thr68 (2661 S, Cell Signaling Technology), phospho-H2AX S139 (9718 S, Cell Signaling Technology), phospho-H2AX S139 (05-636l, Millipore Sigma), phospho-CHK1 Ser345 (2348 S, Cell Signaling Technology), phospho-CHK1 Ser317 (12302 S, Cell Signaling Technology), phospho-RPA32 S4/S8 (A300-245A, Bethyl Laboratories), and CHK2 (6334 S, Cell Signaling Technology).

Techniques: CRISPR, Western Blot, Knock-Out, Clonogenic Cell Survival Assay, Derivative Assay, Glo Assay

Journal: Nucleic Acids Research

Article Title: USP37 counteracts HLTF to protect damaged replication forks and promote survival of BRCA1-deficient cells and PARP inhibitor resistance

doi: 10.1093/nar/gkaf544

Figure Lengend Snippet: Depletion of USP37 results in genomic instability and uncontrolled degradation of nascent replication forks in BRCA1-deficient cells. ( A ) Representative images of γH2AX staining following treatment with olaparib (Ola; 2 μM) for 48 h in WT and USP37 KO HeLa BRCA1 mAID cells. BRCA1 −/− cells were cells with BRCA1 degradation following treatment with 5-Ph-IAA and doxycycline. BRCA1 +/+ were BRCA1 WT cells. NT: no treatment. Scale bar, 10 μm. ( B ) Statistical quantification of γH2AX foci formation per cell from panel (A). Mean number of γH2AX foci per cell was analyzed in >100 cells per sample. P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. **** P < .0001. ( C ) Statistical quantification of percentage of cells with micronuclei from panel (A). At least 100 cells were analyzed per sample over three independent experiments. Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. ** P < .01. ( D ) Representative images of RAD51 staining following treatment with olaparib (2 μM) for 48 h in WT and USP37 KO HeLa BRCA1 mAID cells. Scale bar, 10 μm. ( E ) Quantification of data in panel (D). Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. ns: not significant. ( F ) Top, a schematic of IdU/CIdU pulse-labeling followed by a 4-h treatment with hydroxyurea (HU; 5 mM). Bottom, representative images of ldU and CIdU replication tracks in HU-treated WT or USP37 KO HeLa BRCA1 +/+ and BRCA1 −/− cells. ( G ) Dot plot of CIdU to ldU tract length ratios for individual replication forks in cells from panel (F). The median value of 100 or more IdU and CldU tracts per experimental condition is indicated. Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. * P < .05, ** P < .01, *** P < .001, **** P < .0001.

Article Snippet: Antibodies used in the current study include BRCA1 (sc-6954, Santa Cruz Biotechnology), Vinculin (V9131, Millipore Sigma), Flag (F3165, Millipore Sigma), β-tubulin (T5168, Millipore Sigma), RAD51 (ab63801, Abcam), RPA2 (2208S, Cell Signaling Technology), USP37 (ab190184, Abcam), HLTF (43345S, Cell Signaling Technology), 53BP1 (NB100-304, Novus Biologicals), PCNA (sc-56, Santa Cruz Biotechnology), GAPDH (sc-32233, Santa Cruz Biotechnology), DNA-PKcs (ab70250, Abcam), phospho-DNA-PKcs S2056 (ab18192, Abcam), KAP1 (A300-274A, Bethyl Laboratories), phospho-KAP1 S824 (4127 S, Cell Signaling Technology), phospho-ATM S1981 (13050 S; Cell Signaling Technology), ATM (2873S; Cell Signaling Technology), ATR (2790S; Cell Signaling Technology), phospho-ATR T1989 (30632 S; Cell Signaling Technology), phospho-CHK2 Thr68 (2661 S, Cell Signaling Technology), phospho-H2AX S139 (9718 S, Cell Signaling Technology), phospho-H2AX S139 (05-636l, Millipore Sigma), phospho-CHK1 Ser345 (2348 S, Cell Signaling Technology), phospho-CHK1 Ser317 (12302 S, Cell Signaling Technology), phospho-RPA32 S4/S8 (A300-245A, Bethyl Laboratories), and CHK2 (6334 S, Cell Signaling Technology).

Techniques: Staining, Derivative Assay, Labeling

Journal: Nucleic Acids Research

Article Title: USP37 counteracts HLTF to protect damaged replication forks and promote survival of BRCA1-deficient cells and PARP inhibitor resistance

doi: 10.1093/nar/gkaf544

Figure Lengend Snippet: USP37 is required for cell survival in response to replication stress, and it may mediate RPA deubiquitination. ( A ) 293T cells were transfected with Myc-tagged USP37 and SFB-tagged RPA1, RPA2, or RPA3. Lysates were subjected to co-immunoprecipitation (IP) assays using streptavidin-binding beads and immunoblotted with indicated antibodies. ( B ) HEK293T cells were co-transfected with indicated plasmids for in vivo deubiquitination assay. After 48 h, cells were treated with MG132 for 6 h before harvest. SFB-RPA2 was immunoprecipitated by streptavidin beads. Blots were probed with the indicated antibodies. HU: hydroxyurea; WT: wild type. ( C ) HeLa WT and USP37 KO cells were co-transfected with indicated plasmids for in vivo deubiquitination assay. After 48 h, cells were treated with MG132 for 6 h before harvest. SFB-RPA2 was immunoprecipitated by streptavidin beads. Blots were probed with the indicated antibodies. ( D ) Cell viability assay in response to treatment with ATRi was performed in HeLa USP37 knockout (KO) cells reconstituted with USP37 WT and catalytic dead mutant C350S. Data are presented as means ± SD, n = 3 independent experiments. ( E ) Western blot analysis of various DNA damage signals conducted in HeLa USP37 KO cells reconstituted with USP37 WT and catalytic dead mutant C350S upon treatment with ATRi. NT: no treatment. ( F ) Representative images of RPA2 staining following treatment with ATRi (1 μM) for 48 h in HeLa USP37 KO cells reconstituted with USP37 WT and catalytic dead mutant C350S. Scale bar, 10 μm. ( G ) Quantification of percentage of cells from panel (F) with RPA2 foci >10. At least 100 cells were analyzed per sample over three biologically independent experiments. Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. **** P < .0001, ns: not significant. ( H ) Western blot analysis to evaluate the expression of USP37 WT and catalytic dead mutant C350S in HeLa BRCA1 mAID cells. ( I ) Representative crystal violet images of the clonogenic survival assay conducted in USP37-depleted HeLa BRCA1 mAID cells reconstituted with USP37 WT and catalytic dead mutant C350S. ( J ) Quantification of the data in panel (I). Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. ** P < .01, *** P < .001, ns: not significant.

Article Snippet: Antibodies used in the current study include BRCA1 (sc-6954, Santa Cruz Biotechnology), Vinculin (V9131, Millipore Sigma), Flag (F3165, Millipore Sigma), β-tubulin (T5168, Millipore Sigma), RAD51 (ab63801, Abcam), RPA2 (2208S, Cell Signaling Technology), USP37 (ab190184, Abcam), HLTF (43345S, Cell Signaling Technology), 53BP1 (NB100-304, Novus Biologicals), PCNA (sc-56, Santa Cruz Biotechnology), GAPDH (sc-32233, Santa Cruz Biotechnology), DNA-PKcs (ab70250, Abcam), phospho-DNA-PKcs S2056 (ab18192, Abcam), KAP1 (A300-274A, Bethyl Laboratories), phospho-KAP1 S824 (4127 S, Cell Signaling Technology), phospho-ATM S1981 (13050 S; Cell Signaling Technology), ATM (2873S; Cell Signaling Technology), ATR (2790S; Cell Signaling Technology), phospho-ATR T1989 (30632 S; Cell Signaling Technology), phospho-CHK2 Thr68 (2661 S, Cell Signaling Technology), phospho-H2AX S139 (9718 S, Cell Signaling Technology), phospho-H2AX S139 (05-636l, Millipore Sigma), phospho-CHK1 Ser345 (2348 S, Cell Signaling Technology), phospho-CHK1 Ser317 (12302 S, Cell Signaling Technology), phospho-RPA32 S4/S8 (A300-245A, Bethyl Laboratories), and CHK2 (6334 S, Cell Signaling Technology).

Techniques: Transfection, Immunoprecipitation, Binding Assay, In Vivo, Viability Assay, Knock-Out, Mutagenesis, Western Blot, Staining, Derivative Assay, Expressing, Clonogenic Cell Survival Assay

Journal: Nucleic Acids Research

Article Title: USP37 counteracts HLTF to protect damaged replication forks and promote survival of BRCA1-deficient cells and PARP inhibitor resistance

doi: 10.1093/nar/gkaf544

Figure Lengend Snippet: Depletion of HLTF rescues cell lethality upon USP37 loss in BRCA1-deficient cells, and it prevents MRE11-dependent nascent fork degradation. ( A ) Top: Schematic of the native BrdU assay. Bottom: Model for the native BrdU assay to detect the reversed replication fork. ( B ) Representative images of native BrdU staining in HeLa WT, USP37 KO, HLTF KO, and USP37&HLTF DKO cells with or without the treatment of HU (4 mM) for 3 h. Scale bar, 10 μm. ( C ) Quantification of percentage of cells from panel (B) with BrdU foci. At least 100 cells were analyzed per sample over three biologically independent experiments. Data are represented as means (±SD), n = 3 independent experiments, and P values were derived from a two-way ANOVA with Tukey’s multiple comparisons test. * P < .05, ** P < .01, *** P < .001, **** P < .0001, ns: not significant. ( D ) Western blot analysis to confirm HLTF KO in HeLa BRCA1 mAID WT and USP37 KO cells with or without induction of BRCA1 degradation. ( E ) Representative crystal violet images of the clonogenic survival assay in cells from panel (D) with or without treatment with olaparib (Ola). ( F ) Quantification of the data in panel (E). Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. * P < .05, ** P < .01. ( G ) Western blot analysis to check siRNA knockdown efficiency of USP37 and HLTF in SUM149PT cells. ( H ) Top: Schematic of IdU/CIdU pulse-labeling followed by a 4-h treatment with HU (2 mM). Bottom, representative images of ldU and CIdU replication tracks in HU-treated indicated cells. ( I ) Dot plot of CIdU to ldU tract length ratios for individual replication forks in cells from panel (H). The median value of 100 or more IdU and CldU tracts per experimental condition is indicated. Data are presented as means (±SD), n = 3 independent experiments, and P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. **** P < .0001. ( J ) Top: Schematic of IdU/CIdU pulse-labeling followed by a 4-h treatment with or without Mirin (50 μM) together with HU (2 mM). Bottom, representative images of ldU and CIdU replication tracks in indicated treated cells. ( K ) Dot plot of CIdU to ldU tract length ratios for individual replication forks in cells from ( J ). The median value of 100 or more IdU and CldU tracts per experimental condition is indicated. Data are presented as means (±SD), n = 3 independent experiments, P values were derived from a one-way ANOVA with Tukey’s multiple comparisons test. **** P < .0001. ( L ) Model of the mechanism of synthetic lethality in BRCA1-deficiency and USP37 loss.

Article Snippet: Antibodies used in the current study include BRCA1 (sc-6954, Santa Cruz Biotechnology), Vinculin (V9131, Millipore Sigma), Flag (F3165, Millipore Sigma), β-tubulin (T5168, Millipore Sigma), RAD51 (ab63801, Abcam), RPA2 (2208S, Cell Signaling Technology), USP37 (ab190184, Abcam), HLTF (43345S, Cell Signaling Technology), 53BP1 (NB100-304, Novus Biologicals), PCNA (sc-56, Santa Cruz Biotechnology), GAPDH (sc-32233, Santa Cruz Biotechnology), DNA-PKcs (ab70250, Abcam), phospho-DNA-PKcs S2056 (ab18192, Abcam), KAP1 (A300-274A, Bethyl Laboratories), phospho-KAP1 S824 (4127 S, Cell Signaling Technology), phospho-ATM S1981 (13050 S; Cell Signaling Technology), ATM (2873S; Cell Signaling Technology), ATR (2790S; Cell Signaling Technology), phospho-ATR T1989 (30632 S; Cell Signaling Technology), phospho-CHK2 Thr68 (2661 S, Cell Signaling Technology), phospho-H2AX S139 (9718 S, Cell Signaling Technology), phospho-H2AX S139 (05-636l, Millipore Sigma), phospho-CHK1 Ser345 (2348 S, Cell Signaling Technology), phospho-CHK1 Ser317 (12302 S, Cell Signaling Technology), phospho-RPA32 S4/S8 (A300-245A, Bethyl Laboratories), and CHK2 (6334 S, Cell Signaling Technology).

Techniques: BrdU Staining, Derivative Assay, Western Blot, Clonogenic Cell Survival Assay, Knockdown, Labeling