plenti puro arid1a plasmid  (Addgene inc)

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: Characterization of mutation rate and expression of ARID1A in SCLC. (A) Oncoprint of ARID1A and ARID1B mutations in human primary SCLC. (B to F) Scatterplots of ARID1A expression in SCLC cell lines relative to LUAD cell lines (B; CCLE dataset), in primary SCLC relative to normal lung tissues (C; GEO60052 dataset), in SCLC tissues relative to adjacent normal tissues (paraSCLC) (D; GEO149507 dataset), in 107 paired SCLC and paraSCLC retrieved from the GSA database (E; HRA003419 dataset), and in SCLC blood relative to healthy and benign blood from exoRBase database 2.0 (F). (G) Protein expression of ARID1A in 112 paired SCLC and paraSCLC, retrieved from the OMIX database (OMIX002489). (H and I) Kaplan–Meier survival analysis of the correlations between ARID1A expression and OS (H) and PFS (I) in 41 (H) or 33 (I) patients with SCLC by log-rank tests, respectively. Statistical analysis was performed using 2-tailed unpaired Student’s t tests for (B) to (E), and 2-tailed paired Student’s t tests for (F) and (G).

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Mutagenesis, Expressing

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A suppresses cell viability and clonogenicity in SCLC. (A) RT-qPCR analysis of ARID1A expression in 8 SCLC cell lines and 2 LUAD cell lines. A549 was used as the control for standardization. (B) Detection of ARID1A expression by Western blot across a panel of lung cancer cell lines. β-Actin was used as a loading control. (C) RT-qPCR analysis of ARID1A in DMS273 and DMS53 cells with ARID1A KD and overexpression (OE). (D) Western blot analysis of ARID1A expression following ARID1A KD and OE in DMS273 and DMS53 cells. (E) Representative images of EdU staining in SCLC cells following ARID1A KD and OE (left). Quantitative analysis of the results is shown on the right. (F to I) Cell viability (F and G) and colony formation (H and I) assay after ARID1A KD (F and H)/OE (G and I). The quantitative analysis of the colonies was performed using ImageJ software. Data are shown as the mean ± SEM, n ≥ 3 independent experiments. Statistical analysis was performed using 2-tailed unpaired Student’s t tests. ns, no significance; ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Quantitative RT-PCR, Expressing, Control, Western Blot, Over Expression, Staining, Software

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A restrains tumor growth in vivo . (A) Tumor volume curves of DMS273 control cells and ARID1A depletion cells (top) or overexpression (bottom). (B) Scatterplot representing tumor weights of DMS273 control cells and ARID1A depletion cells (top) or overexpression (bottom) at the endpoint of experiments. (C) Imaging of representative tumors from each group. (D) RT-qPCR analysis of mRNA expression of ARID1A , c-MYC , PARP1 , and RAD51 after KD (top)/OE (bottom) of ARID1A in vivo. (E) IHC analysis of the expression of ARID1A, c-MYC, PARP1, RAD51, and γH2AX in subcutaneous tumor tissues of mice. Scale bars, 100 μm. (F) Quantification of the IHC. The signals were quantified by IOD, equal to optical density by area, by using ImageJ software and indicated as mean ± SEM. Statistical analysis was performed using 2-tailed unpaired Student’s t tests. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: In Vivo, Control, Over Expression, Imaging, Quantitative RT-PCR, Expressing, Software

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A controls SCLC cell survival by inhibiting c-MYC / PARP1 expression. (A and B) Western blot analysis of the indicated proteins in DMS273 and DMS53 cells following ARID1A KD (A) or OE (B). (C) Western blot analysis of the indicated proteins following ARID1A overexpression in ARID1A KD DMS273 and DMS53 cells. (D and E) Clonal formation (D) and cell viability (E) assays following ARID1A overexpression in ARID1A KD DMS273 and DMS53 cells. (F and G) Western blot analysis of the indicated proteins following c-MYC (F) or PARP1 (G) overexpression in ARID1A -overexpressing DMS273 and DMS53 cells. (H to K) Cell viability (H and J) and clonal formation (I and K) assays following c-MYC (H and I) or PARP1 (J and K) overexpression in ARID1A -overexpressing DMS273 and DMS53 cells. Data are shown as the mean ± SEM; n ≥ 3 independent experiments. Statistical analysis was performed using a one-way ANOVA. ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Expressing, Western Blot, Over Expression

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A transcriptionally suppresses the expression of c-MYC and PARP1 . (A and B) RT-qPCR analysis of c-MYC and PARP1 mRNA expression in DMS273 and DMS53 cells following ARID1A KD (A) or OE (B). (C and D) ChIP-PCR analysis of DMS273 cells expressing control or sh ARID1A using antibodies against ARID1A for the promoter regions of PARP1 (C) and c-MYC (D). (E to H) ChIP-PCR analysis of DMS273 cells expressing control or sh ARID1A using antibodies against H3K27Ac (E and F) and H3K27me3 (G and H) for the promoter regions of PARP1 (E and G) and c-MYC (F and H). (I and J) The relative luciferase activities were detected in DMS273 cells with ARID1A KD or OE after transfection with the PARP1 promoter-driven luciferase (I) or the c-MYC promoter-driven luciferase (J) constructs. The firefly luciferase activities were measured and normalized to Renilla luciferase activities (F/R). Data are shown as the mean ± SEM; n ≥ 3 independent experiments. Statistical analysis was performed using 2-tailed unpaired Student’s t tests. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Expressing, Quantitative RT-PCR, Control, Luciferase, Transfection, Construct

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A participates in DDR in SCLC through the c-MYC/PARP axis. (A and B) Western blot analysis of DDR-related gene in DMS273 and DMS53 cells following ARID1A KD (A) or OE (B). (C and D) Representative images of immunofluorescence staining for γ-H2AX (C) and RAD51 (D) in DMS273 and DMS53 cells following ARID1A KD. Cells with more than 5 foci were considered positive. Quantification of γ-H2AX /RAD51 fluorescence intensities from 3 independent experiments was shown as mean ± SD. Statistical analysis was performed using 2-tailed unpaired Student’s t tests. **** P < 0.0001. (E to G) Western blot analysis of the indicated proteins following ARID1A (E), c-MYC (F), or PARP1 (G) overexpression in ARID1A KD DMS273 and DMS53 cells. (H and I) Western blot analysis of PI3K downstream phospho-proteins in DMS273 and DMS53 cells following ARID1A KD (H) or OE (I).

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Western Blot, Immunofluorescence, Staining, Fluorescence, Over Expression

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: The effects of ARID1A on the response to replication stress and DSB induced by HU. (A and B) Western blot analysis of DDR-related genes following the exposure to 2 mM HU (A) and 4 mM HU (B) for the indicated times in ARID1A KD or OE DMS273 cells. (C to H) Cell viability (C and F) and clonal formation (D, E, G, and H) assays following 2 mM HU (C to E) and 4 mM HU (F to H) in ARID1A KD or OE DMS273 cells. n ≥ 3 independent experiments. Data are shown as the mean ± SEM; n ≥ 3 independent experiments. Statistical analysis was performed using a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Western Blot

Journal: Research

Article Title: ARID1A Governs Genomic Stability and Proliferation in SCLC via c-MYC/PARP1 Suppression Driving Vulnerability to BET Inhibitors

doi: 10.34133/research.0908

Figure Lengend Snippet: ARID1A deficiency enhances JQ1 sensitivity and identifies BRD-K98645985 as a novel therapeutic candidate in SCLC. (A and B) Pearson correlation analysis of IC 50 values of JQ1 from the GDSC1 dataset and the mRNA level of ARID1A (A) and MYC family gene (B). (C) Dose–response analysis of JQ1 treatment on cell viability upon ARID1A KD DMS273 and DMS53 cells. (D) Western blot analysis of DDR-related genes following the exposure to JQ1 at indicated concentrations in ARID1A KD DMS273 and DMS53 cells. (E) Tumor volume curves of DMS273 control cells (SCR) and ARID1A KD cells upon exposure to JQ1 in vivo. KD denotes shARID1A-2#. (F) Scatterplot representing tumor weights of DMS273 control cells (SCR) and ARID1A KD cells upon exposure to JQ1 at the endpoint of experiments in vivo. KD refers to shARID1A-2#. (G) Colony formation assays demonstrating the effect of BRD-K98645985 on the cytotoxicity of JQ1 in DMS273 cells. (H) Drug–response curves of BRD-K98645985 and JQ1 combination in DMS273 cells. Cells were treated with escalating doses for 72 h, with viability measured by CellTiter-Glo. (I) Bliss analysis (Combenefit software) showing synergistic effects. (J) Tumor growth curves in xenograft mice treated with JQ1, BRD-K98645985 (BRD), or their combination (JQ1 + BRD). (K) Scatterplot depicting tumor weights from xenograft mice at the endpoints of the experiments. (L) Representative images of xenograft tumors. Data are mean ± SEM, and statistical analysis was performed using a one-way ANOVA. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. (M) Proposed model depicting the function of ARID1A to modulate cell proliferation and genome stability through c-MYC and PARP1 .

Article Snippet: Briefly, 293T cells were transfected with psPAX2 (#12260, AddGene, MA, USA), pMD2.G plasmid (#12259, AddGene), and pLKO.1 plasmid containing nontargeting shRNA (SCR) or ARID1A shRNAs (sh ARID1A ) or pLenti-puro-ARID1A (#39478, AddGene).

Techniques: Western Blot, Control, In Vivo, Software