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. 2021 Jan 6;118(3):e2015808118. doi: 10.1073/pnas.2015808118

Fig. 5.

Fig. 5.

ACTL6A regulates cellular response to cisplatin and DNA repair process through the SWI/SNF complex. (A) Overexpression of both dominant-negative Brg1 and Brm (dn-Brg1/Brm) increases the basal activity of H2AX, and blocks the effect of ACTL6A on the reduction of cisplatin-induced H2AX activation. H1299 cells stably harboring an empty vector or ACTL6A were transiently transfected with an empty vector or both dn-Brg1 and dn-Brm expression vectors. Cells were treated with 10 μM cisplatin for 24 h before lysis for Western blot analysis. (B) Overexpression of both dn-Brg1 and dn-Brm blocks ACTL6A-enhanced DNA repair after cisplatin treatment. Transfected H1299 cells as described in A were treated with vehicle or 10 μM cisplatin for 2 h, and then released and cultured in fresh medium for indicated times. Genomic DNA was isolated and subjected to slot blot assay as described above. (C and D) Overexpression of both dn-Brg1 and dn-Brm inhibits the effect of ACTL6A on enhancing cell viability and cell survival after cisplatin treatment. Transfected H1299 cells as indicated were treated with 10 or 50 μM cisplatin for 48 h (C), or with 1 μM cisplatin for 24 h (D). Cell viability was determined by MTT assay (C). Clonogenic cell survival assay was performed (D). (E) Depletion of BAF155 enhances the basal activity of H2AX and blocks the inhibitory effect of ACTL6A on cisplatin-induced H2AX activation. H1299 cells stably harboring an empty vector or ACTL6A were infected with a lentivirus expressing either shScr or shBAF155 (#1 or #2). Cells were treated with vehicle or 10 μM cisplatin for 24 h followed by Western blot analysis. (F) Depletion of BAF155 blocks ACTL6A-enhanced DNA repair after cisplatin treatment. Transfected H1299 cells as described in E were treated with vehicle or 10 μM cisplatin for 2 h. After release and culture in fresh medium for indicated times, genomic DNA was isolated and subjected to slot blot analysis. (G and H) Depletion of BAF155 inhibits the effect of ACTL6A on enhancing cell viability and cell survival after cisplatin treatment. Transfected H1299 cells as described in E were treated with 10 or 50 μM cisplatin for 48 h (G) or with 1 μM cisplatin for 24 h (H). Cells were subjected to MTT cell viability assay (G) or clonogenic survival assay (H). For cell viability assay, Data shown represent the mean ± SD from at least three or four biological replicates. For colony formation assay, data shown represent the mean ± SD from at least three independent experiments. Representative images are shown on the Left of each figure. *P < 0.05 and **P < 0.01.