Alpha-actnin-4 (ACTN4) selectively affects the DNA double-strand breaks repair in non-small lung carcinoma cells

Figures

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  Fig. 1. The ACTN4 knock-out affects the viability of H1299 cells under genotoxic stress. A Western Blot analysis of ACTN4 expression levels in H1299 (left) and H460 (right) control and ACTN4 knock-out (ACTN4 KO) cells. Three H1299 and two H460 ACTN4 KO clones used in the study are presented. B–D The MTT assay performed on H1299 (B, C) and H460 (D) control, ACTN4 KO and ACTN4 OE cells exposed by different concentrations of cisplatin (B) and etoposide (C, D) for 72 h. Results for three H1299 ACTN4 KO clones are presented in C to ensure the effect. E The H1299 (left) and H460 (right) cells were treated by 50 uM etoposide for 24 h to induce apoptosis. Annexin V (APC)/PI (PE) plots and percentages of cells in quadrants (means of three replicas) are presented. H1299 ACTN4 KO cells display significantly (p < 0.001) lower early apoptosis (AV) than the control cells while H460 cells show the opposite. The data are presented as the mean of at least three replicas ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 compared to untreated cells (multiple Student’s t-test). Each assay was performed at least three times

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  Fig. 2. ACTN4 knock-out does not change cell cycle in H1299 cells but increases G0-G1 phase under genotoxic conditions. H1299 Control and ACTN4 KO cells were treated with 1.5 uM etoposide for 18 (A) or 48 (B) hours. Cell cycle distributions are presented as graphs (upper panels) and flow cytometry plots (lower panels). The data are presented as the mean of at least three replicas in each experiment ± SEM and repeated three times

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  Fig. 3. Depletion of ACTN4 in H1299 and H460 cells leads to more efficient DNA break repairs induced by the topoisomerase II inhibitors. Control, ACTN4 KO and ACTN4 OE cells were treated with 50 uM etoposide (Eto) or 1.5 uM doxorubicin (Doxo) for 40 min. The drug was then replaced with fresh media and 4 or 8 h of cells recovery. A Analysis of gamma-H2AX immunostaining in H1299 control and ACTN4 KO cells. Images of the etoposide-induced foci formation are presented in the left panel. Mean numbers of foci per nucleus were calculated in Eto or Doxo treated cells. The graphs represent the mean gamma-H2AX foci numbers between three replicas (± SE) with at least 500 cells per replica analyzed. Scale bars in panel represent 25 μm. B An alkaline comet assay is performed on H1299 and H460 cells. Images of the DNA comets obtained from H1299 control, ACTN4 KO and ACTN4 OE cells are presented in the left panel. Scale bars in panel represents 250 μm. The graphs represent mean Tail DNA percentages of at least 100 analyzed cells ± SE. ***p < 0.001, *p < 0.05 compared to untreated cells (multiple Student’s t-test). Each assay was performed at least three times. The experiments were performed in triplicate

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  Fig. 4. ACTN4 cells show enhanced NHEJ and suppressed HR. H1299 cells were transfected with NHEJ and HR specific reporter constructs along with a plasmid, encoding dsRed-fused I-SceI endonuclease that performs site-specific cleavage of the reporters. Representative FACS plots display NHEJ and HR specific repair of the reporter GFP as an increase of GFP-positive cells (upper quadrants). The graph represents DNA repair efficiency estimated as the ratio of GFP + /dsRed + cells. Data are presented as mean of three replicas ± SD. ***p < 0.0001compared to untreated cells (Student’s t-test). The assay was performed three times

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  Fig. 5. ACTN4 expression affects the distribution of the key DSBs repair proteins in H1299 cells after the etoposide-induced DNA mage. The H1299 Control and ACTN4 KO cells were treated with 50 uM etoposide for 40 min. The media was then replaced with a fresh one, and cells were incubated for 4 h to recover from the damage. Changes in total intensity and foci number of pATM (A–B), 53BP1 (C–D), pBRCA1 (E–F) proteins were estimated by immunofluorescence. Representative images are presented on B, D and F. Scale bars in panels represent 25 μm. Graphs A, C, E present the mean of three replicas ± SE. Over 500 cells were analyzed per replica. **p < 0.0001, **p < 0.001, *p < 0.05 compared to untreated cells (Student’s t-test)

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  Fig. 6. Possible mechanism of ACTN4 influence on the DSBs repair pathways. ACTN4 may possibly interfere with 53BP1 binding to unprocessed DNA ends, hence suppressing the end processing suitable for NHEJ and promoting assembly of the pBRCA-positive HR complexes. In H1299 ACTN4 KO cells however, DNA binding of 53BP1 is increased, which results in enhancing the NHEJ pathway and the suppression of pBRCA complexes and HR pathways