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. 2016 Feb 4;6:20532. doi: 10.1038/srep20532

Figure 2. The transcript and protein levels of SMARCAL1 vary as a function of DNA damage.

Figure 2

(A) HeLa cells were synchronized using double thymidine block and collected every two hours after release from block. SMARCAL1 expression was analyzed by western blot using polyclonal antibody against the protein. (B) The transcript levels in the synchronized cells after release from the double thymidine block were analyzed by quantitative real-time RT-PCR at time points indicated. (C) Asynchronous population of HeLa cells were treated with 2 μM doxorubicin for 10 minutes and SMARCAL1 levels were compared to the untreated cells by western blot using polyclonal antibody against SMARCAL1. The quantitation was done using ImageJ software. (D) The transcript levels in untreated and doxorubicin treated cells were compared using quantitative real-time RT-PCR (p value  < 0.05). (E) GFP-SMARCAL1 was transiently transfected into HeLa cells and treated with doxorubicin for 10 minutes. The localization of SMARCAL1 and γH2AX was probed using monoclonal anti-GFP antibody and anti-γH2AX antibody. The secondary antibodies were conjugated to FITC and TRITC respectively. Nucleus was stained using Hoechst 33342. Pearson’s coefficient for SMARCAL1-γH2AX co-localization in treated cells was 0.44 ± 0.17 while in untreated cells it was −0.012 ± 0.15. >10 cells were counted in this experiment. (F) HeLa cells were synchronized using double thymidine block and cells were harvested every two hours after release from the block. Prior to harvesting, the cells were treated for 10 minutes with 2 μM doxorubicin. The levels of SMARCAL1 were analyzed by western blot using polyclonal antibody against the protein. (G) The transcript levels in these cells were analyzed by quantitative real-time RT-PCR. (H) Comparison of the transcript levels in untreated and doxorubicin treated cells. The difference between untreated and doxorubicin treated cells was significant with p values  < 0.05 for each of the time point. In all these experiments, GAPDH was used as the internal control. Uncropped western blots are provided in Supplementary Fig. S11.