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. 2022 Oct 3;11:e79128. doi: 10.7554/eLife.79128

Figure 4. S6K1 affects nuclear localization of DNA damage proteins.

(a,b) MCF-10A cells transduced with either empty vector pBABE (pB(-)), S6K1 or Iso-2 were either untreated (control) or treated with UVC 30 J/m2 /sec (UV). Two hours after treatment cells were harvested, fractionated and subjected to western blot analysis. β-tubulin and SRSF1 served as a cytosol (C) and nuclear (N) markers, respectively. Quantification of the blot is shown in b. Data represents means ± SD of three experiments. (c,d) MCF-10A cells transduced with either empty vector (mlp(-)) or S6K1-specific shRNAs (sh1,sh2) were either untreated (control) or treated with UVC 30 J/m2 /s (UV). Two hours after treatment cells were harvested, fractionated and subjected to western blot analysis. β-tubulin and SRSF1 served as a cytosol (C) and nuclear (N) markers, respectively. Quantification of the blot is shown in d. Data represents means ± SD of three biological experiments. (e) U2OS cells transfected with either HT, HT-S6K1 (S6K1) or HT-Iso-2 (Iso-2) were either untreated (cont.) or exposed to UVC 30 J/m2 /sec (UV). After 2 hr, cells were fixed and stained with anti-Halo-tag fluorescence TMR Direct ligand and DAPI. Cells were visually scored using a fluorescent microscope for either nuclear staining (N) or cytoplasmic staining (C). (n=600 cells scored per transfection; 300 for control and 300 for UV treatment). p values were calculated using Student’s t-test (two-tailed) from three biological replicate. (f) U2OS cells were transfected with HT-S6K1 and exposed to UVC 30 J/m2/s and fixed two hours later. Representative photograph of cells stained with anti-Halo-tag fluorescence TMR Direct ligand (red) and anti-γ-H2Ax (green). Arrows point to S6K1 nuclear localization. Images were taken by Nikon-TL (×20). * p<0.05, ** p<0.01. p Values were calculated using Student’s t-test (two-tailed).

Figure 4—source data 1. Western blots of Figure 4.

Figure 4.

Figure 4—figure supplement 1. Total lysates of fractionation experiments.

Figure 4—figure supplement 1.

(a,b) Western blot of MCF-10A cells transduced with either empty vector pBABE (pB(-)), S6K1, or Iso-2 (a) or MCF-10A cells transduced with either empty vector mlp (mlp(-)) or S6K1-specific shRNAs (sh1, 2) (b) with and without UVC 30 J/m2 /sec (UV) treatment. These are total lysates from the fractionation experiment shown in main Figure 4a and c. (c) MCF-10A cells transduced with either empty vector pBABE (pB(-)) or S6K1 were either untreated (control) or treated with UVC 30 J/m2 /sec (UV). Two hr after treatment, cells were harvested, fractionated, and subjected to western blot analysis. Caspase-2 and SRSF1 served as a cytosol (C) or nuclear (N) markers, respectively. (d) U2OS cells were transfected with control Halo-tag vector (HT), HT-S6K1 (HT-S6K1) or HT-Iso-2 (HT-Iso-2) and exposed to UVC 30 J/m2 /s. After 2 hr cells were fixed and stained with anti-Halo-tag fluorescence TMR Direct ligand (red) and DAPI. Images were taken by Nikon-TL (×20). Representative images of experiment summarized in main Figure 4e. (e) Histogram representing the intensity of γ-H2AX fluorescence in U2OS cells expressing HT, HT-S6K1 (S6K1) or HT-Iso-2 (Iso-2) positive cells related to experiment described in main Figure 4f. The intensity was calculated using the NIS-Elements AR software.
Figure 4—figure supplement 1—source data 1. Western blots of Figure 4—figure supplement 1.