Skip to main content
. 2022 Oct 3;11:e79128. doi: 10.7554/eLife.79128

Figure 6. S6K1 overexpression enhances homologous recombination and mismatch repair.

(a) Schematic diagram of DSB reporter plasmid. SceGFP is a GFP gene that contains an I-SceI endonuclease site within the coding region. Cleavage of the I-SceI site in vivo and repair by HDR (Homologous Directed Repair) coupled by the downstream iGFP repeat results in GFP positive cells. (b) U2OS cells expressing DSB reporter plasmid (U2OS-DR-GFP) were co-transfected with ISceI and either Halo-tag empty vector (HT), HT-S6K1 or HT-Iso2. Western blot showing S6K1 expression (top panel). Cells were analyzed by FACS for GFP expression (bottom panel). (c) U2OS-DR-GFP cells knocked-out for S6K1 using CRISPR were transfected with ISceI. Western blot showing S6K1 expression (top panel). Cells were analyzed by FACS for GFP expression (bottom panel). (d) U2OS-DR-GFP cells transfected with either control siRNA (scr) or siRNA specific for Rad51 (siRad51) were co-transfected with ISceI. Western blot showing Rad51 expression (top panel). Cells were analyzed by FACS for GFP expression (bottom panel). (e) U2OS-DR-GFP cells were co-transfected with ISceI and WT CDK1 or mutant CDK1 39 A. Western blot showing HA tag expression (top panel). Cells were analyzed by FACS for GFP expression (bottom panel). (f) U2OS-DR-GFP cells were co-transfected with ISceI and S6K1 with either WT CDK1 or mutant CDK1 39 A. Western blot showing HA tag expression (top panel). Cells were analyzed by FACS for GFP expression (bottom panel). (g,h) Mismatch repair assay using MEF WT (WT) and S6K-/- cells (f) or S6K-/- cells stably expressing either empty vector (pB(-)), S6K1 or Iso-2 (g) transfected with EGFP heteroduplex plasmid. Forty-eight hr after transfection, cells were analyzed by FACS for GFP expression. (i) S6K-/- cells were co-transfected with either Flag-wild type MSH6 (WT), Flag-MSH6 mutant 309 A (309 A) or Flag-MSH6 mutant 309D (309D) and EGFP heteroduplex plasmid. Western blot showing Flag-tagged expression (top panel). Forty-eight hr after transfection, cells were analyzed by FACS for GFP expression (bottom panel). Data represents means ± SD of biological triplicates. *p<0.05 values were calculated using Student’s t-test (two-tailed).

Figure 6—source data 1. Western blots of Figure 6.

Figure 6.

Figure 6—figure supplement 1. Cell cycle analysis of U2OS-DR-GFP cells.

Figure 6—figure supplement 1.

(a–l) Cell cycle FACS analysis of U2OS-DR-GFP cells expressing either HT, HT-S6K1 or HT-Iso2 (a-d, related to main Figure 6b), U2OS-DR-GFP cells expressing Cdk1 WT or Cdk1 39 A (e-h, related to main Figure 6e) or U2OS-DR-GFP co-expressing either Cdk1 WT or Cdk1 39 A with S6K1 (i-l, related to main Figure 6f). Cells were stained with DAPI, anti-phospho-histone H3 Alexa488, anti-Cyclin B1 Alexa647 and anti-Cyclin A PE561. G1, S, and G2 separation displayed according to DAPI staining (b,f,j). Dual analysis of pH3 and DAPI distinguishes M phase (d,h,l). Plots were gated on S/G2/M. Dual analysis of cyclin A and cyclin B1 distinguishess between S, G2, M (c,g,k).