Figure 1.
GSK3A silencing in p53-null colon carcinoma cell lines does not affect proliferation or cell cycle.
A) Western blot on total lysates (30 µg) from HCT116p53KO cells stably infected with retroviral empty (pRS) and GSK3A shRNA-expressing (pRSGSK3A) vectors. B) growth curve of HCT116p53KO-pRS and -pRSGSK3A cells. C) DNA content and percentage of cells in G1, S phase and G2/M of HCT116p53KO-pRS and -pRSGSK3Aas evaluated after PI staning and flow cytometric analysis. D) β-catenin activity as evaluated by reporter assay 48 hrs after co-transfection with a luciferase-encoding vector driven by a β-catenin-dependent promoter. RLU = Relative Light Units. encoding vector (pRSGSK3B).
Figure 2.
Either stable or transient depletion of GSK3A abolishes drug resistance of p53-null colon carcinoma cell lines.
A) colony assay of 5FU-treated HCT116p53KO-pRS and -pRSGSK3A cells. Cells were trypsinized and reseeded at low density 12 hours after 200 µM 5FU treatment. Colony formation was assessed 2 weeks after the reseeding. B) percentage of cell death of HCT116p53KO-pRS, -pRSGSK3A and -pRSGSK3B cells treated for 72 hrs with 200 µM 5FU. Drug sensitive HCT116 cells were used as a positive control. C) percentage of cell death of HCT116p53KO-pRS, -pRSGSK3A and -pRSGSK3B cells treated for 72 hrs with 50 µM Oxaliplatin (OxPt). Drug sensitive HCT116 cells were used as a positive control. D) percentage of cell death of HCT116p53KO upon GSK3A transient silencing and treatment with 200 µM 5FU (72 hrs). In the inset: lysates of HCT116p53KO cells transfected with luciferase (luc)- or GSK3A-specific siRNAs were harvested 24 hs after silencing and GSK3A levels checked by western blot.
Figure 3.
Chemical inhibition abolishes drug resistance of p53-null colon carcinoma cell lines.
A, C, E Lysates of HCT116p53KO (A) and SW480 (C) cells were harvested 24hs after treatment with 1 µM 6-bromoindirubin-3′-oxime (BIO) and 20 µM SB216763 (SB2) GSK3 inhibitors; lysates of HT-29 (E) cells were harvested 24 hs after treatment with 2 µM 6-bromoindirubin-3′-oxime (BIO) and 20 µM SB216763 (SB2) GSK3 inhibitors; specificity of the inhibitor for GSK3A was assessed by checking GSK3A activation/inactivation checked by western blot using a mix of pSer21-GSK3A and pSer9-GSK3B antibodies and antibody cross-reacting with both pTyr279-GSK3A and pTyr216-GSK3B. B, D, F) percentage of cell death of HCT116p53KO(B), SW480 (D) and HT-29 (F) treated with 200 µM 5FU in presence and in absence of the indicated inhibitors (72 hrs).
Figure 4.
GSK3A inhibition abolishes drug resistance of p53-null colon carcinoma cells by affecting the response to DNA damage.
HCT116p53KO cells stably infected with empty (pRS) and GSK3A shRNA-encoding vector (pRSGSK3A) untreated (cnt) or treated for 18 hrs with 200 µM 5FU (5FU) and stained with anti-γH2AX antibody (A) or anti-RPA70 antibody (B) and counterstained with DAPI. HCT116p53KO untreated (cnt), treated for 18 hrs with 200 µM 5FU (5FU) or with 200 µM 5FU+10 µM SB216763 and stained with anti-γH2AX antibody (C) or anti-RPA70 antibody (D) and counterstained with DAPI.
Figure 5.
p53-null, GSK3B-silenced colon carcinoma cells treated with 5FU die by RIP1-independent necroptosis.
A) caspase-3/7 activation in HCT116p53KO-pRS and -pRSGSK3A cells treated with 200 µM 5FU for 72 hrs. HCT116 were used as control. Values indicate the fold increase of enzymatic activity of treated cells relative to the untreated cells arbitrarily set as 1. A representative experiment is shown. B) HCT116p53KO-pRS and -pRSGSK3A treated for 30 hrs with 200 µM 5FU and stained with anti-AIF antibody as well as DAPI. C) percentage of cell death of HCT116p53KO-pRSGSK3A after 72 hs treatment with 200 µM 5FU in presence of Bid inhibitor (20 µM Bi6C9), PARP1 inhibitor (100 µM DiQ), Bi6C9+DiQ or Necrostatin-1 (20 µM Nec1). D) HCT116p53KO-pRSGSK3A treated for 30 hrs with 200 µM 5FU in presence of 100 µM DiQ and stained with anti-AIF antibody as well as DAPI.