Journal: Cell Death and Differentiation

Article Title: Reversible induction of translational isoforms of p53 in glucose deprivation

doi: 10.1038/cdd.2014.220

Figure Lengend Snippet: Physiological relevance of glucose deprivation on SMAR1-p53 IRES association. (a) Western blot of A549-cell extracts after indicated duration of glucose starvation, blotted with CM1 (left panel) or Pab240 antibody (right panel). (b,c) Quantitative PCR of p21, mdm2 and SFN mRNA levels normalized to GAPDH in A549 cells (b) and HCT116 cells (c) following 0, 8 and 30 h of glucose deprivation. (d) Western blot of A549- and HepG2-cell extracts showing the expression pattern of SMAR1 in a time-dependent manner post-glucose deprivation. Glucose was deprived for indicated time in hours and the cells were harvested at the given time points. (e) Western blot analysis of A549-cell extracts transfected with non-targetting (Nsp) or SMAR1 siRNA for 96 h, followed by glucose starvation for 30 h. Δ40p53 levels are indicated by an arrow. (f) Reverse transcriptase-quantitative PCR analysis of RNA extracted from ribonucleoprotein complexes immunoprecipitated from non-starved or 8-h glucose-starved A549 cells using anti-SMAR1, anti-PTB antibodies or IgG-isotype control. Primers corresponding to 1–251 region of p53 RNA were used. Results represented as fold change over and above RNA immunoprecipitated from non-starved cells with IgG control antibody. (g) Reverse transcriptase-quantitative PCR analysis of RNA extracted from ribonucleoprotein complexes immunoprecipitated from non-starved or 8-h glucose-starved H1299 cells transfected with GFP-hp-5'UTR-p53-bicistronic mRNA using anti-SMAR1, anti-PTB antibodies or IgG-isotype control. Primers corresponding to 1–251 region of p53 RNA were used. Results represented as fold change over and above RNA immunoprecipitated from non-starved cells with IgG control antibody. (h) FACS analysis of cell populations in G1, S or G2-M phases in H1299-NS and H1299-S3 cells. The cells were transfected with 14A (top row), 14A-M2 (middle row) or pEGFP (bottom row). Cell cycle was arrested by double-thymidine treatment, then cells were glucose starved for 0, 4, 8, 12, 24 and 30 h and harvested at these time points with corresponding unstarved control cells. (i) Glucose replenishment (rescue) for 12 and 24 h following 8 h of glucose deprivation in H1299 cells transfected with pGFP-hp-p53-5'UTR-cDNA plasmid. Western blots depict SMAR1, p53, Δ40p53 and actin levels. Graphs represent lane-wise densitometric analysis. (j) Glucose replenishment (rescue) for 12 and 24 h following 8 h of glucose deprivation in A549 cells. Western blots depict endogenous p53 and Δ40p53 (shown by an arrow) as well as SMAR1 and actin levels. Graphs represent lane-wise densitometric values. (k) Quantitative PCR for levels of p53-target mRNAs p21/Cip1 (CDK-interacting protein1), SFN (stratifin alias 14-3-3σ), Bax (Bcl-2 associated X), TIGAR (TP53-induced glycolysis and apoptosis regulator), PIDD (P53-induced DNA damage) and Mdm2 (murine double minute 2) in H1299 cells transfected with pGFP-hp-p53-5'UTR-cDNA plasmid, starved of glucose for 8 h (8 h) and then rescued by glucose replenishment for 12 h (8 h/12 h) and 24 h (8 h/24 h). Results represented as fold change in mRNA levels compared to non-starved, transfected cells, n=3

Article Snippet: Samples were then analyzed by western blotting using rabbit-raised CM-1 antibody (kind gift from Professor Robin Fahraeus, INSERM), anti-SMAR1 antibody (Abcam, Cambridge, UK), mouse-raised DO1 and Pab240 antibodies (Santa Cruz Biotechnology, Dallas, TX, USA) or rabbit-raised anti-GFP antibody (IMG5127, Imgenex, Bhubaneswar, Odisha, India) followed by secondary antibody (horseradish peroxidase-conjugated anti-rabbit IgG; Sigma-Aldrich).

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transfection, Reverse Transcription, Immunoprecipitation, Plasmid Preparation