Fig 4. lincRNA-p21 is required for proper apoptotic induction.
(A) Increased cell viability of lincRNA-p21 depleted cells. Relative number of siRNA-transfected MEFs treated with 400nM DOX from 24h after transfection (right) or untreated (left) determined by MTT assay.
(B) Knockdown of lincRNA-p21 with individual siRNAs increases cell viability. Images of MEFs treated with different individual siRNAs after 48h of DOX treatment (72h post transfection).
(C) LincRNA-p21 knockdown doesn't affect cell cycle regulation. Relative cell numbers in each cell cycle phase determined by FACS of BrdU incorporation and PI staining of MEFs treated as in (A). Numbers inside bars represent percentages of cells in each phase.
(D) LincRNA-p21 knockdown causes a decrease in cellular apoptosis. p53-reconstituted p53LSL/LSL MEFs transfected with three individual siRNAs targeting lincRNA-p21 (bottom), two independent control siRNAs (top left and middle) or a siRNA pool targeting p53 (top right). 24h after transfection cells were treated with 400nM doxorubicin and 14h later harvested and subjected to FACS analysis. X-axis represents Annexin-V and y-axis 7-AAD staining. The percentage of cells in each quadrant is indicated.
(E) Decreased apoptosis caused by lincRNA-p21 knockdown. Percentage of Annexin-V positive cells (FACS) at 38h post transfection (14h of 400nM DOX treatment) in MEFs treated as in (A).
(F) LincRNA-p21 knockdown in p53-reconstituted p53LSL/LSL MEFs causes decrease in Caspase 3 cleavage. Levels of cleaved Caspase 3 or control βActin in p53 reconstituted-p53LSL/LSL MEFs treated with the indicated siRNA pools and 500nM DOX for 14h.
(G) Decreased cell viability caused by lincRNA-p21 overexpression. Relative numbers of LKR cells overexpressing lincRNA-p21 or control plasmid determined by MTT assay.
(H) Overexpression of lincRNA-p21 causes cellular apoptosis under DNA damage induction. Percentage of Annexin-V-positive LKR cells overexpressing lincRNA-p21 or control vector treated with 500nM DOX.
(I) LincRNA overexpression doesn't affect cell cycle regulation. Cell cycle analysis of DOX-treated LKR cells overexpressing lincRNA-p21 or control plasmid.
All values are the average of 3 biological replicates (+/-STD). *=P< 0.001 relative to controls.
Also see Figure S4.