PMC

A. A validated shRNA was used to knockdown p16^INK4a in several strains of logarithmically growing HDFs and its effects on p16^INK4a and p18^INK4c were assessed by immunoblotting. B. Lifespan extension in TIG3 cells infected with a retrovirus encoding mouse Bmi1 compared to empty vector (Vec). C. The cells described in panel B were harvested at the indicated days post-infection and the levels of p16^INK4a and p18^INK4c were assessed by immunoblotting. D. Direct comparison of lysates prepared at days 50 and 64 (indicated by the arrows in panel B) from cells infected with Bmi1 virus (+) or empty vector (−).

A. Hs68 cells infected with a retrovirus encoding either shRNA against p18^INK4c or a non-specific shRNA (Con) were passaged until they reached M1 senescence. B. Immunoblotting for p18^INK4c and p16^INK4a in cells infected with p18^INK4c shRNA. C. Expression of H-RAS^G12V in cells transduced with p18^INK4c shRNA caused oncogene-induced senescence accompanied by a further reduction of p18^INK4c levels and enhanced expression of p16^INK4a. D. Lysates from proliferating (P) and senescent (S) fibroblasts were immunoprecipitated with rabbit antibodies against CDK6, CDK4, p18^INK4c and p16^INK4a. The proteins were fractionated by SDS-PAGE and immunoblotted with mouse monoclonal antibodies against the indicated proteins.

A and B. The Hs68 (A) and Leiden (B) strains of HDF were passaged to senescence and the relative levels of INK4c RNA in proliferating (P) and senescent (S) cells were assessed by reverse transcription and quantitative real-time PCR (qRT-PCR). C. Relative levels of INK4c and INK4a RNAs in HDFs (Hs68) infected with a retrovirus encoding H-RAS^G12V or empty vector control (Vec). D. Relative levels of INK4c and INK4a RNAs following addition of tamoxifen (OHT) to HDFs (BF) expressing ER:H-RAS^G12V. E. Relative levels of INK4c RNA in HDFs (Hs68) transduced with retroviral vectors encoding HA-tagged versions of p16^INK4a, p18^INK4c or p21^CIP1. Note that endogenous INK4c RNA was measured using primer sets that do not detect the ectopically expressed INKc cDNA.

A. HDFs (Hs68) treated with the CDK4/6 inhibitor PD0332991 were harvested at the indicated intervals and levels of INK4c, B-MYB and CDC6 RNA were assessed by qRT-PCR and normalized to the untreated control. B. HDFs (TIG3) treated with and without PD0332991 for 14d were stained for SA-β-galactosidase activity and with DAPI to visualize SAHFs. C. Lysates from the cells described in panel B were immunoblotted for p18^INK4c, p16^INK4a, menin, and E2F1 with MEK as a loading control. D. HDFs (BF and Leiden) were infected with a retrovirus encoding H-RAS^G12V or empty vector and the relative levels of INK4c and INK4a RNA were assessed by qRT-PCR and normalized to the vector only control. E. Lysates from the cells described in panel D were immunoblotted for p18^INK4c and p16^INK4a. F. Lysates from Leiden cells infected with a retrovirus encoding H-RAS^G12V or empty vector were immunoblotted for menin, E2F1, p18^INK4c and RAS.

A. HDFs (TIG3) were passaged continuously until they reached replicative senescence (Sen). Cell lysates prepared at the indicated population doublings (PD) were fractionated by SDS-PAGE and immunoblotted for p16^INK4a, p21^CIP1 and p18^INK4c. Right panel shows a direct comparison between proliferating (P) and senescent (S) cells. B. HDFs (TIG3) were infected with a retrovirus encoding H-RAS^G12V or the empty vector control (Vec). Following drug selection, the levels of p18^INK4c and p16^INK4a were assessed by immunoblotting. C. Primary MEFs were infected with a retrovirus encoding H-RAS^G12V or the empty vector control (Vec). The levels of p18^Ink4c and p16^Ink4a were assessed by immunoblotting. D. HDFs (Hs68) were infected with a retrovirus encoding HA-tagged p16^INK4a. The levels of p18^INK4c and p16^INK4a were assessed by immunoblotting. E. MG132 had no effect on p18^INK4c levels in cells transduced with HA-p16^INK4a or empty vector. F. The Leiden strain of p16^INK4a-deficient fibroblasts were passaged until they reached senescence. Samples taken at the indicated PDs were immunoblotted for p18^INK4c, with CDK4 as a loading control.

A. Relative levels of INK4c, INK4a and CDC6 RNAs in Hs68 cells infected with a retrovirus encoding SV40 large T-Ag (LT) or empty vector control (Vec) (left panel). Lysates from the same cells were fractionated by SDS-PAGE and immunoblotted for p18^INK4c and p16^INK4a as indicated (right). B. Survey of p18^INK4c and p16^INK4a expression in a series of human cancer cell lines from different anatomical sites: breast (T47D, BT5499, ZR75-1 MDA-MB468 and MCF7), keratinocyte (HaCaT), bladder (K5637 and T24), cervix (C33A), bone (U2OS) and prostate (DU145 and PC3). The +/− below each lane refers to the functional status of the RB1 gene in these cell lines, based on published literature. CDK4 was used as a loading control. C. Comparison of E2F1 RNA (left) and protein (right) levels in proliferating (P) and senescent (S) HDFs (TIG3). D. Comparison of INK4c and E2F1 RNA expression in HDFs (IMR90) infected with a retrovirus encoding H-RAS^G12V (Ras) or empty vector (Vec). E. Down-regulation of E2F1 protein in HDFs (TIG3) cells transduced with H-RAS^G12V. F. Down-regulation of E2F1 in HDFs infected with a retrovirus encoding HA-p16^INK4a.

A. Diagram of the human INK4c locus with exons depicted as boxes and coding domains shaded. The location of the transcriptional start sites (arrows) and primer sets P1-P6 used for ChIP analyses are indicated. B. ChIP of menin and H3K4me3 at the INK4c locus before and after treatment of IMR90 ER:RAS cells with OHT. Enrichment for each primer set was assessed by qPCR relative to a non-specific IgG and expressed as percentage of input. C and D. Comparison of MEN1 and INK4c RNA levels in proliferating (P) and senescent (S) populations of Hs68 (C) and Leiden (D) fibroblasts (same samples as in ). E. Relative levels of MEN1 and INK4c RNA in cells transduced with retroviral vectors encoding HA-tagged versions of p16^INK4a, p18^INK4c or p21^CIP1, as in . F. Relative levels of INK4c, MEN1 and INK4a RNAs in Hs68 cells infected with a retrovirus encoding H-RAS^G12V or empty vector control (Vec), as in . G. Relative levels of INK4c, MEN1 and INK4a RNAs following addition of tamoxifen (OHT) to BF cells expressing ER:H-RAS^G12V (as in ).