(A) Immunoblots of growing IMR90 cells infected with the indicated shRNAs probed for RB, p107 or p130. Actin was used as loading control. (B) Immunoblots of ras-senescent IMR90 cells. Chromatin-bound fractions were used for the RB, p107 and p130 blots and Histone H3 was used as loading control. Whole cell lysates were used for the p16 and ras blots and actin was used as loading control. (C) SA-β-galactosidase staining. Scale bar=100uM (D) DAPI staining to visualize SAHF. Scale bar=10uM. (E) Quantification of SAHF (red bars), BrdU incorporation (blue bars). Values represent the mean +/− standard error of at least three independent experiments. (V) is empty vector, (S) is senescent, (G) is growing, and (Q) is quiescent.

(A) Heatmap of expression patterns derived from hierarchical clustering of RB responsive genes (1826 probes) highlighting the E2F target genes. (B-E) Magnification of various gene clusters. (B) Immuno-surveillance cluster (C) Cyclin E cluster (D) DNA replication factors clusters. (E) Mitotic cell cycle cluster. See also Figure S1 and Tables S1-S3.

(A) Binding patterns of RB to the MCM3 gene shown as custom tracks on the UCSC genome browser. (B) Histogram comparing binding of RB-as measured by read counts-to DNA replication factors vs other RB targets. Error bars represent the standard deviation of the read counts. (C) Correlation of RB binding to gene promoters and expression of those genes in RB-deficient cells under different growth conditions. See also Figure S2 and Table S4-S5.

Immunoblots and qPCR to measure expression of (A) MCM2, (B) Cyclin A, and (C) Cyclin E1 in growing, quiescent or senescent cells expressing the indicated shRNA. qPCR values are averages of representative experiments done in triplicates. (D) Schematic diagram of the polycistronic shRNAs used to knockdown the indicated members of the RB family. (E) Immunoblots probed for the indicated protein from quiescent IMR90 cells lysates infected with the indicated shRNA. (F) Histograms comparing the binding intensity of p130 at gene promoters before (non-shRB) and after RNAi-mediated suppression of RB (shRB) in quiescent (top) and senescent (bottom) cells. The shift to the right of zero indicates that the p130-specific antibody co-immunoprecipitates more promoter DNA in the absence of RB. (G) Binding patterns of p130 and RB to the MCM5 gene shown as custom tracks on the UCSC genome browser. p130 binding to the MCM5 gene promoter increases 1.8 fold in RB-deficient quiescent cells while binding in senescent cells remain unchanged. See also Figure S3 and Table S6

(A) Immunoblots of chromatin-bound proteins probed for MCM2, MCM3, ORC1, and Cyclin E1. Blots were normalized by Coomassie Blue staining for histones. (B) Time course BrdU incorporation assays of cells undergoing senescence in the absence of RB, p107 or p130 compared to vector control. (S/PS3, S/PS5, S/PS7 stand for post-selection day 3, 5, or 7 respectively). Values represent the mean +/− standard error of a least three independent experiments. (C) Cells cycle profiles of growing (top), quiescent (middle) or senescent (bottom) IMR90 cells expressing the indicated shRNA. .

(A) Immunoblots of lysates from senescent cells expressing a polycistronic shRNA targeting RB and cyclin E1 (Tan depicted on the top of the figure) probed for RB and Cyclin E1. Growing (G), Senescent (S), senescent cells expressing shRNA targeting only RB (shRB/S) or only cyclin E1 (shE1/S) are used as controls. Actin is used as loading control. (B) Cell cycle profiles of vector control senescent cells (V/S), senescent cells expressing shRB (shRB/S) or the tandem shRB/cE1 shRNA (Tan/S). (C) Immunofluorescence of pre-extracted cells to measure proper association of MCM2 with chromatin. Percentage of MCM2 positive cells is indicated in the bottom right corner of the images. DAPI staining was used to visualize the nuclei. At least 200 cells were counted per experiment. Scale bar =100um. (D) Immunoblots of chromatin-bound extracts from growing (G), quiescent (Q), vector control senescent cells (V/S), senescent cells expressing shRB (shRB/S) or the tandem shRB/cE1 shRNA (Tan/S): two independent samples are shown. (E) Immunoblots of lysates from senescent cells expressing the tandem shRB/cE1 shRNA (Tan/S) and shRNA-resistant cyclin E1 cDNA encoding either wild type (WtcE) or kinase-deficient (KDcE) cyclin E1. Lysates from growing (G), vector control senescent cells (V/S), senescent cells expressing shRB (shRB/S) or the tandem shRB/cE1 shRNA (Tan/S) alone are used as controls. See also Figure S4.

(A) Immunoblots of lysates from IMR90 senescent cells expressing the indicated shRNA probed for p21 or actin. (B) Growth curve of ras-infected cells expressing the indicated shRNAs. Counting was initiated at PS7 (post-selection day 7) at which time the vector control cells are fully growth arrested as measure by BrdU incorporation. (C) Analysis of PS7 cells for different proliferation and senescence markers and for the ability to form colonies at low density. Unlike vector control, RB or p21 suppression, suppression of both RB and p21 leads to increase in cell proliferation as measured by BrdU incorporation (20% positive compared to 2% for either shRB or shp21 alone) and colony formation. Scale bars for the micrographs showing SA-β-gal staining and BrdU immunoflourescence=100uM and for DAPI staining=10uM. (D) Cell cycle profiles of ras-infected cells expressing the indicated shRNA. (E) Immunoblots of lysates from ras-infected cells expressing the indicated shRNA probed for the indicated proteins. See also figure S5