Journal: Journal of Biological Chemistry

Article Title: Opposite Functions for E2F1 and E2F4 in Human Epidermal Keratinocyte Differentiation

doi: 10.1074/jbc.m004973200

Figure Lengend Snippet: FIG. 1. Expression of E2F members during in vitro differentiation of human HaCaT keratinocytes. Differentiation was induced by serum starvation as described previously (3), and at the stated times (days) total RNA or protein extracts were obtained and analyzed by Northern (A) or Western (B) blot with the indicated specific probes or specific antibodies (see “Materials and Methods”). Keratin K10 was used to test the induction of differentiation, and 7 S and keratin K14 were used to normalize the loading for Northern and Western analysis, respectively. C, specificity of the E2FzDNA complexes during differentiation binding analyzed by EMSA. The complexes formed using a consensus E2F-specific oligonucleotide (see “Materials and Methods”) and cell extracts from day 4 of differentiation are displaced by a 20-fold excess of the same unlabeled oligonucleotide (w.t. lane) but not by a point-mutated form (mut lane). D, EMSA analysis using extracts from HaCaT cells at the stated days after serum starvation reveals the formation of multiple E2FzDNA complexes. E, pretreatment with 0.1% NaDOC leads to the formation of a single band, corresponding to free E2FzDNA complexes, indicating that the bands observed throughout differentiation are due to the presence of other proteins in the complexes. F, interaction of Rb family proteins with E2F during differentiation. Protein extracts (500 mg), obtained at the indicated days (0–20) after induction of differentiation, were immunoprecipitated with antibodies against the indicated Rb family proteins (Ippt column). Immunoprecipitates were subsequently probed by Western blotting with antibodies specific for E2F1 or E2F4 (wb column). Note that pRb binds E2F1 at early differentiation, and E2F4 only in proliferating cells (time 0). p107 binds E2F4 in proliferating cells and also at days 12 and 16. Finally, p130 binds E2F4 during late differentiation (days 16 and 20).

Article Snippet: Total protein (500 mg; quantified using the Bio-Rad protein assay) was incubated in the presence of 2 ml of antibodies against pRb, p107, or p130 (Santa Cruz) for 12 h at 4 °C.

Techniques: Expressing, In Vitro, Northern Blot, Western Blot, Binding Assay, Immunoprecipitation

Journal: Oncotarget

Article Title: XPO1 inhibition by selinexor induces potent cytotoxicity against high grade bladder malignancies

doi: 10.18632/oncotarget.26179

Figure Lengend Snippet: ( A ) Representative images of p107, p130 and RB (green) IF staining of bladder cancer cells treated with vehicle (V) or selinexor (S) for 48 hours. Tubulin staining (red) and DAPI staining (blue) served to define the cytoplasmic and nuclear compartments, respectively. The inserts are magnifications of the boxed cells. ( B ) Quantification of staining intensity of pocket proteins normalized to DAPI. ( C ) Nuclear and cytoplasmic fractions of cell treated with vehicle or 0.15 uM selinexor (UM-UC-3 and T24 cells), 0.25 uM selinexor (J82) and 0.5 uM selinexor (TCCSUP) for 72 hours were assessed for the expression of RB, p107 and p130. Nup62 and tubulin were used as markers for the nuclear and cytoplasmic fractions, respectively. ( D ) T24 and UM-UC-3 cells transfected with siC or siRB and were treated with vehicle or 0.1 uM selinexor for 72 hours. The results are shown as percent cell viability comparing drug treated to vehicle treated cells. ( E ) Palbociclib reduces T24 and UM-UC-3 bladder tumor cells viability in a dose dependent manner. ( F ) Combined selinexor (0.1 uM) and palbociclib (0.5 uM) treatment is more effective in reducing viability of cells than either treatment alone where the CI = 1.04 for UM-UC-3 cells and 1.02 for T24 cells indicating an additive response. Error bars = ± standard deviation. Student’s t test; * denotes p ≤ 0.05, ** denotes p ≤ 0.01.

Article Snippet: The following antibodies were used: Cell Signaling Technology: PARP (9542s), RB (9309s), tubulin (2125s); Santa Cruz Biotechnology: p27 (sc-528), XPO1 (CRM1) (sc-5595), p130 (sc-317), cyclin A (sc-596), GAPDH (sc-32233), actin (sc-8432) and cdk2 (sc-163); Covance: nup62 (MMS-120P); Novus Biologicals: p107 (NBP2-33735); Bethyl: cyclin B1 (A305-000A).

Techniques: Staining, Expressing, Transfection, Standard Deviation

Journal: Cancer Cell International

Article Title: Association between copy-number alteration of +20q, −14q and −18p and cross-sensitivity to tyrosine kinase inhibitors in clear-cell renal cell carcinoma

doi: 10.1186/s12935-020-01585-1

Figure Lengend Snippet: +20q and resistance to Sunitinib and Cabozantinib. Reproduced from TCGA, shown were a overall survival, b progression-free survival and c clinicopathological parameters of patient with and without +20q; Gene enrichment and functional annotation analysis in TCGA cases with or without +20q using the d NET-GE and e GSEA platforms with f genes of interest shown in heatmap; g Reproduced from TCGA dataset, shown was correlation between copy number against corresponding mRNA expression of the gene of interest; h Reproduced from TCGA and Human Protein Atlas dataset, shown was overall survival of patients with higher and lower RBL1 expression with automatically designated cutoff value; i Reproduced from GEO dataset, shown was RBL1 expression in Sunitinib –untreated and –resistant RCC cells (Data were presented as mean ± standard deviation; *P < 0.05; **P < 0.01; ***P < 0.001)

Article Snippet: Two transcripts were selected for each gene (TRCN0000253673 and TRCN0000253672 for KLHL33; TRCN0000144667 and TRCN0000142196 for ARHGAP28). cDNA clones for overexpression of RBL1 was purchased from Origene (RC207017L3V).

Techniques: Functional Assay, Expressing, Standard Deviation

Journal: The Journal of Clinical Investigation

Article Title: Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice

doi: 10.1172/JCI140903

Figure Lengend Snippet: (A) Either one of the RB-like proteins, p107 or p130, can participate in DREAM assembly by binding to MuvB in WT mice and repressing transcription. In p107D/D p130+/+ mice, the p107D mutation prevents it from binding MuvB, rendering p107D unable to participate in DREAM assembly but still able to form p107-E2F complexes at CHR elements. p130 is now the only RB-like family member able to mediate DREAM assembly in p107D/D p130+/+ mice. In p107D/D p130–/– mice, ablation of p130 (p130–/–) combined with p107D prevents DREAM assembly. The MuvB core now binds to B-MYB to form MYB-MuvB and activates transcription. (B) Protein extracts were prepared from the livers and spleens of 3-month-old p107D/D p130–/– and p107D/D p130fl/fl control mice. The expression of p107D and p130 protein levels was detected by Western blotting, and tubulin served as a loading control. (C) ChIP-qPCR assay to detect p107D and B-MYB binding at the TSS of Mybl2, a known DREAM target. Illustration depicts the primers used for qPCR and the regions of interest: black arrows indicate the negative control 1 kb upstream of the TSS, and red arrows indicate an approximately 100 bp region surrounding the TSS and containing CDE (blue box) and CHR (green) motifs. Chromatin was prepared from livers, and p107 and B-MYB antibodies were used to precipitate associated DNA (n = 4 for each). Graphs show mean quantities of the indicated genome locations precipitated, and error bars indicate 1 SD. Two-way ANOVA was performed for each graph, and significance levels are indicated (**P < 0.01; ****P < 0.0001).

Article Snippet: Antibodies used for blotting were p107 (MyBioSource, anti–p107 rabbit antibody MBS440044), p130 (Santa Cruz Biotechnology, anti–p130 rabbit antibody SC-317), apoA-IV (Cell Signaling Technology, anti–ApoA4 mouse antibody 5700), tubulin (Cell Signaling Technology, anti–tubulin rabbit antibody 2125), and vinculin (Cell Signaling Technology, anti-vinculin rabbit antibody 4650).

Techniques: Binding Assay, Mutagenesis, Control, Expressing, Western Blot, ChIP-qPCR, Negative Control

Journal: The Journal of Clinical Investigation

Article Title: Disrupting the DREAM transcriptional repressor complex induces apolipoprotein overexpression and systemic amyloidosis in mice

doi: 10.1172/JCI140903

Figure Lengend Snippet: (A–D) Chromatin was prepared from livers of 3-month-old p107D/D p130fl/fl and p107D/D p130–/– mice and utilized in ChIP assays to detect p107D, B-MYB, and H2AZ occupancy at promoters (n = 4). For each of Apoa1 (A), Apoa2 (B), Apoa4 (C), and Alb (D) genes, a schematic is shown to illustrate primer annealing sites. Arrows depicting primers are color coded: black represents a neutral location 1 kb upstream of the TSS; red is an approximately 100 bp region encompassing the CHR and/or CDE motifs near the TSS; purple is within the gene body. ChIP protein targets p107, B-MYB, and H2AZ are organized in columns across the top. Bar graphs depict the mean quantity of chromatin associated with each protein target as detected by qPCR and error bars represent 1 standard deviation. Two-way ANOVA was performed for each graph and significance levels are indicated (*P < 0.05; **P < 0.01; ***P < 0.001; and NS, P > 0.05).

Article Snippet: Antibodies used for blotting were p107 (MyBioSource, anti–p107 rabbit antibody MBS440044), p130 (Santa Cruz Biotechnology, anti–p130 rabbit antibody SC-317), apoA-IV (Cell Signaling Technology, anti–ApoA4 mouse antibody 5700), tubulin (Cell Signaling Technology, anti–tubulin rabbit antibody 2125), and vinculin (Cell Signaling Technology, anti-vinculin rabbit antibody 4650).

Techniques: Standard Deviation