Journal: Biochemical and biophysical research communications

Article Title: Loss of DNA replication fork protection by TIMELESS degradation supports oncogene-induced senescence

doi: 10.1016/j.bbrc.2025.152203

Figure Lengend Snippet: (A) Schematic of PARP1-mediated TIM PARylation and PAR-dependent proteolysis. (B-D) WB of U2OS parental or PARP1 KO clones transduced with pBABE-HRAS G12V (vs. EV) for 24 h. Where indicated, 10 μM olaparib (ola) or talazoparib (tal) was co-treated for 20 h before harvest. (E) WB of IMR90 ER:HRAS G12V induced by 100 nM 4-OHT in the presence or absence of 10 μM olaparib. (F, G) WB of BJ-5ta ER:HRAS G12V induced by 4-OHT. For (G), cells were co-treated with indicated inhibitors for 16 h. (H) Anti-Flag immunoprecipitation (IP) of Flag-TIM (pcDNA4) in denaturing conditions followed by anti-pADPr WB in U2OS ER:HRAS G12V induced by 4-OHT and treated with indicated inhibitors for 16 h. (I) As (H), but in cells expressing Flag-TIM wild-type (WT) or PBM1/2. (J) A model depicting the signaling cascade that connects oncogenic RAS to PARP1, prompting PAR-dependent proteasomal degradation of TIM.

Article Snippet: Following antibodies were used: TIMELESS (Bethyl, A300–961A), RB (Santa Cruz, sc-102), HRAS (Proteintech, CL488–18295), Poly(ADP-ribose) (R&D, 4336-BPC-100), PCNA (Santa Cruz, sc-56), Cyclin A (Santa Cruz, sc-271682), and anti-Flag M2 affinity gel (Sigma, A2220).

Techniques: Activation Assay, Clone Assay, Transduction, Immunoprecipitation, Expressing

Journal: Clinical Cancer Research

Article Title: Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C Inhibition

doi: 10.1158/1078-0432.ccr-19-3523

Figure Lengend Snippet: Figure 1. Feedback reactivation of RAS signaling occurs following KRASG12C inhibition. A, Cell lines were treated for 72 hours with a dose titration of ARS-1620 and viability was measured by CellTiter-Glo. B, KRAS-G12C–mutant cell lines were treated with ARS-1620 (1 mmol/L) for 0, 4, 24, 48, and 72 hours. Western blot analysis was performed for phospho- (p)MEK, pERK, pRSK, pAKT, and total MYC with GAPDH as a loading control. C, Densitometry of phospho-ERK normalized to GAPDH for blots in, results represent an average of phospho-ERK across all 8 cell lines (A). D, Cell lines were treated with a dose titration of 0.3–10 mmol/L ARS-1620 for 4 or 48 hours and lysates were subject to a RAF-RBD pulldown and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. E, Densitometry analysis of 4 and 48 hours KRAS-GTP levels normalized to input KRAS and GAPDH loading control in D. F, Densitometry analysis of 48 hours KRAS-GTP, NRAS-GTP, and HRAS-GTP levels normalized to input RAS and GAPDH loading control of blots in D. Densitometry results in E and F represent an average across all 8 cell lines.

Article Snippet: Pulldown samples and whole-cell lysates were resolved on 4%–12% Bis-Tris Gels, and Western blotting was performed using antibodies against KRAS (Sigma), NRAS (Santa Cruz Biotechnology), HRAS (Proteintech), and pan-RAS (Cell Signaling Technology).

Techniques: Inhibition, Titration, Mutagenesis, Western Blot, Control

Journal: Clinical Cancer Research

Article Title: Vertical Pathway Inhibition Overcomes Adaptive Feedback Resistance to KRASG12C Inhibition

doi: 10.1158/1078-0432.ccr-19-3523

Figure Lengend Snippet: Figure 3. SHP2 inhibition enhances the efficacy of KRASG12C inhibition. A, Cell lines were treated with ARS-1620 (1 mmol/L), SHP099 (10 mmol/L), or a combination for 4 or 48 hours and lysates were subject to a RAF-RBD pulldown and blot analysis of KRAS, NRAS, HRAS, and total RAS as well as pERK, pRSK, and GAPDH for input samples. B, KRASG12C-mutant cell lines were treated with ARS-1620 (1 mmol/L), SHP099 (10 mmol/L), or a combination for 0, 4, 24, 48, and 72 hours. Western blot analysis was performed for pMEK, pERK, pRSK, pAKT, total MYC with GAPDH as a loading control. C, Densitometry analysis of pERK normalized to GAPDH was performed for all cell lines. D, Quantification of crystal violet stain of cell lines treated with ARS-1620 (1 mmol/L), SHP099 (10 mmol/L), or a combination for 10–14 days, statistical significance was evaluated by Student t test, where , P < 0.05; , P < 0.01; , P < 0.001; , P < 0.0001.

Article Snippet: Pulldown samples and whole-cell lysates were resolved on 4%–12% Bis-Tris Gels, and Western blotting was performed using antibodies against KRAS (Sigma), NRAS (Santa Cruz Biotechnology), HRAS (Proteintech), and pan-RAS (Cell Signaling Technology).

Techniques: Inhibition, Mutagenesis, Western Blot, Control, Staining