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. Author manuscript; available in PMC: 2026 Jun 19.
Published in final edited form as: Cell Rep. 2018 Dec 26;25(13):3706–3720.e8. doi: 10.1016/j.celrep.2018.12.017

Figure 3. Senescence Hallmarks after VEGFR TKI Resistance Are Model Dependent and Reversible.

Figure 3.

(A) Western blot analysis for cell-cycle regulatory proteins in LM2–4 SuR cell variants (n = 3).

(B) Representative blots (left) and quantification (right) of p21 protein levels in SuR and AxR cell variants by western blot analysis (n = 3).

(C) Senescence-associated β-galactosidase-positive cell quantification for LM2–4, RENCA, and 3T3 SuR cell variants (left) with representative RENCA SuR cell variants pictured (right) (n = 3).

(D) Cell size measurement for SuR and AxR cell variants. Representative pictures and cell area histograms for LM2–4 SuR cell variants (top) and area quantification for each cell (bottom) are shown (n > 2,500 cells).

(E) Cell-cycle analysis for LM2–4 SuR cell variants (left) with heatmap emphasizing the effect on G1 and G2 during resistance and after short- and long-term therapy withdrawal (right) (n = 3).

(F) Proliferation assay for LM2–4 and RENCA SuR cell variants after Su treatment at different concentrations (n = 5–10).

(G) Proliferation assay for LM2–4 and RENCA SuR cell variants after Su withdrawal for different number of days (n = 6).

(H) Heatmap showing the senescent characteristics acquired by SuR and AxR cell lines before and after LT-W (compared to parental controls).

SuR, sunitinib-resistant; AxR, axitinib-resistant; W, withdrawal; ST, short term; LT, long term; SA-β-gal, senescence-associated β-galactosidase; prolif., proliferation; GA, growth arrest; NT, not tested. For (H), closed circles are positive data, and open circles are negative data for senescent characteristics. ST-W equaled 2 days (for A–C and E) and 7 days (for F). For (A)–(C) and (E), Su (5 μM) or Ax (0.5 μM) treatment was for 2 days in P cells. Quantitative data are shown as mean ± SD, except for cell size (mean ± SEM). *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001, compared to parental cell lines or untreated resistant cells, unless noted otherwise. All western blot images have been adjusted equally for brightness. Replicates and unadjusted uncropped images are shown in Data S1. See also Figure S3.