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. 2020 Sep 29;9:e58108. doi: 10.7554/eLife.58108

Figure 6. COX6B2 is essential for tumor cell viability and survival.

(A) Left: Representative images of EdU staining in indicated cell lines. (EdU, green; Hoechst, blue). Scale bar, 100 μm. Right: Quantitation of EdU-positive cells. Bars represent the mean ± SEM (n ≥ 12). p-Value calculated by Student’s t-test. (B) Left: Representative images of colonies in soft agar. Arrows indicate formed colonies. Right: Bars represent relative mean colony numbers ± SEM (n = 6). p-Value calculated by Student’s t-test. (C) Representative immunoblots of whole cell lysates of indicated cells blotted with indicated antibodies. MW markers are indicated. (D) Left: Representative scatter plots of Annexin V staining in indicated cell lines. Bars represent mean fold change + SEM (n = 6). p Calculated by Student’s t-test. (E) Representative phase contrast images of indicated cell lines. Arrowheads indicate the flattened senescence morphology with vacuole accumulation and multinucleation. Scale bar, 100 μm. (F) Representative images of senescence-associated- β-galactosidase staining in indicated cells. Scale bar, 100 μm. Bars represent mean ± SEM (n ≥ 8). p Calculated by Student’s t-test. (G) Whole cell lysates from indicated cell lines were cultured with or without NAC (5 mM) for 7 days were immunoblotted with indicated antibodies. MW markers are indicated. (H) Representative images of senescence-associated- β-galactosidase staining in indicated cell lines cultured with or without NAC (5 mM) for all 11 days. Scale bar, 100 μm. Bars represent mean ± SEM (n = 9). p Calculated by Student’s t-test.

Figure 6.

Figure 6—figure supplement 1. Phenotypes following COX6B2 depletion.

Figure 6—figure supplement 1.

(A) The percentage of EdU-positive cells of COX6B2 depleted by three different sgRNA in HCC515 cells. Bars represent mean ± SEM (n = 7). p-Value calculated by Student’s t-test. ***p<0.001, ****p<0.0001. (B) Representative images of colonies in soft agar. Arrows indicate formed colonies. Graphs represent relative colony numbers in indicated cells. Bars represent mean ± SEM (n = 6). p-Value calculated by Student’s t-test. (C) Left: whole cell lysates from HCC515 + / - COX6B2-V5 were immunoblotted with indicated antibodies. MW markers are indicated. Right: quantitation of immunoblots on left. Bars represent mean ± range (n = 2). (D) Representative images and quantitation of western blots from whole cell lysates of indicated cells and immunoblotted with indicated antibodies. Bars represent mean ± SD (n = 3). (E) The percentage of EdU-positive cells of indicated cells cultured with or without pyruvate (1 mM) and uridine (100 μg/ml) for eight days. Bars represent mean ± SEM (n = 6). p-Value calculated by Student’s t-test. (F) Quantification of senescence-associated- β-galactosidase positive cells in indicated cells cultured with or without pyruvate (1 mM) and uridine (100 μg/ml) for 11 days. Bars represent mean ± SEM (n = 12). p Calculated by Student’s t-test.