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Fig. 3 | Cell Communication and Signaling

Fig. 3

From: Heme oxygenase 1 facilitates cell proliferation via the B-Raf-ERK signaling pathway in melanoma

Fig. 3

Inhibition of HO-1 represses A375 cell proliferation. a The knockdown efficiency of HO-1 in A375 cells was confirmed by Western blotting at 36 h after transfection. Cell viability was evaluated by the CCK-8 assay. b Representative colony formation of A375 cells with HO-1 knockdown. The colony number in each well was determined and statistically analyzed with three independent experiments (n = 3). c The tumor size (mm3) was measured every 5 day for 40 days after injection in HO-1 knockdown group and control group. d Schematic representation of the genomic DNA structure of the HO-1 gene. The sequences, which include the AGG PAM targeted by gRNA with the CRISPR-Cas9 system, is shown in red. e The HO-1 expression in the knockout cell clone candidates was evaluated by Western blotting. f Clone No. 5 had the highest degree of HO-1 reduction and was sequenced, and the sequence was aligned with the wild-type sequence (+: inserted bases; −: deleted bases). g Immunofluorescence staining of F-actin using phalloidin-fluorescein isothiocyanate in scramble and HO-1−/− cells. Scale bar: 10 μm. h The colony number in each well was determined and statistically analyzed to evaluate the clonogenic survival. i CCK-8 assays were used to determine the cell viability of the HO-1−/− cells after treatment. j A375 cells with or without CRISPR/Cas9 HO-1 knockout were subcutaneously injected into the flanks of 6-week-old female SCID mice. Representative tumor images were taken. Tumor volumes (mm3) were measured every 5 days for 45 days. k Tumors were weighed at the endpoint of experiment (Day 45). **P < 0.01; ***P < 0.001 by the t-test

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