Figure 5.

ANXA2 and LC3 knockdown in OSCC xenograft tumors resulted in attenuation of mitophagy

(A) ANXA2, LC3, or scrambled control (SCR) shRNA were stably expressed in UMSCC1A cells. Cells were then injected into the midline flanks of nude mice and allowed to incubate for approximately 23 days before intraperitoneal treatment with LCL768/vehicle control (10 mg/kg, n = 8). Flank tumors were measured using calipers, and measurements indicate mean ± SD of n = 8 independent experiments. Student’s t test was conducted to examine significance in growth response curves (SCR VEH vs. LCL ∗∗p < 0.01, ANXA2KD VEH vs. LCL Ns not significant, LC3KD vs. LCL Ns not significant).

(B) Survival Kaplan-Meier curves of nude mice displaying xenograft tumors induced by injection with UM-SCC-1A OSCC cells stably expressing shRNA for ANXA2, LC3, or scrambled (SCR) control and treated/untreated with LCL768 (n = 8 mice). ∗p < 0.05.

(C) Immunofluorescence was completed on sections of nude mouse xenograft flank tumors stably expressing either ANXA2, LC3, or scrambled (SCR) control shRNA and treated with LCL768 or vehicle control intraperitoneally (10 mg/kg). Quantification of confocal images of LC3 and Tom20, ceramide and LC3, Tom20 and LAMP1, and ceramide and ANXA2 association are shown. Pearson’s correlation coefficient values indicate mean ± SD of n = 3 independent experiments, normalized to uninfected, untreated control. ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

(D) Mouse xenograft flank tumors injected with cells stably expressing ANXA2, LC3, or SCR shRNA and treated/untreated with LCL768 were stained with Ki67 antibody, indicating proliferating tumor cells.

(E) Quantification of Ki67+ nuclei staining in tumor sections was performed in inform Automated Image Analysis software and represents a mean ± SD of n = 3 independent experiments.