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. 2021 Jul 19;9:720111. doi: 10.3389/fcell.2021.720111

FIGURE 3.

FIGURE 3

Nuclear factor of activated T cells inhibitor VIVIT suppresses the increased expression of autophagy-associated genes induced by the overexpression of H-Ras in vivo. (A) Scheme of the in vivo assay: Human keratinocytes (HKCs) were infected with a H-RasG12V expressing or a control retrovirus for 6 h, then were subcutaneously injected into the dorsal skin of NU/NU mice, after which the mice were treated with DMSO, VEET, or VIVIT by intraperitoneal injection. One week later, the grafts were collected for RT-PCR and western-blot analysis of autophagy-associated genes. (B–G) RT-PCR analysis of autophagy-associated genes as indicated from the grafts collected as shown in panel (A). (H) The grafts collected were lysed for immunoblotting analysis of autophagy-associated genes LC3, Beclin-1, p62, Lamp-1, and ATG5-12, GAPDH as a loading control. (I) Quantification of western-blot analysis shown in panel (H) for the relative expression levels of LC3, Beclin-1, p62, Lamp-1, and ATG5-12. Four mice (n = 4) were used in each group, and error bars represent means ± SD; p values are indicated with “*,” *p < 0.05, **p < 0.01, ***p < 0.005 when comparing two corresponding groups indicated with the black lines by Student’s t-test.