Fig. 3

ATF4-mediated degradation of CD58 via Dynamin1 (DNM1). a, b qRT-PCR for CD58 mRNA expression in MHCC-97H and SNU-387 after ATF4 overexpression or silence. (n = 3/group). c Combinatorial analysis of the genes up-regulated in control cells compared with silenced ATF4 and genes down-regulate CD58 protein expression, uncovered 19 common genes including DNM1. d Correlation analysis between ATF4 and DNM1 mRNA expression in human HCC tissues using RNA-seq data from TCGA. e Correlation analysis between ATF4 and DNM1 mRNA expression in human HCC tissues by qRT-PCR. (n=79). f qRT-PCR for DNM1 mRNA expression in MHCC-97H and SNU-387 after ATF4 overexpression or silence. (n=3/group). g Relative luciferase activity of DNM1 promoter plasmid in HEK 293T cells overexpressing ATF4. (n=3/group). h Sequence for ATF4-binding sites in the promoter of DNM1. ChIP for DNA fragment containing the ATF4 binding motif in the DNM1 promoter. P1 and P2 are two binding motifs. (n=3/group). i Western blot for DNM1 and ATF4 expression in MHCC-97H and SNU-387 after ATF4 overexpression or silence. j Western blot for DNM1 and CD58 expression in MHCC-97H and SNU-387 after DNM1 overexpression or treating with dynasore. k Western blot for CD58 and ATF4 expression in ATF4 overexpression SNU-387 along with dynasore treatment. l qRT-PCR for GAMB and IFNG mRNA expression in CD8⁺ T cells co-cultured with ATF4 overexpression SNU-387 along with 10 μM dynasore treatment. (n=3/group). m ELISA of IFNG and GZMB in CD8⁺ T cells cultured with overexpressing ATF4 SNU-387 cells following 10 μM dynasore treatment. (n=3/group). n The left, immunostaining for CD8 (green) and DAPI (blue) in CD8⁺ T cells co-cultured with SNU-387 cells after overexpressing ATF4 along with dynasore treatment. The right is quantification. (n=5/group). Statistical analysis performed using two-tailed t-test (A, B, F, G, H, L-N) and one-way ANOVA (A, B, F). ns for P>0.05, *for P < 0.05, **for P < 0.01, ***for P < 0.001, ****for P < 0.0001