Fig 2: C21orf58 facilitated the activity of wildtype and constitutively mutated STAT3 by forming ternary complex. A) C21orf58 overexpression promoted the interaction of JAK2 on STAT3. B) Attenuated C21orf58 expression decreased the interaction of JAK2 on STAT3. C) In vitro kinase activity assay was performed to verify that C21orf58 promoted the phosphorylation of STAT3 by JAK2. D) After kinase activity assay, proteins were examined by western blot and detected that C21orf58 improved the phosphorylation of STAT3 by JAK2. E) C21orf58 improved the interaction between JAK2 and constitutively activated mutants of STAT3. F) Reduction of C21orf58 expression remarkably declined the phosphorylation of constitutively mutated STAT3. G,H) Downregulation of C21orf58 effectively reduced the interaction of JAK2 on constitutively activated mutants of STAT3.

Fig 3: C21orf58 simultaneously interacted with JAK2 and STAT3 to form a ternary complex. A) The exogenous interaction between C21orf58 and STAT3 in HepG2 cells. B) Truncations of STAT3 were constructed as shown in graphic, NTD: N‐terminal domain; CCD: coiled‐coil domain; DBD: DNA‐binding domain; LD: linker domain; SH2: SH2 domain; TAD: transactivation domain. C) The interaction between C21orf58 and NTD domain of STAT3 was validated by immunoprecipitation. D) The exogenous interaction between C21orf58 and JAK2 in HepG2 cells. E) Truncations of JAK2 were constructed as shown in graphic. F) Interaction domains between JAK2 and C21orf58 was detected by immunoprecipitation, SH2 domain was the binding region of C21orf58 on JAK2. G) Co‐immunoprecipitation showed that C21orf58 simultaneously interacted with JAK2 and STAT3 in HepG2 cells. H) In vitro pull‐down assay was performed to conform that C21orf58 formed a ternary complex with JAK2 and STAT3 in HCC cells by direct interaction.

Fig 4: C21orf58 promoted sorafenib resistance of HCC cells. A) C21orf58 elevated the IC50 value of HepG2 cells. B,C) The clone formation of C21orf58 overexpressed and knockdown HCC cells treated with sorafenib at different concentrations. D) Construction of sorafenib‐resistant Huh7 cells, which were not vulnerable to sorafenib compared with their parental cells. IC50 was the 50% inhibiting concentration. E) The expression of C21orf58 and p‐STAT3(Y705) were increased in sorafenib‐resistant and Huh7 cells. F) Inhibition of C21orf58 expression using siRNA was effectively to repress the cell growth of HCC cells with sorafenib resistance. G,H) The growth curve, volume and weight of tumors derived from sorafenib‐resistant Huh7 cells were suppressed by siC21orf58, tumors treated with siC21orf58 (5 nmol) twice a week. I) After treating with siC21orf58 and negative control siRNA respectively, the expression of p‐STAT3(Y705), STAT3 and C21orf58 proteins in sorafenib‐resistant Huh7‐derived tumors were detected by western blot. siRNA processing condition: tumors were treated with siC21orf58 (5 nmol) or negative control siRNA twice a week. J) Immunohistochemistry was performed to investigate the expression of C21orf58 and Ki‐67 proteins. Scale bar = 100 µm. All *** P<0.001. siNC: negative control siRNA.

Fig 5: C21orf58 accelerated cell cycle of HCC cells and increased the expression of phosphorylated STAT3. A,B) The effects of C21orf58 overexpression and knockdown on cell cycle distribution of HCC cells. C,D) The gene set enrichment analysis (GSEA) plot of IL6‐JAK‐STAT3 signaling pathway based on the RNA seq data from control and C21orf58 knockdown HCC cells (shC21orf58‐1 and shC21orf58‐2, n = 3 per group). NES, normalized enrichment score. E,F) STAT3 and p‐STAT3(Y705) protein levels in C21orf58 overexpression and knockdown HCC cells. G) The expression levels of C21orf58 and p‐STAT3(Y705) proteins in paired clinical HCC tissues (n = 12). The positive correlation between C21orf58 and p‐STAT3(Y705) expression was assessed by linear regression. All * P<0.05, ** P<0.01, *** P<0.001. Scr: Scramble.