Fig 2: A schematic model of TRIM21 regulating OS autophagy and differentiation by interacting with ANXA2.When TRIM21 is overexpressed in OS cells, it competitively binds to ANXA2 and thus facilitating the translocation of ANXA2 toward PM; this enables the disassociation of TFEB from the ANXA2-TFEB complex, allowing TFEB entering into the nucleus. Nuclear TFEB promotes the transcription of autophagy-related genes, including ATG9B, LC3, and P62, and inhibits OS differentiation, presumably, through regulating the Sp1/RUNX2 signaling in OS cells. TRIM21 OE: TRIM21 overexpression. ×: disassociation of ANXA2-TFEB complex.

Fig 3: S100A10 knockout (KO) and AnxA2 KO (resulting in loss of full A2t) does not affect cell proliferation and inhibits multiple high-risk HPV infections in HeLa cells. (a) *Because S100A10 is rapidly degraded in the absence of AnxA2, targeting AnxA2 results in full A2t KO. Here, either dimeric S100A10 or heterotetrameric A2t (S100A10 + AnxA2) was knocked out in wild type (WT) HeLa cells via CRISPR/Cas9 and confirmed by western blot. Western blot images were cropped. See full-length blots with molecular weight standards in Supplementary Fig. 1a. (b,c) WT, S100A10 KO, and A2t KO HeLa cells were seeded with equal cell number, grown for 48 h, and then analyzed for differences in cell proliferation via (b) analyzing cell number and viability via trypan blue exclusion test, and (c) quantification of nucleic acid content via CyQUANT Cell Proliferation Assay Kit (Thermo Fisher) and then analyzed relative to WT HeLa cells. Results (b,c) represent N = 8 ± s.d. and are representative of at least 3 independent experiments. Statistics: 1-way ANOVA with Dunnett’s multiple comparison test. ns = not significant. (d) WT, S100A10 KO, and A2t KO HeLa cells were treated with HPV PsVs (TCID50) carrying a GFP reporter plasmid. Infection (GFP gene transduction) was measured 48 h p.i. via flow cytometry. Neutralizing anti-HPV L1 antibody H16.V5 (neut. Ab) was used as a positive control for infection inhibition, and background from mock infected cells was subtracted. At least 3 independent S100A10 KO and 3 independent A2t KO clones were screened for consistent inhibition of HPV16 infection. Results are representative of at least 3 independent experiments and show the mean %GFP-positive cells ± s.d. (n = 3, normalized to WT). Statistics: 1-way ANOVA with Dunnett’s multiple comparisons test – ns = not significant, *P < 0.05, ***P < 0.001, ****P < 0.0001.

Fig 4: Morphological changes into the epithelial phenotype following ANX2 knockdown in precancerous KC cells. (A) Representative images of KC cells in 3D culture. Spheroid cyst (left panel), irregular cysts (right panel), and spindle shaped cells (lower panel). Scale bar=100 µm. (B) Percentages of spindle-shaped cell formation in KC cells treated with control and ANX2 siRNAs with or without rTNC treatment. (C) Representative images of PANC-1 cells in 3D culture without (left panel) or with (right panel) rTNC treatment. Scale bar=100 µm. (D) Comparative analysis of the expression of ANX2 with or without rTNC between PANC-1 cells treated with control and human ANX2 siRNAs. (E) Percentages of spindle-shaped cell formation in PANC-1 cells treated with control and ANX2 siRNAs with or without rTNC treatment. Error bars represent the standard error of the mean. ANX2, annexin II; siRNA/si, small interfering RNA; rTNC, recombinant tenascin C; 3D, three-dimensional; NS, not significant.

Fig 5: ANXA2 promotes HCC cell proliferation, migration, and invasion in vitro. (A) qRT-PCR and western blot were conducted to measure expression of ANXA2 in ANXA2-overexpressing PLC/PRF/5 cells and ANXA2-silenced MHCC97H cells. GAPDH was used as an internal reference. (B) CCK8 assays showed that ANXA2 overexpression promoted proliferation in PLC/PRF/5 cells (upper panel), whereas ANXA2 silencing inhibited proliferation in MHCC97H cells (down panel). **P < 0.01. (C) Colony-formation results showed that ANXA2 overexpression promoted colony formation in PLC/PRF/5 cells (left panel), whereas ANXA2 silencing inhibited colony formation in MHCC97H cells (right panel). **P < 0.01. (D) Cell-migration assays showed that ANXA2 overexpression promoted migration in PLC/PRF/5 cells (left panel), whereas ANXA2 silencing inhibited migration in MHCC97H cells (right panel). **P < 0.01. Scale bar: 50 µm. (E) Cell-invasion assays showed that ANXA2 overexpression promoted invasion in PLC/PRF/5 cells (left panel), whereas ANXA2 silencing inhibited invasion in MHCC97H cells (right panel). **P < 0.01. Scale bar: 50 µm. (F) qRT-PCR was conducted to measure mRNA levels of ANXA2 in LINC01133-overexpressing PLC/PRF/5 cells and LINC01133-silenced MHCC97H cells. GAPDH was used as an internal reference. (G) Western blot analysis was used to assess protein expression of ANXA2, p-ANXA2, STAT3, p-STAT3, and cyclinD1 following stable upregulation or downregulation of LINC01133. GAPDH was used as an internal control. Data are shown as the mean ± SD and are representative of three independent experiments. (H) Schematic diagram of a proposed model of the regulation of LINC01133 in HCC