(A) Western blot analysis of ANXA3 in Huh7-EVs and HeLa-EVs under serum starvation (SS) after siRNA-mediated ALIX or HRS knockdown. CD63 and TSG101 served as EV markers. (B and C) Polar-SIM images of ANXA3 (red) and CD63-GFP (green) in serum-starved cells after ALIX (B) or HRS (C) knockdown. Nuclei were stained with DAPI (blue). Insets show enlarged views. Scale bar: 10 μm. (D) Quantification of ANXA3⁺ MVB among CD63⁺ MVB in B and C. n = 10 cells. (E) Polar-SIM images of ANXA3 (red) and Rab5Q79L (green) in serum-starved HeLa-Rab5Q79L cells after siALIX or siHRS transfection. Nuclei were stained with DAPI (blue). Scale bar: 10 μm. (F) Quantification of ANXA3 occupancy in Rab5Q79L⁺ endosomes in E. n = 61, 48, 45, 45 Rab5Q79L⁺ endosomes. (G) Reciprocal Co-IP assays in HeLa cells cultured under 10% FBS or SS showing the interaction between ANXA3 and ALIX. Left, IP: ANXA3; right, IP: ALIX. IgG served as a negative control, and whole-cell lysates were used as input. (H) Quantification of Co-IP results in G. Left, ALIX coprecipitated with ANXA3; right, ANXA3 coprecipitated with ALIX, under 10% FBS or SS. n = 3. (I and J) HeLa cells were cotransfected with ALIX-His, and Co-IP (IP: ALIX-His) was performed to detect the ANXA3–ALIX-His interaction under 10% FBS or SS (I). (J) Quantification of ANXA3 coprecipitated with ALIX-His. n = 3. (K and L) PLA showing ALIX–ANXA3 interaction (red puncta) under 10% FBS (left) or SS (right), with nuclei stained by DAPI (blue). Boxed regions are enlarged. Scale bar: 10 μm (K). (L) Quantification of PLA signals. n = 18, 21 cells. Data are presented as mean ± SD. Statistical analysis was performed using ordinary 1-way ANOVA followed by Tukey’s multiple-comparison test. Comparisons between 2 groups were done with 2-tailed Student’s t test. *P < 0.05, ***P < 0.001.