Figure 3.

TSPAN1 interacts with LC3 through conserved LIRs and promotes autophagosome maturation. (A) Co-localization between GFP-LAMP1, GFP-RAB7A and mCherry-TSPAN1 in HeLa cells. (B) Co-localization between GFP-TSPAN1 and mCherry-LAMP1 in HeLa cells. (C) Schematic illustration of TSPAN1 protein and the LIR motifs of TSPAN1 protein localized in the lysosome membrane. (D) Sequences alignment of the predicted LIR motifs and its mutations in the TSPAN1 protein. “M” represents mutation. (E) The interaction between TSPAN1 and wild-type or F52A/L53A mutant LC3. (F) The interaction between LC3 and TSPAN1 was abolished by replacing the phenylalanine and leucine with alanine residues in LIR2 or LIR3. (G) The interaction between LC3 and TSPAN1 was enhanced by CQ (10 μM) treatment. (H) Western blotting analysis of LC3 and SQSTM1 in HEK293T cells transfected with TSPAN1 and its mutants with or without CQ (10 μM) treatment. (I and J) Representative confocal microscopy images of the red-only puncta and the yellow puncta in ASPC-1 cells after co-transfection with TSPAN1 siRNA#2 (targeting the 3ʹUTR of TSPAN1 mRNA) with or without the wild-type TSPAN1 or TSPAN1 M2/3 mutant under nutrient-rich and EBSS starvation conditions. The numbers of the red-only puncta and yellow puncta were quantified (n=10). Scale bars, 10 µm. (K) Excised tumors in different groups were shown. (L) Weights of the excised tumors in each group. (M) Growth curve showing the changes in the tumor volume in mice in different groups every 5 d from the injection. Data were represented as mean ± SD, *P < 0.05; **P < 0.01; ***P < 0.001