Fig 2: Low pH triggers MakA-induced membrane damage and non-canonical autophagy—Supplemental immunofluorescence data. (A) Representative confocal images of MEF cells treated with 250 nM Alexa568-MakA for 16 h and co-stained for endogenous IST1, CHMP2A, or ALIX, as indicated. Nuclei were counterstained with DAPI. Scale bars, 10 µm. Alexa568-MakA refers to Alexa568 labeled monomeric MakA. (B) Representative confocal images of HeLa cells treated with 125 nM Alexa568-MakA for 16 h and co-stained for endogenous ALIX and Gal3 or Gal8, as indicated. Nuclei were counterstained with DAPI. Scale bars, 10 µm. Alexa568-MakA refers to Alexa568 labeled monomeric MakA. (C) Representative confocal images of HeLa cells transfected with mCherry or mCherry-SopF and treated with 250 nM FITC-MakA for 16 h. Cells were co-stained for endogenous LC3 (left) or ALIX (right). Scale bars, 10 µm. FITC-MakA refers to FITC labeled monomeric MakA.

Fig 3: Immunofluorescence images of Gal3 and LAMP-2 expression in AZM and LPZ-treated cells for assessing lysosomal membrane permeability. Fluorescence images of galectin-3 puncta and LAMP-2 expression and distribution in A549 cells treated with AZM (50 µM) or LPZ (100 µM) for 24 h. A549 cells treated with 1 mM LLOMe for 3 h were used as LMP-positive control as previously described (29). Scale bar, 10 µm. *P<0.05 vs. untreated A549 cells. The summarized Gal3-positive cell ratio in 6 fields is presented in the bottom right panel. (n=6; mean ± SD). *P<0.05 vs. the control. Gal3, galectin-3; LAMP-2, lysosome-associated membrane protein 2; AZM, azithromycin; LPZ, lansoprazole; LLOMe, L-Leucyl-L-Leucine methyl ester (hydrochloride); LMP, lysosomal membrane permeabilization.

Fig 4: GAL3-PODXL is a mutually antagonistic ligand-receptor pair.(A and B) Western blot and quantitation, PC3-high expressing Scramble or GAL3 shRNAs for GAL3 or GAPDH (loading control). Lines, not adjacent in gel. Normalized to Scramble, means ± SD, n = 3 independent experiments. (C and D) Phase images and quantitation of (A). Cysts, yellow outlines. Outlines colored by time in single frames and overlaid. Heatmap, proportions of round, spread, and tunnel forming, z-score–normalized. n = 3 independent experiments, 4 technical replicates per condition, 1063 to 1789 cysts per condition per experiment. (E and F) Phase images and quantitation of PC3-high cysts (control) with Re:GAL3. Cysts, yellow outlines. Outlines colored by time in single frames and overlaid. Heatmap, proportions of round, spread, and tunnel forming, z-score–normalized. n = 3 independent experiments, 3 technical replicates per condition, 297 to 577 cysts per condition per experiment. (G and H) Western blot and quantitation, PC3 overexpressing GFP-PODXL WT, coexpressing either TagRFP-T, TagRFP-T-GAL3 (WT or R186S mutant), and Scramble or GAL3 shRNA for RFP, GAL3, GFP, or GAPDH (loading control for RFP). Combined band intensity of GAL3 relative to control. Means ± SD, n = 4 independent experiments. (I and J) Phase images and quantitation of cysts from (G). Cysts, yellow outlines. Outlines colored by time in single frames and overlaid. Heatmap, proportions of round, spread, and tunnel forming, z-score normalized. n = 2 independent experiments, 3 technical replicates per condition, 287 to 551 cysts per condition per experiment. (K and L) Representative plot and geometrical mean of surface PODXL levels in Scramble, PODXL, or GAL3 shRNA cells. Bar chart: Dots, experiments; midline, mean; boundaries, minimum and maximum values; n = 3 independent experiments. Heatmap P values, Cochran-Mantel-Haenszel test, Bonferroni-adjusted, comparing proportion to control, grayscale as indicated. Scale bars, 300 μm. P values, unpaired Student’s two-tailed t test; *P ≤ 0.05, **P ≤ 0.005, ***P ≤ 0.0005, and ****P ≤ 0.0001.

Fig 5: PODXL cortical levels regulate metastasis in vivo.(A to R) PT and metastasis in mice injected with PC3 (A to K) expressing Scramble or PODXL shRNA, n = 12 mice per condition or (L to R) sorted for low or high surface PODXL, n = 10 mice per condition. (A and L) PT incidence. In mice with PT, (B and M) prostate volume by ultrasound, (C and N) prostate weight, (D and P) the number of mice with metastases, (E and Q) and the number of metastasis-containing organs per mouse; in mice with metastasis, (F and R) metastasis site tropism. (G and H) Heatmap, weighted histoscore for PODXL or GAL3 across six control mice (Scramble) and for PT (prostate) or metastatic sites. (I and J) Pearson correlation of histoscore from PT of six control mice (Scramble) with the number of organs bearing macrometastasis. (K) Serial sections from mouse ID = 3 of the PT (prostate) and metastasis (abdominal wall), stained for H&E, PODXL, or GAL3. Scale bars, 100 μm. Arrowhead, cortical PODXL. (O) Images at intermediate magnification (H&E; scale bars, 200 μm) with high-magnification inserts (H&E; scale bars, 100 μm). Low–surface PODXL tumors (i and ii), black arrows, solid growth with rounded contours; black arrowheads, geographic necrosis with sparse islands of viable tumor around capillaries. High–surface PODXL tumors (iii and iv), yellow arrowheads, looser architecture, infiltrative growth with frequent lymphovascular space invasion; yellow arrow, infiltrative focus. Box-and-whiskers plots, dots, values from individual mice; +, mean; midline, median; boundaries, quartiles; error bars, minimum to maximum values; n = 3 to 10 mice that developed PT. P values (chi-square test on raw counts for contingency tables or unpaired Student’s two-tailed t test for box-and-whiskers plots..