Supplementary Table 4. The Chi-squared and Fisher's Exact tests of metastases in the group of mice with orthotopic tumor.

Figure S10. (A) Evaluation of MGAT5-mediated Integrin αv glycosylation in the orthotopic tumor samples. Tissue samples were co-stained with Integrin αv (green) and Cy3 labeled PHA-L lectin (red). Representative Z-stack images were collected by SIM and reconstructed using Imaris for clarity. (B) Mander's colocalization between red and green in the samples from A. Kruskal-Wallis test; n indicates the number of foci counted. For all statistics: median ± SD, ****p<0.0001, ***p<0.001, **p<0.005, *p<0.05. (C) Evaluation of MGAT3-mediated Integrin αv glycosylation in the orthotopic tumor samples. Tissue samples were co-stained with Integrin αv (green) and Cy3 labeled PHA-E lectin (red). Representative Z-stack images were collected by SIM and reconstructed using Imaris for clarity; bars, 3 µm. (D) Mander's colocalization between red and green in the samples from C. Kruskal-Wallis test; n indicates the number of foci counted; mean ± SD, ****p<0.0001, **p<0.01, and *p<0.05.

PC-3 cell treated with water, stained for Golgi (GRASP65, green) and MGAT3 (red). Surfaces were reconstructed by Imaris.

Figure S3. (A) Representative images of triple IHC staining of Integrin αvβ6 (green), Gal-3 (brown), and Na+/K+-ATPase (red) in normal prostate and tumor tissues from PCa patients with different grades. Deconvoluted images of Integrin αvβ6 are shown on the right; bars, 100 µm. (B) Quantification of Integrin αvβ6 H-score at PM from samples in A. Dunn Test (1964) Kruskal-Wallis multiple comparisons, p-adjusted using Benjamini-Hochberg; ****p<0.0001 and **p<0.01, median ± SD. (C) Quantification of Integrin αvβ6 and Gal-3 colocalization at PM in normal prostate and PCa, grades 2-5. Mann Whitney test; ***p<0.001, mean ± SD.

Figure S2. (A) Representative images of triple IHC staining of Integrin αvβ5 (green), Gal-3 (red), and E-cadherin (brown) in the normal prostate and tumor tissues from PCa patients with different grades. Deconvoluted images of Integrin αvβ5 are shown on the right; bars, 100 µm. (B) Quantification of Integrin αvβ5 H-score at PM from samples in A. Dunn Test (1964) Kruskal-Wallis multiple comparisons, p-adjusted using Benjamini-Hochberg; ****p<0.0001 and ***p<0.001, median ± SD. (C) Quantification of Integrin αvβ5 and Gal-3 colocalization at PM in normal prostate and PCa, grades 2-5. Kruskal-Wallis test; ***p<0.001, mean ± SD.

Supplementary Table 1. Combinations of Abs used for IHC experiments.

Figure S9. (A) Left Panels: representative images of triple IHC staining of Na+/K+-ATPase (green), Integrin αv (red), and Gal-3 (brown) in the orthotopic tumor samples from the indicated group of mice; bars, 100 µm. Middle panel: IF of Golgi stained by GM130 in the prostate tumor foci; bars, 10 µm. White boxes indicate areas enlarged at the right. (B) Quantification of Golgi fragments per cell in tumor foci from samples in A; median ± SD. (C) ATF6 IF in the orthotopic tumor samples from the indicated group of mice; bars, 20 µm. Areas of mice prostate epithelium is highlighted by dotted lines. (D) Quantification of ATF6 IF intensity from samples in C; median ± SD. For all graphs: pairwise comparisons using Wilcoxon rank sum exact test, p-adjusted using Benjamini-Hochberg; ****p<0.0001, **p<0.01, and *p<0.05; n indicates the number of foci counted. (E) IF staining of the prostate tumor foci from the indicated group of mice to visualize Na+/K+-ATPase (magenta), Integrin αv (red), and Gal-3 (green); bars, 10 µm. (F, G) Quantification of IF intensity of Integrin αv (F) and Gal-3 (G) at the PM in samples from C; median ± SD.

PC-3 cell treated with HCQ, stained for Integrin αv (red), Gal-3 (green), EEA1 (magenta). Areas of 3-color colocalization are highlighted with white. Surfaces were reconstructed by Imaris.

Supplementary Methods and Materials

Figure S6. (A) Immunostaining of Gal-3 (green) and Na+/K+-ATPase (red) in control and HCQ-treated PC-3 cells; bars, 20 µm. (B) Quantification of Gal-3 integrated intensity at PM in cells from A. Mann Whitney test; *p<0.05, mean ± SD; n indicates number of cells.

Supplementary Table 3. The detailed information about orthotopic surgery, tumor sizes, and metastases.

Figure S7. (A, B) Alcohol dehydrogenase (ADH1A) and Aldehyde dehydrogenase (ALDH2) W-B of the lysate of PC-3 (A) and DU145 (B) cells. Asterisks indicate the bands corresponding to the appropriate proteins.

Figure S8. AST (A) and ALT (B) serum activity from the indicated mice. ALT and AST activity were examined using the ALT Assay Kit (Abcam, ab241035) and the AST Assay Kit (Abcam, ab105135), respectively, per the manufacturer's protocol. Kruskal-Wallis test, all p>0.05, mean ± SD; n represents number of mice tested.

The following provides a summary of the 16th World Congress on Inflammation (WCI 2024), which took place in Québec City (Canada) from July 21 to 24, 2024. At the meeting, researchers from around the world presented their latest findings which transcended fundamental and clinical science in the field of inflammation. Participants shared original work which covered a multitude of topics and discussed practical aspects of their research including the application of new diagnostic strategies and medical therapeutic technologies. Among the main topics discussed at the congress were: immunometabolism, immunity, infectious diseases, pathology of the lungs, skin and musculoskeletal system, microbiota, mucosal inflammation, neuro-inflammation, pain, targets in the treatment of inflammation and much more. The overarching theme of the event was "Inflammation in Health and Disease: A Balancing Act".

Figure S4. (A) Representative images of Golgi in DU145 cells treated with 60 µM HCQ for 72 h and stained by GM130 (green); bars, 20 µm. (B) Quantification of Golgi spots per cell in samples from A. Unpaired t test; **p<0.01, mean ± SD; n represents a number of cells counted. (C) Representative images of Golgi in DU145 cells treated with control or ATF6α siRNAs and stained by GM130 (green); bars, 20 µm. (D) Quantification of Golgi spots per cell in samples from C. Unpaired t test; ***p<0.001, mean ± SD; n represents number of cells counted. (E) ATF6 W-B of the lysates of DU145 cells transfected with control or ATF6α siRNAs; β-actin is a loading control. (F) Representative images of Golgi in PC-3 cells treated with control or ATG5 siRNAs and stained by GM130 (green); bars, 20 µm. (G) Quantification of Golgi spots per cell in samples from F. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (H) ATG5 W-B of the lysates of PC-3 cells transfected with control or ATG5 siRNAs; γ-tubulin is a loading control. (I) Representative images of Golgi in DU145 cells treated with control or ATG5 siRNAs and stained by GM130 (green); bars, 20 µm. (J) Quantification of Golgi spots per cell in samples from I. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (K) ATG5 W-B of the lysates of DU145 cells transfected with control or ATG5 siRNAs; γ-tubulin is a loading control. (L) Representative images of Golgi in PC-3 cells treated with Bafilomycin A1 (10 µM for 72 h) or appropriate amount of DMSO and stained by GM130 (green); bars, 20 µm. (M) Quantification of Golgi spots per cell in samples from L. Unpaired t test; ****p<0.0001, mean ± SD; n represents number of cells counted. (N-R) GRASP65 (N), GM130 (O), Giantin (P), Golgin-245 (Q), and TGN46 (R) W-Bs of the lysates of PC-3 cells treated with HCQ; β-actin is a loading control. All data presented are representative of at least three independent experiments.

Figure S1. (A) Representative images of triple IHC staining of Integrin αvβ3 (green), Gal-3 (red), and E-cadherin (brown) in normal prostate and tumor tissues from PCa patients with different grades. Deconvoluted images of Integrin αvβ3 are shown on the right; bars, 50 µm. (B) Quantification of Integrin αvβ3 H-score at PM from samples in A. Dunn Test (1964) Kruskal-Wallis multiple comparisons, p-adjusted using Benjamini-Hochberg; ****p<0.0001 and *p<0.05, median ± SD. (C) Quantification of Integrin αvβ3 and Gal-3 colocalization at PM in normal prostate and PCa with grades 2-5. Kruskal-Wallis test; *p<0.05, mean ± SD.

Supplemental Movie 1 PC-3 cell treated with water, stained for Golgi (GRASP65, green) and MGAT3 (red). Surfaces were reconstructed by Imaris. Supplemental Movie 2 PC-3 cell treated with HCQ, stained for Golgi (GRASP65, green) and MGAT3 (red). Surfaces were reconstructed by Imaris. Supplemental Movie 3 PC-3 cell treated with water, stained for Integrin αv (red), Gal-3 (green), EEA1 (magenta). Areas of 3-color colocalization are highlighted with white. Surfaces were reconstructed by Imaris. Supplemental Movie 4 PC-3 cell treated with HCQ, stained for Integrin αv (red), Gal-3 (green), EEA1 (magenta). Areas of 3-color colocalization are highlighted with white. Surfaces were reconstructed by Imaris.

PC-3 cell treated with HCQ, stained for Golgi (GRASP65, green) and MGAT3 (red). Surfaces were reconstructed by Imaris.

Figure S5. (A, B) Representative EM images of PC-3 (A) and DU145 (B) cells. Note phagophore nucleation from Golgi (arrowheads) and autophagosomes localizing close to the Golgi (arrow). Orange boxes indicate the areas magnified at the right. (C) WIPI2 W-B of the Golgi fractions isolated from LNCaP, PC-3, and DU145 cells. Samples were normalized by GM130.

PC-3 cell treated with water, stained for Integrin αv (red), Gal-3 (green), EEA1 (magenta). Areas of 3-color colocalization are highlighted with white. Surfaces were reconstructed by Imaris.