Fig. 6. Interaction of Gal-9 with poly-LacNAc is critical for functionality of lysosome.

a Schematic illustration of N-glycosylated moieties with the indicated colored carbohydrates. Specific enzymes required for each N-glycan synthesis step are shown in red. Gal-9 binding targets are indicated by shaded green area, while epitopes for natural lectin binding are indicated by dotted rectangle. β-1,3 N-acetyl-glucosaminyl-transferase (β3GnT) and β-1,4 galactosyl-transferase (β4GalT), key enzymes for the synthesis of poly-N-acetyl-lactosamine (poly-LacNAc), are indicated. b Western blot analysis of β3GnT2 and β4GalT1 proteins in gene-knockdown (KD) CMT93 cells. c Flow cytometry analysis of tomato lectin (LEA) binding efficiency to LacNAc in β3gnt2^KD and β4galt1^KD CMT93 cells. d Co-immunoprecipitation of Gal-9 and Lamp2 in β3gnt2 or β4galt1 gene-knockdown CMT93 cells. e Flow cytometry analysis of monodansylcadaverine (MDC⁺) autophagic vacuoles in the indicated CMT93 cells, cultured with or without exogenous recombinant mouse Gal-9 (rmGal-9). f Western blot analysis of autophagy, ER stress, and apoptosis markers in the indicated CMT93 cells, cultured with or without exogenous rmGal-9. g Immunofluorescence analysis of the indicated CMT93 cells, cultured with or without exogenous rmGal-9, for degradation of endocytosed fluorochrome-labeled EGF. Cells were pulsed with Alexa Flour 555-conjugated EGF for 5 min and confocal images were captured after 1 h post fixation. Data shown are representative or combined (e) results from two independent reproducible experiments. Statistical significance (p value) is indicated (c, e: Unpaired two-tailed t-test). Data are presented as mean ± SD. Source data are provided as a Source data file.