Gal-9 depletion causes morphological and ciliogenesis defects. (A–C) A highly sensitive, electrochemiluminescence-based MSD technique was used to quantitate Gal-9 protein levels in retroviral shRNA-mediated knockdown cells. shRNA-susceptible canine and shRNA-resistant human Gal-9–EGFP constructs were stably expressed in MDCK cell lines to test the specificity of shRNA-mediated down-regulation. Samples were normalized to amounts of GAPDH. A one-tail, unpaired t test was used to generate P values. Error bars indicate SD. The data represent two mock-infected and shRNA cell pairs from three independent experimental groups. (D) Acetylated tubulin stains ciliary axoneme and basal body; ZO-1 reveals TJ areas. shRNA cells are enlarged, fusiform, and flat and show complete lack of cilium with occasional staining of the basal body (white arrowheads). (Scale bars: 10 μm.) The x–z view reveals TJs at varying heights.

Gal-9 depletion causes transport defects that are rescued with recombinant Gal-9 protein. Adenoviruses expressing the apical cargo protein HA-M2-GFP or basolateral cargo protein VSVG-SP–GFP were harvested for total cell-surface MSD-based transport assays. (A and B) HA surface signal was fourfold lower, and VSV-G surface signal was sixfold higher, after 45 min of cargo release from TGN as compared with the mock-infected cells. (C) shRNA cells were treated with 0.15 μM recombinant Gal-9 for 5 d. The HA transport assay showed surface delivery comparable to mock-infected conditions. Samples in A–C are normalized to levels of GFP in lysates used to indicate transfection efficiency. (D) Gal-9–treated shRNA cells showed a recovery of TER comparable to levels in mock-infected cells. The data represent three independent experiments conducted in duplicate. P values were generated from a one-tailed, unpaired t test. Error bars indicate SD.

FGL is a receptor for Gal-9 on the apical surface of MDCK cells. (A) Fully polarized MDCK epithelial cells were incubated with varying concentrations of anti-FGL antibody (μg/mL) on ice for 45 min. Biotinylated recombinant Gal-9 (0.01 μM) was allowed to bind for 1 h on ice. Cells were fixed, immunostained with anti-biotin antibody without permeabilizing the cells, and were counterstained with DAPI. (Scale bars: 10 μm.) (B) The unbound fraction was collected, and the bound fraction was stripped from the cellular membrane with a pH drop. Biotin–Gal-9 in each sample was quantitated by MSD assay. Rat IgG was used to negate any nonspecific antibody-mediated effects. Green bars indicate the sum of bound and unbound fractions. The data represent results from three independent experimental sets performed in duplicate. Error bars indicate SD.

Gal-9 depletion causes mislocalization of protein markers for apical and basolateral polarity. (A, C, E, and G) Mock-infected MDCK cells that were filter-grown for 5 d show E-cadherin localized to the basolateral membrane and CEA localized to the apical membrane. Top, merge of the three 0.5-μm slices farthest from the filter support used to grow the cells. Bottom, merge of three 0.5-μm slices near the filter support (attached surface). (B, D, F, and H) Gal-9 shRNA cells show E-cadherin and CEA expression in all planes, x–z views below C–D and G–H respectively. E-cadherin also showed an intracellular punctate staining (white arrowhead in D); (Scale bars: 10 μm.).

Recombinant Gal-9 rescues ciliogenesis and steady-state expression of apical and basolateral marker proteins. (A) Filter-grown mock-infected and Gal-9–treated shRNA cells were immunostained with anti-acetylated tubulin to visualize the cilium. (Scale bar: 10 μm.) (B and C) The number of cells positive for either a ciliary base or cilia in Gal-9–treated shRNA cells is comparable to the number in mock-infected cells, but the Gal-9–treated shRNA cells show a twofold increase in ciliary length. (D) Mock-, shRNA-, and Gal-9–treated shRNA cells were grown on a filter for 5 d, biotinylated from both sides of the filter, and subjected to MSD-based assay. (E) Mock- and Gal-9–treated shRNA cells were biotinylated from either the apical or the basolateral side and subjected to MSD assay to generate a steady-state profile of relative amounts of CEA and E-cadherin Samples were normalized to amounts of GAPDH detected in cell lysates. The data represent the results of three independent experimental sets conducted in duplicate. ap, apical membrane; AU, arbitrary unit. bl, basolateral membrane. P values are generated from a one-tailed, unpaired t test. Error bars indicate SD.

Internalization and recycling of recombinant biotin–Gal-9. Biotinylated recombinant Gal-9 (0.01 μM) was bound to the apical membrane on ice, and the internalization to different cellular regions was followed over time. (A–C) After 10 min, Gal-9 was found inside the cells and colocalized with the marker of early endosome, EEA1. No significant colocalization was detected with the lysosomal marker Lamp1, but some colocalization was observed with recycling endosome (Rab11) compartments at around 30 min of internalization. Within 60 min, a major fraction of Gal-9 was detected in the TGN compartment, revealed through colocalization with furin-convertase staining. Gal-9 was seen at the base as tubular extensions emanating from the TGN (indicated by white arrowheads in the Furin/Gal-9/Merge, Bottom Right). After 2 h, biotin–Gal-9 localized primarily with FGL on the apical membrane (SI Materials and Methods). (Scale bars in A and B: 10 μm.) (D) Time-course analysis, using the MSD assay, shows the percentage of biotin–Gal-9 reaching the apical surface and the amount remaining in the cell lysates. Graph shows relative intensity of signal detected at the apical surface and in the cell lysates normalized to the amount of phospholipids in each sample. Individual values for per cent of arrival at each time point are indicated. Error bars in C and D indicate SD. Details on quantitation are given in SI Materials and Methods.