Galectin-12 and VPS13C are co-upregulated during adipocyte differentiation.

<p>Expression was assayed by quantitative real-time RT-PCR for mRNA levels (<b>A and B</b>) and by immunoblotting for protein levels (<b>C and D</b>) in subconfluent 3T3-L1 fibroblasts, or at different time points of adipocyte differentiation. Adipocyte differentiation was induced at day 0, when cells were three days post confluence, following an established adipogenic regimen [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153534#pone.0153534.ref022" target="_blank">22</a>]. Bar graphs present data (means ± s.e.) from three experiments.</p

VPS13C is required for galectin-12 protein stability in adipocytes.

<p>3T3-L1 cells were stably transduced with a doxycycline-inducible conditional knockdown system for <i>LacZ</i> (control), galectin-12, or <i>Vps13c</i>. Cells were then stimulated to undergo adipocyte differentiation for 7 days and then treated with doxycycline for 3 days to induce gene knockdown. Protein levels and galectin-12 mRNA levels were determined by immunoblotting (<b>A</b>) and quantitative RT-PCR (<b>B</b>), respectively. (<b>C</b>) 3T3-L1 adipocytes transduced with the above system were treated with doxycycline for 3 days in the absence or presence of the lysosome inhibitor chloroquine or the proteasome inhibitor MG-132 before immunoblot assay with galectin-12 or tubulin antibodies. Results are representative of three experiments. Asterisks denote statistical significance.</p

VPS13C colocalizes with lysosomes and galectin-12.

<p>(<b>A</b> and <b>B</b>) HeLa cells transfected with Myc-tagged VPS13C were first stained with MitoTracker Deep Red, fixed, permeabilized and immunostained with mouse anti-Myc tag and rabbit anti-calnexin antibodies. (<b>C</b>) HeLa cells transfected with Myc-tagged VPS13C were immunostained with mouse anti-LAMP1 and rabbit anti-Myc antibodies. (<b>D</b>) HeLa cells were transduced with a retrovirus encoding 3xFLAG-tagged galectin-12 and transfected with Myc-tagged VPS13C. Cells were then immunostained with mouse anti-FLAG and rabbit anti-Myc antibodies and appropriate fluorescence-labeled secondary antibodies. Deconvolved image stacks were analyzed for colocalization of the red and green signals with the Coloc 2 plugin of ImageJ. Scale bar, 10 μm. Results are representative of three experiments.</p

Like galectin-12, VPS13C is required for adipocyte differentiation.

<p>3T3-L1 cells were stably transduced with a doxycycline-inducible conditional knockdown system for <i>LacZ</i> (control), galectin-12, or <i>Vps13c</i>. Cells were then stimulated to undergo adipocyte differentiation following an established regimen for 10 days, in the continuous absence or presence of doxycycline. Adipocyte differentiation was assayed by Oil-Red-O staining of neutral lipids (<b>A</b> and <b>B</b>), by quantification of triglycerides with AdipoRed (<b>C</b>), and by immunoblotting of indicated adipocyte proteins (<b>D</b>). Asterisks denote statistical significance (*P < 0.05). Results are representative of three to four experiments.</p

VPS13C knockdown sensitized cells to proteasome inhibition.

<p>3T3-L1 fibroblasts engineered with the doxycycline-induced LacZ or VPS13C knockdown system were treated for three days without or with doxycycline in the presence of chloroquine, MG-132, or 3-MA. Cell viability was then analyzed using MTS assay[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153534#pone.0153534.ref058" target="_blank">58</a>]. Results are representative of three experiments. Asterisks denote statistical significance.</p

Identification of VPS13C as a galectin-12-binding protein.

<p>(<b>A</b>) Lysates from 3T3-L1 adipocytes transduced with a control retrovirus (Ctr) or one that expresses 3xFLAG tagged mouse galectin-12 (3Fm12) were immunoprecipitated with either an anti-FLAG or anti-galectin-12 antibody, as indicated. Immunoprecipitates were further immunoblotted with a galectin-12 antibody. (<b>B</b>) Immunofluorescence of 3T3-L1 adipocyte expressing 3Fm12 with anti-FLAG antibody (red). Cells were co-stained with Bodipy 493/503 and Hoechst 33342 to reveal lipid droplets (LD, green) and the nucleus (blue), respectively. Scale bar, 10 μm. (<b>C</b>) Lysates of control 3T3-L1 adipocytes or those expressing 3Fm12 were immunoprecipitated with anti-FLAG antibody and the immunocomplexes were analyzed by LC-MS/MS. (<b>D</b>) Lysates from 293T cells transducedwith 3xFLAG-tagged LC3 (control), full-length galectin-12 or truncated mutants lacking the N- or C-terminal CRD were immunoprecipitated with anti-FLAG M2-agarose beads, and total lysates or immunoprecipitates were analyzed by immunoblotting with anti-VPS13C or anti-FLAG antibodies. H and L denote the heavy and light chains of IgG, respectively. (<b>E</b>) Cytosol and lipid droplet fractions of 3T3-L1 adipocytes were immunoblotted with indicated antibodies. Results are representative of three experiments.</p

RNAi design for specific knockdown of indicated genes with amiRs.

<p>RNAi design for specific knockdown of indicated genes with amiRs.</p

<i>Vps13c</i> knockdown does not affect bulk autophagy.

<p>Both human VPS13C (<b>A</b>) and ATG2 (<b>B</b>) carry a Chorein_N and a ATG_C domains. cc, coiled coil. Data from the Pfam protein families database [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153534#pone.0153534.ref057" target="_blank">57</a>]. (<b>C</b>) An ATG-C-nearby sequence in ATG2A (1723–1829), essential for autophagy and required for ATG2A localization to both the autophagic membrane and lipid droplets [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0153534#pone.0153534.ref033" target="_blank">33</a>], is also conserved in VPS13C. (<b>D</b>) 3T3-L1 fibroblasts engineered with a doxycycline-regulated knockdown system for control (LacZ) or <i>Vps13c</i> gene were treated for 3 days with or without doxycycline. Cells were then cultured 3 h under basal conditions, or in a medium depleted of amino acid and serum (starvation), in the absence or presence of chloroquine. Cells were lysed and analyzed by immunoblotting for indicated proteins. Results are representative of three experiments.</p

DNA oligos used for Q-PCR.

<p>DNA oligos used for Q-PCR.</p

Impaired Vps13 function leads to defects in protein homeostasis.

<p>(A) Percentage of isogenic control and <i>Vps13</i> mutant flies that eclosed at increasing temperatures. (B) Percentage of homozygous <i>Vps13</i> mutant flies and excision line flies that eclosed at 29°C. (C) Percentage of flies of various genotypes that eclosed at 29°C. Two independent deficiency lines (lacking a genomic area containing the <i>Vps13</i> gene) were crossed with <i>Vps13/ CyO</i> heterozygous flies. Eclosion rate of the following genotypes was analyzed: <i>Vps13/+</i>, <i>Df #7535/+</i>, <i>Vps13/Df #7535</i>, <i>Df #7534/+</i> and <i>Vps13/Df #7534</i>. (D) Percentage of <i>Vps13</i> flies that eclosed at 22°C on food with increasing concentrations of L-canavanine. (E) Western blot analysis of lysates of 1 day old control and <i>Vps13</i> mutant fly heads. Ubiquitylated proteins, K48 ubiquitylated proteins and K63 ubiquitylated proteins were detected. All quantifications show the mean and SEM of at least three independent experiments per condition. For statistical analysis a two-tailed students T-test was used in combination with a Welch’s correction if necessary. P<0.05 is *, P<0.01 is ** and P<0.001 is ***.</p