Fig 2: Comparison of morphologic profile annotations to known biology and identification of novel morphologic interactions. (A) Morphologic correlations overlaid with known gene annotations. Shown is a circular dendrogram relating the morphologic profiles obtained from perturbing 133 genes, non-targeting controls, and empty wells. The distal leaves of the dendrogram (individual genes) are labeled and color-coded according to their relation to known genetic disease associations and cellular biology. The first three major branches of the dendrogram are highlighted in color and named “control-like” (blue; inclusion of non-targeting controls), “lethal” (red; inclusion of empty wells) and “lipocluster” (black; inclusion of many known lipodystrophy genes). (B) Cell number and lipid accumulation in sets of genes according to known disease/biological associations or morphologic similarity. Boxplots show median, 25th and 75th percentiles of cell number (top panels) or lipid droplet number (bottom panels) in the 133 gene knockouts grouped by known disease/biological associations (left panels) or by morphologic profile similarity (as shown in 2A). (C) Scatterplot comparing 425 morphologic features (Z-scores) identified from human adipocytes ablated for BSCL2 or PLIN1. Pearson r = 0.89, p < 2.2 × 10−16. (D) Scatterplot comparing 425 morphologic features (Z-scores) identified from human adipocytes ablated for CEBPA or AGPAT2. Pearson r = 0.85, p < 2.2 × 10−16.

Fig 3: Ablation of BSCL2 in HepG2 cells does not alter lipid accumulation. (A) Schematic representation of CRISPR/Cas9 gRNA editing sites in exon 2 of BSCL2. (B) PCR identification of BSCL2 exon 2 deletion by CRISPR/Cas9 in a mixed population of HepG2 cells. KO, knockout; NTC, no template control; WT, wild type. (C) PCR identification of single-cell HepG2 colonies containing BSCL2 exon 2 deletion isolated by serial dilution. (D,E) PCR analysis of clones selected for expansion (D) and DNA sequencing (E) of PCR products confirming BSCL2 exon 2 deletion in HepG2 cells. (F) Western blot analysis confirming the ablation of BSCL2 protein in HepG2 single cell clones with BSCL2 exon 2 deletion. (G) Representative images of Oil Red O-stained HepG2 cell clones (scale bars: 50 µm) and quantification of the eluted stain from biological replicates (n=8); data presented are from two independent experiments. Data are presented as the mean±s.e.m., *P<0.05 vs Ctrl #7.

Fig 4: Protein–protein interaction of BSCL2 and PLIN1 at the lipid droplet surface. (A) Histogram of Z-scores for variation in lipid droplet size in SGBS adipocytes ablated for 133 genes. Variation in lipid droplet size is quantified by automated image analysis and feature extraction and summarized as the standard deviation for all cells exposed to targeting constructs for a gene. Adipocytes targeted with constructs against BSCL2 or PLIN1 result in the most extreme Z-scores for variation in lipid droplet size among the 133 genes. (B) 20× brightfield and fluorescence (488 nm, DAPI stained) images of SGBS cells exposed to gene targeting and non-targeting CRISPR/CAS9 constructs and subsequently differentiated. Residual large lipid droplets are observed with BSCL2 or PLIN1 ablation when compared to control or PPARG ablation. (C) Co-immunoprecipitation and western blotting of seipin and perilipin in human adipocytes. SGBS adipocytes were transduced with Myc-seipin and differentiated for seven days. Endogenous perilipin was immunoprecipitated and co-immunoprecipitation of exogenous Myc-seipin is observed. Cyclophilin A was blotted as a cell loading control. (D) 3T3-L1 cells were induced to differentiate and co-transfected with FLAG-seipin-YFPn and Myc-perilipin-YFPc on day 2 of differentiation. Following a temperature shift to induce the formation of reconstituted YFP, cells were fixed on day 5 of differentiation and stained for FLAG-seipin (top panels) or Myc-perilipin (middle panels) and DAPI to label nuclei. The direct interaction between seipin and perilipin is indicated by the presence of YFP signal. Identically transfected cells were used to co-immunostain for FLAG-seipin-YFPn and Myc-perilipin-YFPc (bottom panels). Note that these cells were not temperature shifted to prevent formation of YFP, which would confound the Alexa Fluor 488 fluorescence used to detect the FLAG epitope. Top and middle panels, individual images are shown in grayscale and merged images show overlay of YFP (yellow) and FLAG-seipin or Myc-perilipin (red). Bottom panels, individual images are shown in grayscale and merged image shows overlay FLAG-seipin (green) and Myc-perilipin (red), inset boxes show zoomed images. Scale bars, 10 μm. (E) AFM analysis of the interaction of perilipin with seipin. FLAG-perilipin and seipin-Myc were co-expressed in tsA 201 cells and proteins were isolated using anti-Myc immunoaffinity chromatography. AFM images of isolated seipin dodecamers (large particles) singly (upper panels) or doubly (lower panels) decorated by smaller perilipin particles. Scale bar, 25 nm; height scale, 0–2 nm. Histograms show the frequency distribution of volumes of seipin particles bound to perilipin (n = 100) and of perilipin particles bound to seipin particles (n = 100). The curve indicates the fitted Gaussian function. The peak of the distribution (±SEM) is indicated. (F) HEK293 cells were transfected with FLAG-perilipin in the absence or presence of either wild-type Myc-seipin (WT) or mutants lacking the N terminus (ΔNT), first transmembrane domain (ΔTM1), ER luminal loop region (Δloop), second transmembrane domain (ΔTM2), or the C terminus (ΔCT). Lysates or anti-FLAG immunoprecipitates were immunoblotted for FLAG, Myc and the ER membrane protein calnexin. Quantification of the interaction of mutant vs. wild-type seipin from replicate experiments is shown in the lower panel. Data are means ± SEM (n = 3). ** indicates p < 0.01 versus co-immunoprecipitation with wild-type seipin.

Fig 5: Evaluation of AAV vector tissue specificity in SKO mice.Relative human BSCL2 gene expression levels (A) and representative antibody staining of human BSCL2 protein (B) from livers of AAV-CMV, AAV-aP2 and AAV-TBG treated mice 8 weeks after AAV administration. Gonadal white adipose tissue (gWAT) weights (C) and representative H&E sections of gWAT (D) from WT, AAV-CMV, AAV-aP2 and AAV-TBG treated mice. E Circulating serum levels of leptin and adiponectin in WT, AAV-GFP, AAV-aP2 and AAV-TBG injected mice fasted for 5 h. Western blot tissue panel analysis of human BSCL2 protein levels in AAV-aP2 (F) and AAV-TBG (G) injected mice. Data in (A, C, E) are biological replicates presented as the mean ± SD, n = 5 (AAV-CMV), 5 (AAV-aP2) and 5 (AAV-TBG) mice per group for (A), n = 44 (WT), 5 (AAV-CMV), 5 (AAV-aP2) and 5 (AAV-TBG) mice per group for (C), n = 12 (WT), 10–11 (AAV-GFP), 5 (AAV-aP2) and 5 (AAV-TBG) for (E). **p < 0.01 vs. AAV-CMV and ##p < 0.01 vs. AAV-aP2 for (A), *p < 0.05 and ****p < 0.0001 vs. WT, ##p < 0.01, ###p < 0.001 and ####p < 0.0001 vs. AAV-GFP for (C, E) by one-way ANOVA, ntd no tissue dissected. Scale bars represents 50 µm.