PMC

GO-based functional classification ([A] GO: molecular function; [B] GO: cellular component, and [C] GO: biological process) of genes resulting in lipid understorage phenotypes when targeted by dsRNAs (208 genes). For details, see main text and legend of .

GO-based functional classification ([A] GO: molecular function; [B] GO: cellular component, and [C] GO: biological process) of genes resulting in lipid overstorage phenotypes when targeted by dsRNAs (318 genes). For details, see main text and legend of .

(A–F and M–R) AML12 or 3T3-L1 cells (G–L), with (B–F and H–R), or without (A and G) oleic acid and stained for nuclei (Hoechst 33342) and lipid (BODIPY493/503). Cells were transfected with ALLStars negative control siRNA (control) or the siRNAs targeting the indicated genes. Drosophila homologs are given (parentheses). Scale bar in (A) represents 50 μm.

(A) Schematic representation of the COPI trafficking pathway (center) directing traffic from the Golgi (right) to the ER (left). COPII members (not detailed) direct traffic from the ER to the Golgi. Members showing increased lipid storage following knockdown in the Drosophila primary screen are highlighted (red).
(B) Primary screen results for COPI and COPII components are tabulated. The average B-score for the given gene (higher B-scores indicate more stored lipid, see ) as well as the average false discovery rate (FDR)-corrected p-value are given.

(A–F) Drosophila Kc₁₆₇ cells grown in standard medium (A, C, and E) or medium supplemented with 400 μM oleic acid complexed to 0.4% BSA (B, D, and F). Cells were treated with dsRNA targeting brummer (bmm; [C and D]) or midway (mdy; [E and F]). The corresponding mouse genes are Atgl and Dgat-1. Nuclei are shown in blue (stained with Hoechst 33342), and lipid droplets in green (stained with BODIPY493/503). Scale bar in (A) represents 10 μm.

(A) Relative activity [(experimental/ALLStars negative control (control)) − 1] in radiolabel assays for TG esterification (nM) (grey bar) and NEFA release (nM) (black) relative to total protein concentration in cells treated with siRNAs targeting the indicated transcripts. Significance at p < 0.01, impaired t-test, is shown (indicated by an asterisk [*]). Standard error is indicated by the bars.
(B) Relative activity [(experimental/ALLStars negative control (control) and DMSO) − 1] in radiolabel assays for NEFA release (nM) relative to total protein concentration in cells treated with siRNAs targeting the indicated transcripts in the presence of DMSO only (open bar), BFA (5 μM) in DMSO (light-grey bar), or Exo1 (5μM) in DMSO (dark-grey bar). Significance at p < 0.01, impaired t-test, is shown (indicated by an asterisk [*]). Standard error is indicated by the bars.

Mouse AML12 cells store little lipid in the absence of exogenous NEFA (A), whereas addition of NEFA to the growth medium induces lipid droplet deposition (B). Knockdown effects on cellular lipid droplets of the mouse genes encoding Uqcrq (CG7580 in Drosophila), an electron transport chain member, and Smarca4 (Brahma in Drosophila), a chromatin-associated protein (C and D). Nuclei are shown in blue (stained with Hoechst 33342), lipid droplets in green (stained with BODIPY493/503). Scale bar in (A) represents 50 μm.

Mouse AML12 cells were treated with ALLStars negative control siRNA (control) (A) or siRNAs targeting γCOP (B) for 4 d, respectively. Cells were incubated with 400 μM oleic acid for 12 h prior to staining with antibodies detecting TIP47 (green channel) or ADRP (red channel). Images of the separate channels as well as a merged image are shown. (C–H) Merged channel images of similarly treated mouse AML12 cells. Targeted genes are indicated. Scale bar in (A) represents 50 μm.

COPI and COPII trafficking systems shuttle proteins and lipids between ER and Golgi. Small TIP47-coated lipid droplets are born at the ER and reside in the cytosol. The bigger lipid droplets are coated by ADRP, and TIP47 is returned to the ER. ATGL can translocate between the cytosol and the lipid droplet surface, where it begins the conversion of TG to DG and eventually NEFA. Other TG lipases may also be present (indicated by the second arrow liberating TG from the hydrophobic lipid droplet core). Wild-type Golgi–ER transport function of COPI might be necessary for proper lipid droplet biogenesis at the ER. COPI could directly function at the lipid droplet surface, mediating a similar ER-targeted transport function as reported for the Golgi–ER COPI transport. COPI is a positive regulator of ATGL with a unresolved mechanism (indicated by the question mark) and a negative regulator of TIP47 lipid droplet localization. ADRP and TIP47 are negative regulators of ATGL activity at the lipid droplet surface.

GO-based functional classification (GO: biological process) of genes resulting in lipid storage phenotypes upon targeting by dsRNAs of the present (526 genes) as well as a concurrent study (227 genes; []). GO terms were organized into networks using the VLAD software (A and B). Node labels include the associated GO-term identifier and GO-term description as well as the associated p-value reflecting enrichment as compared to the whole-genome annotation reference set. The number of genes associated with the respective GO term included in the test set as well as the complete-genome annotation is also shown. A darker node color reflects lower p-values. Yellow stars indicate GO-terms shared by the two genome-wide RNAi screen datasets.

(A and B) Regions of original images from the screen of cells stained with DAPI (A) or the lipid droplet-specific dye BODIPY 493/503 (B).
(C and D) Image segmentation results of the images shown in (A and B). Nuclei and lipid droplet boundaries are depicted.
(E) Sample off-line lipid droplet quantification results by image segmentation. Cells were grown with or without oleic acid and treated with the indicated dsRNAs. Significant differences between dsRNA treated and untreated cells at p < 0.05 (two-sided t-test) are shown (indicated with an asterisk [*]). Error bars indicate standard deviation.
(F) Rank-order plot of the normalized screen results where positive B-scores indicate overstorage and negative B-scores indicate understorage.
(G) Performance of controls incorporated in the on-line screening plates. Black bars represent overstorage (B-score > 2.0), and light grey bars represent understorage phenotypes (B-score < −1.7).

(A and B) Mouse AML12 cells were grown in the presence of NEFA (400 μM oleic acid) overnight and incubated in DMSO (A) or 5 μM BFA in DMSO (B) for 6 h, prior to staining with antibodies detecting TIP47 (green channel) or ADRP (red channel). Separate images for the different channels as well as a merged image are shown.
(C) Western blots of lipid droplet protein preparations. Lipid droplets from AML12 cells grown in the presence of 400 μM oleic acid and treated with either DMSO (lanes one and three) or 5 μM BFA in DMSO (lanes two and four) were purified by sucrose gradient ultracentrifugation, and equal amounts of the associated proteins were subjected to western blot. Two separate experiment replicates are represented. Band density ratios (±BFA) are given. Scale bar in (A) represents 50 μm.

(A–F) Drosophila cells with or without oleic acid stained with BODIPY493/503 to detect lipid. Control cells not treated with dsRNA (A and D) or cells incubated with dsRNAs targeting Arf79F (an Arf1 homolog) (B and E) or αCop (C and F) are shown.
(G) All COPI, and several COPII members as well as additional Arfs, were retested using independent dsRNAs and gave similar results. Results and number of dsRNAs present in the primary screen (including oleic acid) and retests are given.
(H) Dose response of Drosophila S3 cells to Exo1 (structure inset) showing the %-activity derived either from the lipid specific signal (filled circles [•]) or the lipid/cell ratio (open circles [○]). Percent activity refers hereby to the changes of lipid storage relative to Triacsin C treatment, which decreases lipid storage by blocking TG synthesis (this is analogous to the mdy controls in RNAi experiments, see Material and Methods). Increased activity indicates increased lipid storage, which increased with concentration.
Scale bar in (A) represents 10 μm.