Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a Knockout strategy for the Grasp55 locus. The reporter-neo cassette replaced all 11 exons of mouse Grasp55 . Red triangles indicate the location of loxP sites. More detailed diagrams are provided in the Supplementary Fig. . b Expressions of GRASP55 and GRASP65 in major organs were examined by immunoblotting. The amount of β-actin protein was monitored as a cytosolic protein loading control. c Survival curves of wild-type ( Grasp55 +/+ ) and Grasp55 −/− male mice over 40 weeks ( n = 15). d Photographs of Grasp55 +/+ and Grasp55 −/− mice at postnatal day 28. e Body weight curves of male mice fed normal diet (ND, left) and high-fat diet (HFD, right) at 1–30 weeks of age ( n = 15). The arrow indicates the initiation time of HFD at 6 weeks of age. The body weight curve of the Grasp55 +/+ mice fed ND was replotted in the HFD graph (open circle, right) for comparison. f The relative fat mass ratio (fat/body weight) of 16-week-old male mice fed ND and after HFD for 4 weeks ( n = 5–7). g – j Gross and histological examinations of epididymal white adipose tissue (EWAT). Ventral view ( g ) and wet weights ( h ) of EWAT (right side) of male mice at 18 weeks of age fed on ND and after HFD for 12 weeks ( n = 6). Arrows indicate epididymal fat tissues. Representative light microscopic tissue images (H&E staining) are presented in ( i ). Quantitative analyses of the H&E images, that inversely correlate with fat contents of EWAT by counting the number of adipocyte nuclei in a light microscopic field (450 × 340 μm), are depicted in ( j , n = 10). Unprocessed blots are presented in Supplementary Fig. . Data are shown as mean ± SEM. Scale bars: 50 μm. ** p < 0.01. All p values were calculated by unpaired two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Knock-Out, Western Blot, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a , b Analyses of energy intake ( a ) and energy expenditure ( b ) were conducted in 12-week-old Grasp55 +/+ and Grasp55 −/− mice fed normal diet (ND) or high-fat diet (HFD). Energy expenditure differences between Grasp55 +/+ and Grasp55 −/− mice are plotted in ( b inset). Data are shown as average values from a 2-day observation ( n = 6). c , d Plasma leptin levels of 12-week-old Grasp55 +/+ and Grasp55 −/− mice that were fasted for 16 h and fed ND ( n = 6). Plasma leptin levels normalized to body fat mass of mice are presented in d ( n = 6). e , f Fasting plasma levels of insulin ( e ) and glucose ( f ) were measured in 12-week-old Grasp55 +/+ and Grasp55 −/− mice fasted for 16 h ( n = 6). g An insulin tolerance test was performed in mice fasted for 4 h. The relative ratios of blood glucose levels to basal glucose levels were determined at the indicated time points following intraperitoneal administration of human insulin (0.75 U kg −1 body weight) in 12-week-old Grasp55 +/+ and Grasp55 −/− mice ( n = 6). Data are shown as mean ± SEM. n.s.: not significant, * p < 0.05, ** p < 0.01. All p values were calculated by unpaired two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a – c H&E and Oil Red O staining images of jejunal epithelia. Intestinal tissues were taken from 12-week-old Grasp55 +/+ and Grasp55 −/− mice that were fasted for 16 h ( a ) or 4 h after oral gavage of olive oil ( b , 10 μl g −1 of body weight). Quantification of stained lipid droplets (LDs) was performed by measuring sample absorbances at 510 nm ( c , n = 7). The readings were normalized to the background value obtained from a non-induced fasting control. Scale bars: 50 μm. d Intestinal triglyceride (TG) contents were measured in jejunums of 12-week-old Grasp55 +/+ and Grasp55 −/− mice that were fasted for 16 or 4 h after olive oil bolus (olive oil, 10 μl g −1 of body weight, n = 6). e Electron-microscopic (EM) images of mouse jejunum 4 h after oral gavage of olive oil (10 μl g −1 of body weight). Arrows indicate cytosolic LDs. Quantitative analyses of LD diameters are summarized in ( f ) ( Grasp55 +/+ , n = 313 from five mice; Grasp55 −/− , n = 307 from five mice). Dashed circle represents the supersized LD (diameter, >5 μm). GRASP55 deficiency increased the LD size from 0.74 ± 0.02 μm to 3.19 ± 0.16 μm ( p < 0.01). Scale bars: 5 μm. g , h EM images showing chylomicrons of mouse intestinal epithelial cells. Mouse jejunums were prepared 2 h after oral gavage of olive oil. Arrows indicate chylomicron particles. Quantitative analyses of chylomicrons are summarized in ( h ). The number of chylomicron particles in each EM field (3.72 × 3.72 μm) of the Golgi region was determined ( n = 50 from five mice each). Scale bars: 500 nm (enlarged: 200 nm). Data are shown as mean ± SEM. n.s.: not significant, ** p < 0.01. All p values were calculated by unpaired two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a An oral fat tolerance test was performed with 12-week-old male mice after oral gavage of olive oil (10 μl g −1 of body weight). Tyloxapol (Triton WR-1339) was administered 20 min after the olive oil gavage (arrow, 0.5 g kg −1 intravenous injection) to inhibit lipoprotein catabolism. Plasma triglyceride (TG) concentrations were measured at the indicated time points ( n = 6). b Photograph of blood plasma taken from WT and Grasp55 −/− male mice 2 h after application of olive oil. c , d Fractionation analysis of plasma lipids from mice using fast performance liquid chromatography. Blood samples were taken 2 h after oral garage of olive oil. TG concentrations were determined in each fraction. The area under curve (AUC) of plasma lipoprotein fractions were measured using ImageJ software and summarized in ( d , n = 3 independent experiments from pooled blood plasma samples of 3–4 mice). e Schematic diagram of the in vitro two-chamber system with differentiated Caco-2 cells. f Immunoblot results showed that the short-hairpin RNA against GRASP55 ( shGRASP55 ) specifically depleted endogenous GRASP55 protein expression and GRASP55 with a COOH-terminal Myc-tag ( GRASP55-Myc ) induced exogenous GRASP55 protein. The filled arrowhead and the open arrowhead indicate levels of exogenous GRASP55 and endogenous GRASP55, respectively. Aldolase A was monitored as a cytosolic protein loading control. g Transepithelial lipid transport of Caco-2 monolayers were determined using 14 C-labeled oleic acid. The majority of the exogenous oleic acids were incorporated into TGs and secreted as TGs in Caco-2 cells . Basolateral (B) to apical (A) transport of lipids (left, B to A) was much smaller than that of apical to basolateral transport (middle, A to B), and was not affected by GRASP55 depletion or overexpression. Specific transepithelial lipid absorption was determined by subtracting values of (B to A) from those of (A to B) in each paired experiment (right, n = 5). GRASP55 depletion reduced lipid absorption by 49.2 ± 14.5%. GRASP55 overexpression rescued the lipid absorption defect in GRASP55-depleted Caco-2 cells. CPM, counts per min. CM, chylomicron, VLDL, very-low-density lipoprotein; IDL, intermediate density lipoprotein; LDL, low density lipoprotein; HDL, high density lipoprotein. Unprocessed blots can be found in Supplementary Fig. . Data are shown as mean ± SEM. n.s.: not significant, * p < 0.05, ** p < 0.01. P values were calculated by unpaired ( a , c ), paired ( d ) two-tailed Student’s t tests or ANOVA followed by Tukey’s multiple comparison tests ( g ). Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Injection, Fractionation, Liquid Chromatography, Software, In Vitro, Western Blot, shRNA, Expressing, Labeling, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a Subcellular localization of ATGL and MGL in the intestinal epithelial cells of Grasp55 +/+ and Grasp55 −/− mice was determined using LD floating ultracentrifugation (for Fraction 1), followed by subcellular fractionation assay (for Fractions 2–9; using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g −1 of body weight]) as described in Methods. RAB18/ADRP, Aldolase A, Giantin, ERGIC53, and calreticulin were used as organelle markers of the LDs, cytosol, Golgi, the ER-Golgi intermediate compartment (ERGIC), and the ER, respectively. Immunoblotting results with a full list of proteins involved in lipid metabolism are shown in the Supplementary Fig. . The Golgi localizations of ATGL and MGL were reduced by GRASP55 deficiency in mouse intestinal cells (red boxes). Three independent experiments showed similar results. b , c The protein expressions of ATGL, phospho-HSL (pHSL), HSL, MTP, and ADRP were analyzed by immunoblotting. Jejunum tissues were prepared from Grasp55 +/+ and Grasp55 −/− mice fasted for 16 or 4 h after olive oil bolus (olive oil, 10 μl g −1 body weight). Representative immunoblots are shown in ( b ). Densitometric analysis of ATGL are shown in ( c , n = 5). A summary of pHSL, HSL, MTP, and ADRP is shown in Supplementary Fig. . The level of β-actin was monitored as a cytosolic protein loading control. d , e Measurements of ATGL in the lipid droplet (LD) fraction of intestinal epithelial cells (using pooled jejunum tissue from four male mice 4 h after olive oil bolus [olive oil, 10 μl g −1 of body weight]). Representative immunoblots are shown in ( d ) and a summary of multiple experiments is shown in ( e , n = 3). TM, total membrane. f , g Intracellular localization of ATGL and ADRP, a marker protein of LD, in jejunal epithelia of mice 4 h after olive oil bolus. Representative images are shown in ( f ) and quantitative analyses of colocalization between ATGL and ADRP are presented in ( g , n = 5). MCC, Manders’ colocalization coefficient. Unprocessed blots can be found in Supplementary Fig. . Data are shown as mean ± SEM. Scale bars: 20 μm. ** p < 0.01. P values were calculated by unpaired ( c , g ) or paired ( e ) two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Fractionation, Western Blot, Marker, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a , b Measurements of ATGL in the lipid droplet (LD) fraction of Caco-2 cells after treatment with 400 μM oleic acid for 16 h. Representative immunoblots are shown in ( a ) and a summary of multiple experiments is shown in ( b , n = 3). c Presence of ATGL on the LD surface was examined by co-staining with anti-ATGL antibodies and BODIPY 493/503 (an LD marker) in Caco-2 cells, in which endogenous GRASP55 was depleted ( shGRASP55, shG55 ) and/or exogenously supplemented ( GRASP55-Myc, G55-Myc ). Cells were treated with 400 μM oleic acid for 4 h before immunostaining. Arrows indicate the location of ATGL, which is targeted to the peripheral regions of LDs. Four independent experiments showed similar results. d , e ATGL protein stability was examined in Caco-2 cells, in which GRASP55 was depleted ( shGRASP55 , open arrowhead) and/or exogenously supplemented ( GRASP55-Myc , filled arrowhead). Cytosolic proteins were collected 0, 2, and 4 h after treatment with cycloheximide (0.1 mg ml −1 ), an inhibitor of protein biosynthesis. Aldolase A was used as a cytosolic protein loading control. Representative immunoblot is shown in ( d ), and the results of multiple experiments are summarized in ( e , n = 3). f Immunoblot results of Caco-2 monolayers, in which GRASP55 was depleted ( shGRASP55 ) and exogenous ATGL ( ATGL-Myc ) was supplemented. Levels of endogenous ATGL (open arrowhead) and exogenous ATGL (filled arrowhead) are shown. A high level of ATGL-Myc supplementation restored LD-associated ATGL levels in GRASP55-depleted cells, although the LD transport efficiency was much lower (compare endogenous and exogenous ATGL levels in lanes 3 and 4). g Transepithelial lipid transport of Caco-2 monolayers were determined using 14 C-labeled oleic acid. The basolateral (B) to apical (A) transport of oleic acid (left, B to A) was much smaller than that of apical to basolateral transport (middle, A to B), and it was not affected by GRASP55 depletion or ATGL overexpression. Transepithelial lipid absorption was determined by subtracting values of (B to A) from those of (A to B) in each paired experiment (right, n = 4). ATGL overexpression rescued lipid absorption in GRASP55-deficient Caco-2 Cells. Unprocessed blots are presented in Supplementary Fig. . CPM, counts per min. TM, total membrane. Data are shown as mean ± SEM. Scale bars: 10 μm. n.s.: not significant, * p < 0.05. P values were caluculated by paired ( b ), unpaired ( e ) two-tailed Student’s t tests or ANOVA followed by Tukey’s multiple comparison tests ( g ). Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Western Blot, Staining, Marker, Immunostaining, Labeling, Over Expression, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a – d Coimmunoprecipitation experiments with anti-Myc antibodies were performed in Caco-2 cells. For the induction of ATGL protein expression, some cells were treated with 400 μM oleic acid for 16 h. A representative coimmunoprecipitation assay using GRASP55 with a COOH-terminal Myc-tag ( GRASP55-Myc ) is shown in ( a ), and the results of multiple experiments are summarized in ( b , n = 3). A representative coimmunoprecipitation assay using ATGL with a COOH-terminal Myc-tag ( ATGL-Myc ) is shown in ( c ), and the results of multiple experiments are summarized in ( d ) ( n = 3). Aldolase A was used as a cytosolic protein loading control. e , f Pull-down assays were performed with GRASP55 and ATGL fragments. The domain structures of His 6 -tagged GRASP55 and GST-tagged ATGL constructs used in this study are shown in ( e ), and a representative result from the pull-down assay is shown in ( f ). Expression of control GST and each GST-fusion protein is visualized by Ponceau S staining ( f lowermost panel). An asterisk indicates the band of each GST-tagged protein. One microgram of each His 6 -tagged protein was loaded as an input control. The PDZ1 domain of GRASP55 interacted strongly with the ATGL-patatin domain. Three independent experiments showed similar results. Unprocessed blots can be found in Supplementary Fig. . Data are shown as mean ± SEM. n.s.: not significant, * p < 0.05, ** p < 0.01. All p values were calculated by paired two-tailed Student’s t tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Expressing, Co-Immunoprecipitation Assay, Construct, Pull Down Assay, Staining, Two Tailed Test

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: a – d Immunoblot analysis of ATGL levels in Caco-2 cells, in which GRASP55 was depleted ( shGRASP55 ) and/or exogenously supplemented ( GRASP55-Myc ). Cytosolic proteins were collected in the absence or presence of the proteasomal inhibitor MG132 (0.5 μM, 16 h). For the induction of ATGL protein expression, cells were treated with 400 μM oleic acid for 16 h. Representative immunoblots of cytosols and LDs are shown in ( a ) and ( c ), and the results of multiple experiments are summarized in ( b ) and ( d ), respectively ( n = 3 each). Aldolase A and RAB18/ADRP were used as loading controls for cytosolic and LD proteins, respectively. MG132 reversed the decreased cytosolic levels of ATGL by GRASP55 depletion, but did not significantly affect the ATGL levels in LDs. In contrast, the add-back of GRASP55 restored ATGL levels in both the cytosol and LDs. e , f Coimmunoprecipitation (IP) experiments with anti-GRASP55 antibodies were performed in Caco-2 cells. The ER-to-Golgi trafficking was blocked by dominant-inhibitory forms of SAR1 (SAR1-T39N) and ARF1 (ARF1-T31N). For the induction of ATGL protein expression, cells were treated with 400 μM oleic acid for 16 h. A representative IP assay is shown in ( e ), and the results of multiple experiments are summarized in ( f , n = 3). Aldolase A was used as a cytosolic protein loading control. MG132 does not rescue the dissociation of GRASP55 and ATGL induced by SAR1-T39N and ARF1-T31N. Unprocessed blots can be found in Supplementary Fig. . Data are shown as mean ± SEM. ** p < 0.01, difference from control without MG132. ## p < 0.01, difference from control. All p values were calculated by ANOVA followed by Tukey’s multiple comparison tests. Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Western Blot, Expressing

Journal: Nature Communications

Article Title: Grasp55 −/− mice display impaired fat absorption and resistance to high-fat diet-induced obesity

doi: 10.1038/s41467-020-14912-x

Figure Lengend Snippet: Wild-type ( Grasp55 +/+ ) and Grasp55 −/− mice were crossed with transgenic mice overexpressing GRASP55-Myc ( TgGrasp55 ). a , b Photographs ( a ) and a summary of body weight ( b , n = 6) of 12-week-old male mice. c , d Photographs ( c ) and a summary of weight ( d , n = 6) of epididymal white adipose tissues (EWAT, right side). e , f Representative light microscopic images (H&E) of EWAT are shown in ( e ). Quantitative analyses of the H&E images, that inversely correlate with fat contents of EWAT by counting the number of adipocyte nuclei in a light microscopic field (450 × 340 μm), are presented in ( f , n = 6). g H&E and Oil Red O staining of mouse jejunum 4 h after olive oil bolus (10 μl g −1 body weight). TgGrasp55 (+) reduced the postprandial lipid accumulation of jejunal epithelia in Grasp55 −/− mice. Arrows indicate supersized LDs. h , i The protein amount ATGL of mouse jejunum was analyzed by immunoblotting. Representative immunoblots are shown in ( h ). In some experiments, jejunum tissues were prepared 4 h after olive oil bolus (10 μl g −1 body weight). The filled arrowhead and open arrowhead indicate levels of the exogenous GRASP55-Myc and endogenous GRASP55, respectively. A blot with a longer exposure time of GRASP55 is shown in the left four lanes to better visualize the presence and absence of endogenous GRASP55. The results of multiple experiments ( n = 4) are summarized in ( i ). TgGrasp55 (+) increases the protein levels of ATGL in jejunal epithelia of Grasp55 −/− mice. Unprocessed blots can be found in Supplementary Fig. . Data are shown as mean ± SEM. Scale bars: 50 μm. n.s.: not significant, ** p < 0.01. P values were calculated by unpaired two-tailed Student’s t tests ( b ) or ANOVA followed by Tukey’s multiple comparison tests ( d , f , i ). Source data are provided as a Source Data file.

Article Snippet: To generate stable GRASP55 knockdown, GRASP55 overexpression, and ATGL overexpression cell lines, Caco-2 and HeLa cells were transduced with lentiviral particles produced from HEK293T cells that had been transfected with the psPAX2 packing plasmid, pMD2.G envelope plasmid, and pLKO.1 short-hairpin RNA (shRNA) plasmid (control shRNA: #SHC001, GRASP55 -specific shRNA: #TRCN0000129489; Yonsei Genome Center, Seoul, Korea) or Lenti ORF clone ( GRASP55 : #RC202946L3, ATGL: #RC205708L3; Origene, Rockville, MD, USA).

Techniques: Transgenic Assay, Staining, Western Blot, Two Tailed Test