hig2 7 11  (Addgene inc)

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 2. HIG2 interacts with ATGL. (A) HCT116 cells transfected with control vector or FLAG-ATGL vector were incubated under hypoxia for 24 hr followed by immunoprecipitation with anti-FLAG gels. Then the elution was separated on the 10–20% SDS-PAGE gel and stained with Coomassie Blue. The arrow indicates FLAG-ATGL. (B) The numbers of peptides from (A) recovered by mass spectrometry. (C) Combined coverage map of HIG2 peptides from (B). Detected peptides are in red. (E) Protein sequence alignment of HIG2 and G0S2. (E) HIG2 and ATGL in samples from (A) were detected by immunoblotting with anti-HIG2 and anti-FLAG antibodies, respectively. (F) HeLa cells were co-transfected with Myc-ATGL vector (N- terminal Myc tag) along with vector alone, HIG2-FLAG WT or HIG2-FLAG D7–11 vector (C-terminal FLAG tag). Immunoprecipitation was performed by anti-FLAG gels. HIG2 and ATGL proteins were detected by anti-FLAG and anti-Myc antibodies, respectively. (G) HeLa cells were co-transfected with HIG2-FLAG vector along with vector alone, Myc-ATGL or mutant vectors. ATGLDPT and ATGLDHD are two internal deletion mutants that lack the patatin domain (residues 10–178) and the hydrophobic domain (residues 259–337), respectively. Immunoprecipitation was performed by anti-Myc gels. HIG2 and ATGL proteins were detected by anti-FLAG and anti-Myc antibodies, respectively. DOI: https://doi.org/10.7554/eLife.31132.003

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Transfection, Control, Plasmid Preparation, Incubation, Immunoprecipitation, SDS Page, Staining, Mass Spectrometry, Sequencing, Western Blot, FLAG-tag, Mutagenesis

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 3. HIG2 inhibits ATGL enzymatic activity. (A, B) HIG2 from in vitro translation was added to extracts of HeLa cells transfected with human ATGL vector (hATGL) (A) or mouse ATGL vector (mATGL) (B), and TG hydrolase activity was determined. n = 2 biologically independent experiments. *p<0.05 vs. hATGL +Vector or mATGL +Vector. (C) 10 ml of recombinant proteins purified from E. coli were separated on a 10% SDS-PAGE gel and stained with Coomassie Blue. (D) 4 mg of recombinant proteins from (c) were added to extracts of HeLa cells transfected with mouse ATGL vector, and TG hydrolase Figure 3 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Activity Assay, In Vitro, Transfection, Plasmid Preparation, Recombinant, Purification, SDS Page, Staining

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 4. Lipolytic inhibition by HIG2 causes TG accumulation under hypoxia. (A–C) HCT116 or HeLa KO clone cells created by CRISPR/Cas9 method were incubated under normoxia or hypoxia for 24 hr, and then cellular TG was detected by TG kits or by BODIPY 493/503 (green indicates lipids and blue indicates DAPI-stained nucleus). n = 3 biologically independent experiments for (A); n = 4 biologically independent experiments for (B).*p<0.05, **p<0.01, ***p<0.001 vs. Normoxia WT; DDp<0.01, DDDp<0.001 vs. Hypoxia WT; ###p<0.001 vs. Hypoxia HIG2 KO. (D) Following an overnight transfection with DNA vectors, HCT116 cells were incubated under normoxia or hypoxia for 24 hr, and then TG was measured. n = 4 biologically independent Figure 4 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Inhibition, CRISPR, Incubation, Staining, Transfection

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 5. Lipolytic inhibition by HIG2 prevents cell apoptosis under hypoxia. (A, B) Cell number was determined by counting viable cells at indicated times. n = 4 biologically independent experiments. **p<0.01 vs. Hypoxia WT. (C, D) After 48 hr of incubation under normoxia or hypoxia, apoptosis in HCT116 clone cells was assessed by immunoblotting (C) or by staining with Annexin V for Flow Cytometry (D). n = 3 biologically independent experiments. ***p<0.001 vs. Normoxia WT; DDDp<0.001 vs. Hypoxia WT; ###p<0.001 vs. Hypoxia HIG2 KO. (E, F) Following an overnight transfection with Figure 5 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Inhibition, Incubation, Western Blot, Staining, Flow Cytometry, Transfection

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 6. Enhancement of lipolysis in the absence of HIG2 increases PPARa activity, FAO rate and ROS production under hypoxia. (A–C) After 36 hr of incubation under normoxia or hypoxia, mRNA levels (A), FAO (B) and ROS levels (C) were measured in HCT116 clone cells. Acaa2, acetyl-CoA acyltransferase 2; Cpt1a, carnitine palmitoyltransferase Ia; Cpt1b, carnitine palmitoyltransferase Ib; Mcad, Medium-chain acyl-CoA dehydrogenase; Vlcad, very-long-chain acyl-CoA dehydrogenase. n = 4 (WT, HIG KO) or 3 (ATGL KO, HIG2/ATGL KO) biologically independent experiments for (A); Figure 6 continued on next page

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Activity Assay, Incubation

Journal: eLife

Article Title: Inhibition of intracellular lipolysis promotes human cancer cell adaptation to hypoxia

doi: 10.7554/elife.31132

Figure Lengend Snippet: Figure 8. The antilipolytic signal is upregulated in human cancers. (A) Heat map of gene expression in colon adenocarcinoma (COAD) and kidney renal clear cell carcinoma (KIRC). Pan-cancer normalized expression scores were further Z-score normalized, and fold changes of expression were reported as Log2. Vegfa, vascular endothelial growth factor A; Glut1, glucose transporter 1; Ldha, lactate dehydrogenase A. (B) Protein expression and TG content were examined in kidney tissues from human with renal cell cancer. T = kidney tumor, N = adjacent normal kidney tissue. (C) The proposed model illustrating the protective role of HIF-1-dependent HIG2 expression and lipid storage against oxidative stress under hypoxia. DOI: https://doi.org/10.7554/eLife.31132.016

Article Snippet: DOI: https://doi.org/10.7554/eLife.31132 17 of 24 Production and purification of bacterially expressed proteins The human HIG2 or HIG2 7–11 was subcloned by standard PCR into pET His6 MBP vector (addgene, #29708) producing fusion protein with a His6-MBP tag at the Nterminal end.

Techniques: Gene Expression, Expressing