Journal: Protein Science : A Publication of the Protein Society

Article Title: Cysteine residues contribute to the dimerization and enzymatic activity of human nuclear dUTP nucleotidohydrolase (nDut)

doi: 10.1002/pro.3481

Figure Lengend Snippet: Primers for cloning and mutagenesis

Article Snippet: The resulting products were cloned into the NdeI‐HindIII restriction sites of the pCW‐LIC vector (Addgene plasmid 26098).

Techniques: Clone Assay, Sequencing, Mutagenesis

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A Epitopes regulated by AZD8186 treatment in MDA‐MB‐468 cells. Representation of the results from RPPA analysis performed with 300 antibodies: comparison of the lysates from MDA‐MB‐468 treated with vehicle or AZD8186 250 nM for 2 (left) or 28 h (right). The dot‐plot shows the log 2 fold change in signal between vehicle and treated conditions vs the P ‐value (single sample t ‐test for non‐zero coefficient in the regression model) of the difference between the two conditions (biological triplicates) for each antibody. B Inducible expression of flagged‐Cas9 in MDA‐MB‐468. Cells transduced with the doxycycline‐inducible Cas9 (iCas9) construct were selected with hygromycin and single‐cell cloned. The MDA‐MB‐468 iCas9 clone was then treated with vehicle (ethanol) or doxycycline (DOX) for the indicated times and cell lysates probed with the indicated antibodies. Another cell line previously tested for inducible expression of Cas9 was used as a control (Ctrl), and predicted molecular weight of Cas9 is marked on the blot by an arrow. C Frequency and types of genetic modifications induced by a single sgRNA construct in MDA‐MB‐468 iCas9 clone. The cells were transduced with a lentiviral construct codifying for an sgRNA that targets the gene H1F0, whose inactivation is known to do not impact cell proliferation. The target genetic locus was sequenced and the frequency of the genetic modifications reported in the bar graph. D MDA‐MB‐468 parental and iCas9 clone showed similar sensitivity to PI3K pathway inhibitors. MDA‐MB‐468 parental cells or iCas9 clone was treated with serial dilutions of the indicated drugs for 4 days. Mean ± SD of triplicates and representative of two independent experiments. E–G KO efficiency in MDA‐MB‐468 iCas9 clone. Cells were transduced with a mix of five lentiviral constructs codifying for not overlapping non‐target sgRNAs (sgCTRL) or five sgRNAs designed to target GNB2 (E), EGFR (F), or ULK1 (G). Transduced cells were treated with doxycycline for the indicated times or for 8 days where not stated and cell lysates were probed with antibodies against the protein codified by the targeted gene or loading control. Quantification of the bands was performed by ImageLite software. H Inactivation of the essential gene PLK1 in MDA‐MB‐468 was used here as a killing control to validate the system. MDA‐MB‐468 iCas9 clone was transduced with a mix of five lentiviral constructs encoding control sgRNAs or sgRNAs against PLK1, and cell viability was measured by cell titer blue after 4 days of treatment with doxycycline. Mean ± SD of two independent experiments. I Scatter plot of raw phospho‐S6 signals from CRISPR‐Cas9 screening. Color corresponds to individual genes knocked‐out by sgRNAs, and each dot is associated with an individual measurement from a biological and technical replicate. Technical replicates are highlighted by different shapes and positioned in separate columns within each treatment condition. Solid black line indicates the mean level across all observations per drug treatment, and dashed horizontal black lines indicate standard deviation of the same. The horizontal cyan line indicates the threshold for lower outliers. Overlap between outliers can be observed between replicates and treatment conditions J Results of CRISPR‐Cas9 screening in combination with MK2206. The dot‐plots show for each gene knocked‐out by sgRNAs the fold change (log 2 ) between vehicle‐ and MK2206‐treated condition in the fluorescence signal (anti‐phosphoS6 Immunofluorescence) vs the P ‐value of the difference calculated by two‐sided t ‐test. The plots represent means of biological triplicates. Genes for which it was calculated a P < 0.0001 and Log 2 (fold change) > 0.25 are reported and highlighted in green; genes having a 0.0001 < P < 0.05 and a Log 2 (fold change) > 0.25 are reported and shown in red. K MDA‐MB‐468 iCas9 cells transduced with a mix of four GNB2 sgRNAs were single‐cell cloned after Cas9 induction and screened by immune‐fluorescence (IF) with an anti‐GNB2 antibody. GNB2‐expressing MDA‐MB‐468 cells were used as a positive control (+ ctrl), and three independent clones GNB2‐negative were identified (Clones 1–3). Signal from GNB2 was detected through Alexa Fluor‐488 conjugate secondary antibody (green), while nucleus staining was performed by DAPI (blue signal).

Article Snippet: Anti‐EGFR1 (from Francis Crick Institute Cell Service) was used in the Immuno‐precipitation experiments (5 μg antibody/1 mg protein). pLenti_BSD_sgRNA plasmid was a generous gift of Paola Scaffidi, and it was generated through replacement of GFP cassette with BSD cassette into the pLenti‐sgRNA‐Lib from Wei lab (Addgene Plasmid #53121). pCW‐Cas9 vector generated in David Sabatini's lab was obtained from Addgene (#50661).

Techniques: Expressing, Transduction, Construct, Clone Assay, Molecular Weight, Software, CRISPR, Standard Deviation, Fluorescence, Immunofluorescence, Positive Control, Staining

Journal: EMBO Molecular Medicine

Article Title: Combined targeting of G protein‐coupled receptor and EGF receptor signaling overcomes resistance to PI 3K pathway inhibitors in PTEN ‐null triple negative breast cancer

doi: 10.15252/emmm.202011987

Figure Lengend Snippet: A, B Results of CRISPR‐Cas9 screening in combination with AZD8186 100 nM (A) or GDC0941 400 nM (B). The dot‐plots show for each gene knocked‐out by sgRNAs the fold change (log 2 ) between treated conditions (AZD8186 or GDC0941, respectively) and vehicle in the fluorescence signal (anti‐phosphoS6 immunofluorescence) vs the P ‐value of the difference calculated by two‐sided t ‐test. The plots represent means of biological triplicates. Genes for which it was calculated a P < 0.0001 and Log 2 (fold change) > 0.25 are reported and highlighted in green; genes having a 0.0001 < P < 0.05 and a Log 2 (fold change) > 0.25 are reported and shown in red. Gefitinib combined with AZD8186 represents the positive control of the experiment (A). C Box and whisker plot showing the fold change in fluorescence signal between vehicle and GDC0941‐treated conditions for MDA‐MB‐468 cells transduced with non‐target control, GNB2, or GNG5 sgRNAs ( N = 2 or 3). Data presented in a box and whisker plot with the central band indicating the median, the upper, and lower extremes of the box or hinge being the third and first quartiles, respectively, and the whiskers extending to the most extreme data values within 1.5 times the inter‐quartile range. Statistical significance of unpaired t ‐test **** P < 0.0001. D Biochemical analysis of GNB2 KO cells. Cell lysates of MDA‐MB‐468 parental cells and three MDA‐MB‐468 GNB2 KO clones were probed with the indicated antibodies. Quantification of the bands was performed by ImageLite software. Source data are available online for this figure.

Article Snippet: Anti‐EGFR1 (from Francis Crick Institute Cell Service) was used in the Immuno‐precipitation experiments (5 μg antibody/1 mg protein). pLenti_BSD_sgRNA plasmid was a generous gift of Paola Scaffidi, and it was generated through replacement of GFP cassette with BSD cassette into the pLenti‐sgRNA‐Lib from Wei lab (Addgene Plasmid #53121). pCW‐Cas9 vector generated in David Sabatini's lab was obtained from Addgene (#50661).

Techniques: CRISPR, Fluorescence, Immunofluorescence, Positive Control, Whisker Assay, Transduction, Clone Assay, Software

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