Journal: Nature Communications

Article Title: MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

doi: 10.1038/s41467-022-31331-2

Figure Lengend Snippet: a Stable isotope tracing of FA synthesis and FA uptake in SK-N-AS MYCN-ER ™ cells with or without 4-OHT (500 nM) for 24, 48, and 72 h. Mean ± SD ( n = 3); two-sided unpaired t -test per time point. b Viability of NB and normal cells upon 72 h treatment with FA synthesis inhibitors (A939572, orlistat) and FA uptake inhibitors (CB16.2 and CB5). IC 50 calculated in GraphPad Prism (7.01). Mean ± SD ( n = 3). c Top, cell viability in complete media, delipidized media, and delipidized media supplemented with 0.025% FAs. MNA: LAN5 (0–6 day), IMR32 (0–4 day) and SK-N-BE(2c) (0–6 day); non-MNA: SHEP (0–6 day) and SK-N-AS (0–6 day). Mean ± SD ( n = 3); two-way ANOVA with Dunnett’s multiple comparisons test. Bottom, Caspase 3/7 activity in complete media, delipidized media, and delipidized media supplemented with 0.025% FAs for 4 days. Mean ± SD ( n = 3); one-way ANOVA with Dunnett’s multiple comparisons test. FC fold change, CM complete media, DLM delipidized media. Source data are provided in the Source Data file.

Article Snippet: Drugs: A939572 (APExBIO, TX, USA, B3607), orlistat (Sigma, O4139), CB16.2 (ChemBridge, CA, USA, 5830995), CB5 (ChemBridge, 5674122), VP16 (Selleck Chemicals, TX, USA, S1225) and TMZ (Selleck Chemicals, S1237).

Techniques: Activity Assay

Journal: Nature Communications

Article Title: MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

doi: 10.1038/s41467-022-31331-2

Figure Lengend Snippet: a Silencing SLC27A2 in LAN5 cells. Two sh SLC27A2 GIPZ vectors tested, with empty GIPZ vector as control. Mean ± SEM ( n = 3). b FA uptake in LAN5 shCTRL and sh SLC27A2 cells. Cells stained with the FA analog BODIPY ™ 558/568 C12 and quantified as CTCF by ImageJ2. Mean±SD ( n = 3). c Cell growth in LAN5 shCTRL and sh SLC27A2 cells. Mean ± SD ( n = 3). d Clonogenic assay in LAN5 shCTRL and sh SLC27A2 cells. Mean ± SD ( n = 3); e – h LAN5 shCTRL and sh SLC27A2 orthotopic xenograft model. e Tumor volumes at weeks 3 and 4 post-implantation. CTRL = 15; sh SLC27A2 = 14; two-way ANOVA with Sidak’s multiple comparisons test. f Tumor weights at week 5 post-implantation. Mean ± SEM (CTRL = 13, sh SLC27A2 = 12); two-sided unpaired Mann–Whitney test. g Oil Red O staining of intratumoral lipids. Mean ± SEM (CTRL = 6, sh SLC27A2 = 8); two-sided unpaired Mann–Whitney test. h Lipidomics profiling of shCTRL and sh SLC27A2 tumors ( n = 8 each). Lipids (FDR < 0.25, absolute FC > 2) are shown in the heatmap (color scaled by Z-score: red = upregulated; blue = downregulated). MGDG monogalactosyldiacylglycerol, DGDG digalactosyldiacylglycerol, dhCER dihydroceramides, CL cardiolipin, SM sphingomyelin, PA phosphatidic acid, lyso.PC lysophosphatidylcholine, lyso.PE lysophosphatidylethanolamine, plasmenyl.PC plasmenylphosphatidylcholine, all other abbreviations consistent with Fig. . i FA uptake following CB5 treatment in LAN5 and SHEP cells (0–15 µM, 5 min). Cells stained with BODIPY ™ 500/510 C1, C12 and quantified as CTCF by ImageJ2. Mean ± SD ( n = 3). j Caspase 3/7 activity of NB and normal cells after CB5 treatment (0–10 µM, 24 h). Mean ± SD ( n = 3). k Apoptosis, p53/p21, and MYCN/c-MYC protein expression in LAN5, IMR32, and SHEP with CB5 (0–20 µM, 16–24 h). VP16 (10 µM, 24 h) = positive CTRL. Representative blots from three independent experiments are shown. FC fold change, Arb. Unit arbitrary unit. a , b , d , and i , one-way ANOVA with Dunnett’s multiple comparisons test; c and j , two-way ANOVA with Dunnett’s multiple comparisons test. Source data are provided in the Source Data file.

Article Snippet: Drugs: A939572 (APExBIO, TX, USA, B3607), orlistat (Sigma, O4139), CB16.2 (ChemBridge, CA, USA, 5830995), CB5 (ChemBridge, 5674122), VP16 (Selleck Chemicals, TX, USA, S1225) and TMZ (Selleck Chemicals, S1237).

Techniques: Plasmid Preparation, Staining, Clonogenic Assay, MANN-WHITNEY, Activity Assay, Expressing

Journal: Nature Communications

Article Title: MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

doi: 10.1038/s41467-022-31331-2

Figure Lengend Snippet: a – c NB cell line-derived orthotopic xenograft model. a LAN5 or SK-N-AS cells were orthotopically implanted in NCr nude mice. Two weeks later mice were treated with vehicle or CB5 (25 mg/kg, b.i.d ., 6 days/week) for 2 weeks. b LAN5 tumor sizes (IVIS) and weights after treatment. Mean±SEM (CTRL = 6, CB5 = 6); two-way ANOVA with Sidak’s multiple comparisons test (left); two-sided unpaired Mann–Whitney test (right). c SK-N-AS tumor volumes (MRI) and weights after treatment. Mean ± SEM (CTRL = 8, CB5 = 8); two-way ANOVA with Sidak’s multiple comparisons test (left); two-sided unpaired Mann–Whitney test (right). d – g TH-MYCN +/+ -derived orthotopic allograft model. d Cells from one TH-MYCN +/+ tumor were orthotopically implanted in NCr nude mice. Two weeks later mice were treated with vehicle or CB5 (25 mg/kg, b.i.d ., 6 days/week) for 2 weeks. e Tumor volumes (MRI) on treatment days 1 and 14. Tumors were framed and quantified; representative images and mean ± SEM are shown (CTRL = 10, CB5 = 9). f Tumor weights at treatment day 14. Mean ± SEM (CTRL = 10, CB5 = 9); two-sided unpaired Mann–Whitney test. g Oil Red O staining of intratumoral lipids. Mean±SEM (CTRL = 6, CB5 = 5 responsive tumors); two-sided unpaired Mann–Whitney test. h Cells from one TH-MYCN +/+ tumor were orthotopically implanted in syngeneic 129 × 1/svj wild-type mice. Two weeks later mice were treated with vehicle or CB5 (30 mg/kg, b.i.d ., 6 days/week) for 2 weeks. Tumors were weighed on treatment day 14. Mean ± SEM (CTRL = 14, CB5 = 13); two-sided unpaired Mann–Whitney test. i Cells from one MNA patient tumor (P6) were orthotopically implanted in NCr nude mice, and 2 weeks later mice were treated with vehicle or CB5 (25 mg/kg, b.i.d ., 6 days/week) for 6 weeks. Tumor incidence analyzed by Fisher’s exact test (CTRL = 8, CB5 = 9). Kaplan–Meier survival analyzed by log-rank test. Arb. Unit arbitrary unit, Px treatment period. Source data are provided in the Source Data file.

Article Snippet: Drugs: A939572 (APExBIO, TX, USA, B3607), orlistat (Sigma, O4139), CB16.2 (ChemBridge, CA, USA, 5830995), CB5 (ChemBridge, 5674122), VP16 (Selleck Chemicals, TX, USA, S1225) and TMZ (Selleck Chemicals, S1237).

Techniques: Derivative Assay, MANN-WHITNEY, Staining

Journal: Nature Communications

Article Title: MYCN-driven fatty acid uptake is a metabolic vulnerability in neuroblastoma

doi: 10.1038/s41467-022-31331-2

Figure Lengend Snippet: a Synergy analyses in MNA (LAN5 and IMR32) and non-MNA (SHEP) cells treated with CB5 (0.4–2.5 µM), VP16 (5–50 nM) and their combination for 72 h. Heatmap represents mean Bliss scores from three independent experiments. Bliss score > 10 indicates synergy. b Cell viability and Caspase 3/7 activity after single and combination treatments. Cells were treated with CTRL, CB5 (3 µM), VP16 (80 nM), or their combination for 72 h. Mean ± SD ( n = 3); one-way ANOVA with Tukey’s multiple comparisons test. c Anti-tumor activity of CB5 + VP16 combination therapy in LAN5-derived orthotopic xenografts. LAN5 cells were implanted in the renal capsule of NCr nude mice. Two weeks after implantation, mice were treated with CTRL (vehicle), CB5 (15 mg/kg, b.i.d . 6 days/week), VP16 (8 mg/kg daily, 3 days/week) or their combination for 2 weeks. Tumor weights after 2 weeks of treatment are shown. Mean ± SEM (CTRL = 11, CB5 = 11, VP16 = 10, CB5 + VP16 = 12); two-sided unpaired Mann–Whitney test. d Survival analysis in patient-derived orthotopic xenografts. Cells prepared from one MNA patient tumor (P8) were implanted in the renal capsule of NCr nude mice. Five and half weeks after implantation, mice were treated with CTRL (vehicle), CB5 (15 mg/kg, b.i.d . 6 days/week), VP16 (6 mg/kg daily, 3 days/week), or their combination for 5 weeks. Survival was plotted as a Kaplan–Meier curve and analyzed by the log-rank test (CTRL = 12, CB5 = 10, VP16 = 10, CB5 + VP16 = 11). Px treatment period; FC fold change. Source data are provided in the Source Data file.

Article Snippet: Drugs: A939572 (APExBIO, TX, USA, B3607), orlistat (Sigma, O4139), CB16.2 (ChemBridge, CA, USA, 5830995), CB5 (ChemBridge, 5674122), VP16 (Selleck Chemicals, TX, USA, S1225) and TMZ (Selleck Chemicals, S1237).

Techniques: Activity Assay, Derivative Assay, MANN-WHITNEY

Journal: Autophagy

Article Title: Mass spectrometry proteomics reveals a function for mammalian CALCOCO1 in MTOR-regulated selective autophagy

doi: 10.1080/15548627.2020.1719746

Figure Lengend Snippet: CALCOCO1 has a role in reticulophagy. (A) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and 2 sgCALCOCO1 KO cell lines, treated with Veh, 100 nM MLN, or glucose (Glc) starved. GST-tagged proteins/peptides in cell lysates were captured by glutathione agarose affinity purification. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. CALCOCO1 in cell lysates is shown. “B2” and “C3” represent different targets for guide RNA. *nonspecific fragment (B) GSTLSCSGFP-cb5 reticulophagy assay in HEK293 control and sgCALCOCO1 KO cells, treated with Veh, MLN, 10 ug/ml tunicamycin (Tun), or with galactose (Gal). Top panel. GST-tagged proteins/peptides in the LMF. Ratio of the bottom fragment to the full-length fusion protein is shown, normalized to MLN in control cells. Bottom panel. Immunoblot analyses of CALCOCO1 in LMF and high-speed supernatant (HSS) fractions. *nonspecific fragment (C) Immunoblot analysis of GST-Keima-cb5 assay in HEK293 cells, treated with Veh, MLN, and 5 ug/ml Tun as labeled. Ratio of 25 kD fragment to the full-length fusion protein is shown, normalized to MLN in control cells. (D) Relative protein abundances in sgCALCOCO1 KO HEK293 cells compared to sgGFP (GFP) control WT cells (mean, n = 5). Cells were treated for 24 h with 100 nM MLN0128. Proteins were quantified by label-free mass spectrometry. P-values for the protein abundance changes were determined by a two-sided Student’s t-test. (E) GST-BHMT autophagy assay in control and sgCALCOCO1 KO clones, with treatments as in Figure 6A. Ratio of BHMT fragment to GFP, normalized to MLN in control cells, is shown. Immunoblots were probed with antibodies as described. (F) Immunoblot analysis of WT MB231 and 3 sgCALCOCO1 KO cell lines, with antibodies, as shown. Cells were treated for 48 h with Veh, 100 nM MLN, and 20 nM bafilomycin A1, as shown

Article Snippet: GST - RFP-cb5 and GST-Keima-cb5 (Addgene, 137754 and 137755; deposited by Carol Mercer) were made by PCR of RFP or Keima from existing plasmids, using sequence-specific primers with recognition sites for NotI and XbaI restriction enzymes (RE).

Techniques: Control, Affinity Purification, Western Blot, Labeling, Mass Spectrometry, Quantitative Proteomics, Clone Assay