Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: VCP/p97-governed proteostasis is tied to metabolic pathways and nutrient availability. a Network graph of leading-edge enriched genes from the top three most highly enriched KEGG pathways. Edge weight and size indicate strength of correlation between genes/nodes. Nodes are coloured by the pathways each gene contributes to. b GSEA plots of the KEGG term Metabolic Pathways, with genes ranked by correlation with VCP/p97 expression, in the Cancer Cell Line Encyclopedia (CCLE) and in patient-derived tumour cells in the Myeloma IX study. The correlation value of individual genes is indicated by black bars (left axis) and running enrichment score is highlighted by red line (right axis). NES normalised enrichment score. c Relative mRNA levels of the indicated genes in A549 cells stably expressing non-targeting control (NTC) or one of two IPTG-inducible VCP/p97 shRNAs after 48 h of IPTG treatment in complete medium (25 mM glucose, 2 mM glutamine), followed by 24 h maintenance in complete, low glucose (1 mM glucose, 2 mM glutamine) or no glutamine (25 mM glucose, 0 mM glutamine) medium supplemented with 500 μM IPTG. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). d Relative mRNA levels of the indicated genes in A549 cells grown under different conditions of nutrient availability and treated with CB5083 (1 μM) as indicated for 16 h. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). e Proportion of non-viable A549 cells as determined by Annexin V and 7-AAD staining after treatment with CB5083 under the indicated conditions of nutrient availability for 48 h. Data shown are mean and SEM, n = 3 (two-way ANOVA with Sidak’s multiple comparisons test). f VCP/p97 mRNA expression in the indicated cell lines grown in complete, low glucose or no glutamine medium for 16 h (A549), 24 h (DU145), or 48 h (A172). Data shown are mean and SEM, n = 3 (Mann–Whitney test)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Expressing, Derivative Assay, Stable Transfection, Control, Staining, MANN-WHITNEY

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: Metabolite responses to VCP/p97 inhibition. A549 cells were grown in complete (25 mM glucose, 2 mM glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium for 16 h followed by intracellular metabolite extraction and gas chromatography-mass spectrometry. Data show the effects of CB5083 treatment (1 μM) for 16 h. a Volcano plots indicating metabolites with significant changes (coloured). The horizontal dashed line represents a multiple testing-adjusted p -value of 0.05, and the vertical dashed lines represent log 2 fold changes of 0.67 and 1.5, respectively. b Bar graphs show the effect of treatment with CB5083 on individual metabolites (fold change of treatment to control for each of the three media compositions). Comparisons with significant effects (two-way ANOVA, n = 4 per experimental condition) are outlined in red

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Inhibition, Extraction, Gas Chromatography, Mass Spectrometry, Control

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: GCN2 modulates proteotoxic stress and ERK signalling in response to VCP/p97 inhibition. a Immunoblot for GCN2 and β-tubulin on extracts from untransduced A549 cells and A549 cells stably expressing targeting (shGCN2) or non-targeting (shNTC) shRNA . b-e A549 cells stably expressing non-targeting control (shNTC) or one of two shRNAs targeting GCN2 (shGCN2) were grown in complete (25 mM glucose, 2 mM l -glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium. CB5083 was used at 1 μM. b Proportion of non-viable A549 cells as determined by Annexin V-FITC and 7-AAD staining after 48 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). c mRNA levels relative to shNTC expressing cells grown in complete medium after 16 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). d Immunoblot analyses on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative phosphorylated ERK1/2 immunoblot band intensities (mean and SEM) compared to control cells. e Proportion of non-viable A549 cells as determined by Annexin V and 7-AAD staining after 24 h of experimental treatment with AZD6244, U0126, and CB5083 for 24 h. Mean and SEM of n = 3 experiments is shown (two-way ANOVA and Tukey’s multiple comparisons for effects of AZD6244 and U0126 compared to vehicle-treated or CB5083-treated cells; two-way ANOVA and Bonferroni’s multiple comparisons test for effects of shGCN2 compared to shNTC under the same experimental conditions (grey asterisks); exact p values for all comparisons are shown in Supplementary Figure )

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Inhibition, Western Blot, Stable Transfection, Expressing, shRNA, Control, Staining

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: GCN2 attenuates autophagy in response to metabolic and proteotoxic stress. A549 cells stably expressing non-targeting control (shNTC) or one of two shRNAs targeting GCN2 (shGCN2) were grown in complete (25 mM glucose, 2 mM l -glutamine), low glucose (1 mM glucose, 2 mM glutamine), or no glutamine (25 mM glucose, 0 mM glutamine) medium. CB5083 was used at 1 μM. a GSEA plot of KEGG autophagy pathway with genes ranked by correlation with VCP/p97 expression. The correlation value of individual genes is indicated by black bars (left axis) and running enrichment score is highlighted by red line (right axis). NES normalised enrichment score. b mRNA levels relative to shNTC expressing cells grown in complete medium after 16 h of experimental treatment. Data shown are mean and SEM, n = 3 (two-way ANOVA with Tukey’s multiple comparisons test). c Immunoblot analyses on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative LC3BII and p62 immunoblot band intensities, compared to control cells (mean and SEM). d Immunoblot analyses on whole-cell extracts from A549 cells treated with CB5083 (1 μM) for 24 h and bafilomycin A1 (100 nM) for 16 h. e Propoof non-viable A549 cells treated with CB5083 (1 μM) for 48 h and bafilomycin A1 (100 nM) for 40 h. Data shown are mean and SEM, n = 3 (two-way ANOVA and Tukey’s multiple comparisons test)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Stable Transfection, Expressing, Control, Western Blot

Journal: Oncogene

Article Title: The coordinated action of VCP/p97 and GCN2 regulates cancer cell metabolism and proteostasis during nutrient limitation

doi: 10.1038/s41388-018-0651-z

Figure Lengend Snippet: Effects of GCN2 on metabolites and protein synthesis. a Bar graphs show metabolite levels in shGCN2 A549 cells compared to shNTC cells in different media (fold change for each of the three media compositions (16 h), and metabolite levels in shGCN2(1) cells (fold change to shNTC cells) after treatment with CB5083 (1 μM, 16 h). Comparisons with significant effects are outlined in red ( n = 4, two-way ANOVA). b , c Immunoblot analyses for the indicated proteins on whole-cell extracts from A549 cells (three independent experiments) stably expressing shNTC or shGCN2(1). Bar graphs show relative immunoblot band intensities, compared to control cells (mean and SEM)

Article Snippet: A549 cells stably expressing inducible control shRNA or inducible shRNA targeting VCP/p97 (clones 4252–2 and 4252–3) were described before and were a kind gift from Dan Anderson (Cleave Biosciences) [ ].

Techniques: Western Blot, Stable Transfection, Expressing, Control