Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet: (A) Schematic representations of the shRNA and CRISPR constructs used to knock down and knock out CD97, respectively. (B) Histograms of CD97 surface staining following knockdown or overexpression of isoform 3 of CD97 in a PDGC. (C) Bar graph visualizing tumorsphere formation following knockdown of CD97 and followed by rescue with a dox-inducible and a shRNA-resistant form of CD97 isoform 3 (n = 5 for each PDGC; two-way ANOVA F 3,36 = 9.795, p < 0.0001; Tukey’s multiple comparisons test: empty vector (EV) SCRsh vs. EV CD97sh: ***p < 0.001; EV CD97sh vs. overexpressed (OE) CD97sh: *p < 0.01; OE SCRsh vs. OE CD97sh: ns, p > 0.05). (D) Representative wells from the tumorsphere formation assay quantified in (C) before and after dox-induced rescue. (E) ELDA plots for three PDGCs are displayed. (F) Summary plot showing calculated clonogenic frequencies for all tested PDGCs after CD97 knockdown based on ELDA assays. Paired samples are connected with a line (n = 3 per PDGC; two-way ANOVA F 1,28 = 75.24, p < 0.0001). (G) Graph showing compromised viability of PDGCs infected with three separate gRNAs against CD97 (orange) compared to a control gRNA against the human homolog of ROSA26 (black) in a cell competition assay (n = 3 per gRNA; two-way ANOVA F 4,40 = 10.37, p < 0.001; Tukey’s multiple comparisons test: ROSA26 vs. CD97: days 4, 8, and 12: ns, p > 0.05; days 16 and 20: ***p < 0.001). (H) Bioluminescent images taken with IVIS of intracranial GBM xenografts 90 days after injection. (I) Immunofluorescent images of mouse brains injected with PDGCs lentivirally infected with the indicated shRNA construct, the TagRFP fluorophore, and luciferase. White arrows indicate the resulting tumor at the site of injection. (J) Graph depicting the average bioluminescent radiance captured by IVIS for mice harboring PDGC xenografts (n = 9 mice per group; unpaired t test; *p < 0.05). (K) Kaplan-Meier survival curve showing significantly increased survival of mice xenografted with a PDGC harboring CD97 shRNA (n = 5 mice per group; log-rank test; **p < 0.01). Error bars indicate SEM.

Article Snippet: Short hairpin RNAs were cloned cloning into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289, Addgene) shRNA expression vector.

Techniques: shRNA, CRISPR, Construct, Knockdown, Knock-Out, Staining, Over Expression, Plasmid Preparation, Tube Formation Assay, Infection, Control, Competitive Binding Assay, Injection, Luciferase

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet: (A) Volcano plot derived from bulk RNA-seq data collected from PDGCs lentivirally infected with the SCR or CD97 shRNA (n = 3 biological replicates for each). Genes involved in canonical glycolysis and glucose transport are represented as large yellow points. Genes involved in the TCA cycle and OXPHOS (cytochrome oxidase subunits) are represented as large green points. CD97 is represented by an orange point. (B) The top ten enriched and depleted pathways determined by the largest fold enrichment among downregulated genes using GO PANTHER pathway enrichment analysis. Stars indicate metabolic pathways. (C) Correlation matrix from bulk RNA-seq data collected from PDGCs following knockdown or overexpression of CD97 shows high correlation between CD97 and glycolysis-related genes and anti-correlation with TCA cycle-related genes. HK2 (hexokinase 2) and SLC2A3 (glucose transporter 3 [GLUT3]) transcripts are both included because they have been implicated in Warburg metabolism. (D) Bar graph showing decreased lactate production after knockdown of CD97 (n = 6 per PDGC; two-way ANOVA F 1,15 = 27.24, p < 0.001) and increased lactate production after CD97 overexpression in PDGCs (n = 3 per PDGC; two-way ANOVA F 1,6 = 26.07, p < 0.01). (E) Steady-state metabolomic data reveal depletion of glycolytic metabolites after knockdown of CD97 in PDGCs (PN [proneural], n = 2–3 [one replicate was removed for technical reasons]; CL [classical], n = 3). (F) Depleted (red) and enriched (green) heavy-labeled glycolytic and TCA cycle metabolites after a heavy-labeled glucose tracing experiment. (G) Representative Seahorse Cell Energy Phenotype graph showing OCR and ECAR changes before (baseline) and after (maximal) addition of mitochondrial stressors. (H and I) Bar graphs quantifying baseline and maximal ECAR (n = 6 per PDGC; baseline: two-way ANOVA F 1,10 = 14.06; **p < 0.01; maximal: two-way ANOVA F 1,10 = 17.87, p < 0.01). (J and K) Bar graphs quantifying baseline and maximal OCR (n = 6 per PDGC; baseline: two-way ANOVA F 1,10 = 2.820, p > 0.05; maximal: two-way ANOVA F 1,10 = 5.474; *p < 0.05). Error bars indicate SEM.

Article Snippet: Short hairpin RNAs were cloned cloning into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289, Addgene) shRNA expression vector.

Techniques: Derivative Assay, RNA Sequencing, Infection, shRNA, Knockdown, Over Expression, Labeling

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet: (A) Schematic of possible CD97 signaling mechanisms tested. Listed in red are the methods for testing. (B) Phosphoproteomic data collected from GBM samples identify five phosphorylation sites on the CD97 cytosolic C terminus. The heatmap depicts the percentage of GBM samples with the detected phosphorylation site. (C) Homogenous time-resolved fluorescence (HTRF) ratios from a β-arrestin recruitment assay performed after overexpression of WT CD97 or the ΔPS mutant. Two PDGCs (n = 2 for each) are compiled (n = 4 per condition; unpaired t test; ns p > 0.05; *p < 0.05; **p < 0.01). (D) Immunoblot for p-ERK1/ERK2 after overexpression of WT CD97 or the ΔPS mutant. (E) Bar graphs displaying densitometry ratios from (D) (n = 7; paired t test; ns p > 0.05; *p < 0.05). Two PDGCs (n = 3–4 for each) are compiled. (F) Bar graphs depicting the number of tumorspheres in a tumorsphere formation assay after CD97 knockdown followed by overexpression of shRNA-resistant forms of WT CD97 or the ΔPS mutant. The SCR shRNA groups used for normalization are not shown (n = 4 per PDGC; ANOVA F 5,12 = 80.79, p < 0.0001; Tukey’s multiple comparisons test: EV vs. WT: ***p < 0.001; EV vs. ΔPS: ns p > 0.05; WT vs. ΔPS: *p < 0.05). (G‒I) Single-cell RNA-/ATAC-seq data from showing expression of CD97 , CD55 , and THY1/CD90 . (J) A bar graph depicting the percentage of positive cells measured by flow cytometry after surface staining of PDGCs with fluorescein isothiocyanate (FITC)-conjugated antibodies against CD55, THY1/CD90, and an IgG control (n = 3 for each PDGC; two-way ANOVA F 2,20 = 95.11, p < 0.0001; Tukey’s multiple comparisons test: IgG vs. CD55: **p < 0.01; IgG vs. CD90: ****p < 0.00001; CD55 vs. CD90: ****p < 0.01). (K) Immunoblot for p-ERK1/ERK2 from CD97-overexpressing PDGCs plated on laminin or recombinant forms of putative ligands CD55 and THY1/CD90. (L) Quantification of densitometry ratios from immunoblots in (K) (n = 2–4 per PDGC; EV p-ERK/ERK: two-way ANOVA F 2,6 = 0.9450, p > 0.05; EV ERK/GAPDH: two-way ANOVA F 2,6 = 1.047, p > 0.05; CD97 overexpression p-ERK/ERK: two-way ANOVA F 2,16 = 12.48, p < 0.001; Tukey’s multiple comparisons test: laminin vs. CD55: ns, p < 0.05; laminin vs. CD90: ***p < 0.001; CD97 overexpression ERK/GAPDH: two-way ANOVA F 2,16 = 2.257, ns p > 0.05). Error bars indicate SEM.

Article Snippet: Short hairpin RNAs were cloned cloning into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289, Addgene) shRNA expression vector.

Techniques: Phospho-proteomics, Fluorescence, Over Expression, Mutagenesis, Western Blot, Tube Formation Assay, Knockdown, shRNA, Expressing, Flow Cytometry, Staining, Control, Recombinant

Journal: Cell reports

Article Title: The expression profile and tumorigenic mechanisms of CD97 (ADGRE5) in glioblastoma render it a targetable vulnerability

doi: 10.1016/j.celrep.2023.113374

Figure Lengend Snippet:

Article Snippet: Short hairpin RNAs were cloned cloning into the pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Plasmid #28289, Addgene) shRNA expression vector.

Techniques: Recombinant, Modification, Disruption, Activation Assay, Knock-Out, shRNA

Journal: Molecular Cancer Research

Article Title: Metabolic Regulator IAPP (Amylin) Is Required for BRAF and RAS Oncogene-Induced Senescence

doi: 10.1158/1541-7786.mcr-20-0879

Figure Lengend Snippet: Figure 7. Chromatin modifications affected by IAPP knockdown following BRAF activation. A, Western blots of indicated histone modifications from vector control, IAPP or ERK2 -specific shRNAs in BRER cells following 5 days BRAF-ER activation. B, Quantification of relative expression (relative to tubulin expression) from A. Each data point represents an individual immunoblot. Line represents the mean and error bars represent SEM. Where differences reached significance, it is noted. Significance was determined using Tukey’s multiple comparisons test. C, Immunoblot for senescence-related histone marker H3K9me3 from cells treated as in A. D, Location of H3K9me3 relative to SAHF on Day 5 after BRAF activation. Percentage of nuclei with SAHF was quantified as in methods. Error bars represent SEM.

Article Snippet: DNA encoding shRNA to IAPP (shIAPP-1: sequence: 50-ACCGGGCTGGTACTAAGAGGTTATTTgttaatattcatagcAAATAGCCTCTTAGTACCAGCTTTT) and MAPK1 (ERK2, sequence: 50-ACCGGTATTACGACCTGAGTGATGAGgttaatattcatagcCTCGTCACTCGGGTCGTAATATTTT) were PCR amplified (fwd: 50-ATATATCTTGTGGAAAGGAAGACACACCGG, rev: 50-ATATCTCGTATGCCGTCTGAAGACTGCGAAAAAAc) and subcloned into pRSI9-U6-(sh)-UbiC-TagRFP-2A-Puro (Addgene, 28289) using BbsI.

Techniques: Knockdown, Activation Assay, Western Blot, Plasmid Preparation, Control, Expressing, Marker