Journal: iScience

Article Title: Mitochondria-targeted cancer analysis using survival and expression: Prioritizing mitochondrial targets that alleviate pancreatic cancer cell phenotypes

doi: 10.1016/j.isci.2024.110880

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Article Snippet: Mouse monoclonal anti-ERK1/2 , Cell Signaling Technology , Cat#9107; RRID: AB_10695739.

Techniques: Recombinant, Modification, Ligation, Transfection, Infection, Electron Microscopy, Protease Inhibitor, Negative Control, shRNA, Sequencing, Plasmid Preparation, Software

Journal: Cell reports

Article Title: Regulation of MYC by CARD14 in human epithelium is a determinant of epidermal homeostasis and disease

doi: 10.1016/j.celrep.2024.114589

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: ERK (rabbit) (WB (1:1000)) , Cell Signaling Technologies, Danvers, MA , Cat# 4695; RRID: AB_390779.

Techniques: FLAG-tag, Plasmid Preparation, Recombinant, Modification, Transfection, Staining, Protease Inhibitor, Blocking Assay, Western Blot, Stripping, XF Assay, Reporter Assay, Activity Assay, Bicinchoninic Acid Protein Assay, Sequencing, Cloning, Mutagenesis, Software, Membrane

Journal: Cell reports

Article Title: CRISPR/Cas9 Screens Reveal Multiple Layers of B cell CD40 Regulation

doi: 10.1016/j.celrep.2019.06.079

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Article Snippet: Rabbit monoclonal anti-p38 antibody Clone D13E1 , Cell Signaling Technology , Cat# 8690s; RRID:AB_10999090.

Techniques: Ubiquitin Proteomics, Virus, Recombinant, Protease Inhibitor, SYBR Green Assay, Purification, Gel Extraction, Reverse Transcription, Quantitative RT-PCR, Plasmid Preparation, Isolation, Cell Culture, Immunoprecipitation, Gene Expression, CRISPR, Software, Sequencing, Modification

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Boxplots of growth phenotypes for PC9-Cas9-mCherry cells expressing the indicated pgRNA compared to PC9-Cas9-GFP cells expressing a double-safe-targeting control pgRNA. Boxes indicate mean ± SEM of six biological replicates, which are shown as overlaid points. Growth phenotype is defined as the log 2 -scaled ratio of mCherry:GFP cell counts at the late time point compared to the day 1 mCherry:GFP cell counts. Expected DKO phenotypes are the sum of single KO growth phenotypes. The expected and observed DKO phenotypes were compared using a one-tailed t test. Data shown are for the time point with the most extreme difference between expected and observed DKO growth phenotypes, termed the late time point: CCNL1/CCNL2 (day 12), CDK4/CDK6 (day 7), MEK1/MEK2 (day 11), and OXSR1/STK39 (day 10). Full time course data are shown in . (B) Fluorescence microscopy images of competitive fitness assays on early (day 1) and late time points as indicated above for (A). Scale bar, 100 μM. (C) Western blot validation of single KO and DKO pgRNA-induced gene inactivation. For CCNL1, pie charts of percent mutant alleles based on next-generation sequencing are shown due to lack of a suitable CCNL1 antibody for western blotting. Additional genomic DNA-level validation data are presented in . See also and and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Expressing, Control, One-tailed Test, Fluorescence, Microscopy, Western Blot, Biomarker Discovery, Mutagenesis, Next-Generation Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet: (A) Rank plot of target-level GI scores in HeLa cells. Table insert, top synthetic lethal paralogs based on GI score. (B) Volcano plot of target-level GI scores in HeLa cells. FDR indicates the multiple hypothesis-adjusted p values from a two-tailed t test . Blue, synthetic lethal paralog GIs with GI < −0.5 and FDR < 0.1; red, buffering paralog GIs with GI > 0.25 and FDR < 0.1. (C) Scatterplot of target-level GI scores for paralog pairs in PC9 versus HeLa cells. Blue, synthetic lethal paralog pairs with GI < −0.5 and FDR < 0.1 in either PC9 or HeLa cells; gray, all paralog pairs with GI ≥ −0.5 or FDR ≥ 0.1. (D) CRISPR scores for representative synthetic lethal paralog pairs identified in the PC9 and HeLa cell screens. Top row: data shown are the mean CRISPR score for each single KO or DKO target across three biological replicates with replicate data shown in overlaid points. Shared synthetic lethal paralogs (e.g., CCNL1/CCNL2 and MEK1/MEK2 ) have FDR < 0.1 in both cell lines; PC9-specific paralogs (e.g., CDK4/CDK6 and OXSR1/STK39 ) have FDR < 0.1 in PC9 only; and HeLa-specific paralogs (e.g., GFTP1/GFPT2 and SOS1/SOS2 ) have FDR < 0.1 in HeLa only. Dashed lines indicate CRISPR score < −0.5. Bottom row: paralog gene expression in PC9 and HeLa cells from RNA-seq analysis. Dashed lines indicate log 2 (TPM) = 1, the threshold for gene expression. (E) Boxplots comparing the effect of CRISPR-mediated KO of the indicated gene in DepMap cell lines with high (top quartile) compared to low (bottom quartile) copy number of its paralogous gene. For boxplots, the middle line, hinges, notches, and whiskers indicate the median, 25th/75th percentiles, 95% confidence interval, and data points within 1.5× the interquartile range from the hinge, respectively. p values were computed using a two-tailed Wilcoxon rank-sum test. CRISPR score and copy number data were obtained from DepMap. (F) As in (E), but for gene expression. (G) Bar plot indicating the p values (computed using a two-tailed Wilcoxon rank-sum test) obtained by comparing the effect of a single paralog KO to the copy number (as in E) or gene expression (as in F) of its pair across human cancer cell lines profiled by DepMap. Bar color indicates whether each pair was synthetic lethal in PC9 only, HeLa only, or both cell lines in the pgPEN screens. Dashed line indicates p = 0.05. See also and , , and .

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: Two Tailed Test, CRISPR, Gene Expression, RNA Sequencing

Journal: Cell reports

Article Title: Discovery of synthetic lethal and tumor suppressor paralog pairs in the human genome

doi: 10.1016/j.celrep.2021.109597

Figure Lengend Snippet:

Article Snippet: Primary antibodies used for western blotting: CCNL2 (Novus Biologicals #NB100–87009, 1:2000), MEK1 (Cell Signaling Technology #2352, 1:1000), MEK2 (Cell Signaling Technology #9147, 1:1000), OXSR1 (alias OSR1, Cell Signaling Technology #3729, 1:1000), STK39 (alias SPAK, Cell Signaling Technology #2281, 1:500), CDK4 (Cell Signaling Technology #12790, 1:1000), CDK6 (Cell Signaling Technology #13331, 1:1000), vinculin (Sigma #V9264, 1:10,000).

Techniques: CRISPR, Recombinant, Plasmid Preparation, Software

Journal: Cell reports

Article Title: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR-activated lung cancer

doi: 10.1016/j.celrep.2022.111827

Figure Lengend Snippet: (A) Phosphorylation levels altered by GDH1 and RSK2 knockdown. Human phosphorylation pathway profiling array results were obtained using 55 antibodiesdetecting AKT, JAK/STAT, MAPK, NF-κB, and TGF-β signaling in A549 lysates. (B) A549 and H157 cells with GDH1 and RSK2 knockdown were cultured under attached or detached conditions and assayed for CREB, AKT, and ERK1/2 phosphorylation by immunoblotting. GDH1, RSK2, and β-actin blots were obtained from attached conditions, and similar stable knockdown efficacy was observed in detached conditions. (C) Effect of RSK2 and GDH1 knockdown on CREB activity was assessed by CREB transcription factor assay. Nuclear extracts from the detached A549 and H157 cells were incubated with a specific CRE consensus sequence, and the activated CREB-CRE complex was quantified by phospho-CREB S133 ELISA. (D) RNA levels of CREB transcription targets PTK6, ING3, and Fascin-1 in RSK2 and GDH1 knockdown cells were determined by quantitative RT-PCR. (E) Effect of CREB phosphorylation-mimetic mutant S133D (SD) or -deficient mutant S133A (SA) expression on cell invasion and anoikis resistance in GDH1 and RSK2 knockdown cells. GDH1 and RSK2 double knockdown cells were overexpressed with myc-tagged CREB SD or SA mutants, and invasive and anoikis resistant potentials were determined by Matrigel cell invasion assay and annexin V staining. (F) Effect of p38 or CREB inhibitors on p38 and CREB activity. A549 cells were treated with 5 μM BIRB 796 (p38 inhibitor) or 100 nM 666–15 (CREB inhibitor) for 24 h. The activities of p38 and CREB were assessed by p38 T180/Y182 and CREB S133 phosphorylation. (G and H) Effect of CREB S133D overexpression or 10 μM of p38 activator U-46619 on cell invasion and anoikis resistance in fmk- and R162-treated cells. A549 cells were treated with CREB S133D and/or U-46619 for 24 h, and invasive and anti-anoikis potentials were determined as in (E). Western blot results shown are representative of four (B) and two (F) independent biological experiments. Error bars represent ±SD from two replicates for (A) and three replicates for the others. p values were obtained by one-way ANOVA (ns, not significant; *0.01 < p < 0.05; **p < 0.01). See also – .

Article Snippet: Rabbit polyclonal anti-p38 antibody , Cell Signaling Technology , Cat#9212; RRID: AB_330713.

Techniques: Phospho-proteomics, Knockdown, Cell Culture, Western Blot, Activity Assay, Transcription Factor Assay, Incubation, Sequencing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Mutagenesis, Expressing, Invasion Assay, Staining, Over Expression

Journal: Cell reports

Article Title: EGFR-phosphorylated GDH1 harmonizes with RSK2 to drive CREB activation and tumor metastasis in EGFR-activated lung cancer

doi: 10.1016/j.celrep.2022.111827

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Article Snippet: Rabbit polyclonal anti-p38 antibody , Cell Signaling Technology , Cat#9212; RRID: AB_330713.

Techniques: Microarray, Recombinant, Purification, Membrane, SYBR Green Assay, Viability Assay, Phospho-proteomics, Kinase Assay, Reverse Transcription, Transcription Factor Assay, Enzyme-linked Immunosorbent Assay, Multiplex Assay, Extraction, Isolation, shRNA, Sequencing, Plasmid Preparation, Software

Journal: eLife

Article Title: Human Erbb2-induced Erk activity robustly stimulates cycling and functional remodeling of rat and human cardiomyocytes

doi: 10.7554/eLife.65512

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Article Snippet: Antibody , p44/42 MAPK (Erk1/2) (Total Erk) (Rabbit polyclonal) , Cell Signaling Technology , 9102 , WB (1:1000).

Techniques: Control, Recombinant, Flow Cytometry, Imaging, Plasmid Preparation, Software, Sequencing

Journal: Developmental cell

Article Title: Enhanced Dendritic Actin Network Formation in Extended Lamellipodia Drives Proliferation in Growth-Challenged Rac1 P29S Melanoma Cells

doi: 10.1016/j.devcel.2019.04.007

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit polyclonal anti-MEK 1/2 , Cell Signaling , Cat#9122; RRID: AB_823567.

Techniques: Recombinant, Mutagenesis, Blocking Assay, Plasmid Preparation, Immunodetection, Staining, Imaging, Flow Cytometry, CRISPR, Sequencing, Knock-Out, Software, Immunofluorescence