Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: a Proteome profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA) probed with serum-free-conditioned medium (SF-CM) of C33a-FL-CA IX or C33a-NS-CA IX cells incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h. Spot doublets of corresponding proteins with differential intensities are framed and indicated by arrows. Medium pooled from five independent experiments was used for the array. Spot intensities representing b pro-oncogenic basigin, and c anti-angiogenic thrombospondin-1 were analysed in culture medium samples of C33a-FL-CA IX versus C33a-NS-CA IX cells. Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD. d Validation of the differential levels of basigin and thrombospondin-1, respectively, by Western blotting analysis of medium precipitates (MP) and cell lysates (CL) of normoxic (NO) and hypoxic (HY) cells. (* P < 0.05, ** P < 0.01, *** P < 0.005).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Incubation, Biomarker Discovery, Western Blot

Journal: British Journal of Cancer

Article Title: Impairment of carbonic anhydrase IX ectodomain cleavage reinforces tumorigenic and metastatic phenotype of cancer cells

doi: 10.1038/s41416-020-0804-z

Figure Lengend Snippet: Analysis of the differential expression of a IGFBP-2 and b IGFBP-3 in SF-CM samples from C33a-FL-CA IX and C33a-NS-CA IX incubated under normoxic (NO) and hypoxic (HY) conditions for 48 h and evaluated by Proteome Profiler Human XL Cytokine Array (ARY022, R&D Systems, MN, USA). Abundance of the proteins was assessed according to spot intensity using densitometry. Data are shown as mean ± SD of five independent samples. c Western blotting analysis of CA IX, IGFBP-2 and IGFBP-3 protein levels in medium precipitates (MP) of C33a-FL-CA IX and C33a-NS-CA IX cells. Impairment of shedding in NS-CA IX is associated with increased IGFBP-2 and reduced IGFBP-3 secretion to culture medium. Quantitative PCR analysis of relative mRNA levels of d IGFBP-2, e IGFBP-3 and f IGF1 in C33a-FL-CA IX versus C33a-NS-CA IX cells normalised to β-actin mRNA. (*** P < 0.005, ns non-significant).

Article Snippet: PPA analyses were performed using the Human XL Cytokine Array Kit and Human Soluble Receptor Array Non-Hematopoietic panel kit (R&D Systems, MN, USA).

Techniques: Quantitative Proteomics, Incubation, Western Blot, Real-time Polymerase Chain Reaction

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: SP140 expressed heterogeneously in HNSCCs and its high expression in TAMs is associated with a favorable survival and high tumor mutation burden. (A) SP140 expression in primary HNSCC tumors and normal adjacent tissue in TCGA data (n=520 HNSCC, n=44 normal). (B) SP140 expression versus TP53 mutation status in patient with HNSCC tumors in the TCGA dataset (n=327, TP53 mutant, and n=175, TP53 non-mutant). (C) SP140 expression in primary HPV+ HNSCC tumors, HPV− HNSCC tumors, and normal adjacent tissue in the TCGA data (n=44, normal; n=80, HPV+ HNSC, and n=434, HPV− HNSCC). (D) Representative images of SP140 expression in HNSCC TAMs (CD68+) of normal tissues and HNSCC tumor tissues. (E) Mean fluorescent intensity was calculated in TAMs (CD68+) by quantification of SP140 expression using QuPath software and compared between controls, patients with HNSCC with unfavorable 5-year survival, and patients with HNSCC with favorable 5-year survival. (F) Correlation of SP140 expression and number of non-silent mutations in 497 HNSCCs in the TCGA dataset. (G) Correlation of SP140 expression and number of non-silent mutations in over 9766 cancers in the TCGA dataset. (A–C) Box and whisker plots, with whiskers representing minimum and maximum for the panel. (E) Scatter plot (median and 95% CI). P values (A–C) were calculated by Student’s t-test and corrected for multiple comparison by Bonferroni correction. The correlation between SP140 and mutation burden was quantified with Pearson’s correlation coefficient and corrected for multiple comparison. ***P< 0.001, ****P< 0.0001 after correction for multiple comparisons. The scale bars indicate 100 μm in the left images and 30 μm in the right images. HNSCC, head and neck squamous cell carcinoma; HPV, human papillomavirus; RSEM, RNA-Seq by Expectation-Maximization; SP140, speckled protein 140; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Expressing, Mutagenesis, Software, Whisker Assay, Comparison, RNA Sequencing

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: High levels of SP140 in HNSCC tumors are associated with higher expression of proapoptotic markers and favorable survival in HNSCC. (A–D) HNSCC TCGA tumors with available protein expression data (n=212) were divided to two groups of SP140 high and SP140 low based on the median of SP140 expression. The protein levels of LCK, CASP7, CDKN1B, and PXN were compared between the two groups by Student’s t-test, and the p value was calculated after Benjamini-Hochberg correction. (E) Disease-specific survival of patients with high levels of SP140 (n=261) and low levels of SP140 (n=261) in TCGA HNSCC. The p value was calculated based on the log-rank test. **P<0.01, ***P<0.001 after correction for multiple comparisons. HNSCC, head and neck squamous cell carcinoma; RPPA, reverse phase protein array; SP140, speckled protein 140; TCGA, The Cancer Genome Atlas.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Expressing, Protein Array

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: High levels of SP140 in the pan-cancer TCGA tumors are associated with IFN-γ-dominant tumors and high infiltration of proinflammatory TAMs (M1) and CD8 T cells. (A) Association of SP140 gene expression with immune infiltrate subtypes in the TCGA pan-cancer cohort (n=12 839). (B, C) TCGA pan-cancer gene expression data were used to dichotomize SP140 expression based on the median. M1 macrophage and T-cell infiltrations were estimated for each cancer type in SP140 high and SP140 low tumors. Data are presented as scatter plots. The statistical significance is annotated by the number of stars. *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001. IFN-γ, interferon gamma; M1, proinflammatory phenotype; SP140, speckled protein 140; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Gene Expression, Expressing

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: (A, B) Correlation of SP140 expression with M1 macrophage and CD8 T-cell infiltration levels HNSCC (n=497). (C, D) Genes whose expression significantly correlated with SP140 expression were analyzed by GSEA in HNSCC samples (n = 522). The plot shows the top positively enriched pathways, hallmark inflammatory response, IFN-γ response, and T-cell activation. The Y-axis is the GSEA enrichment score. The X-axis is a list of genes ranked by differential expression correlation with SP140, with black bars representing genes in each gene set. GSEA, gene set enrichment analysis; HNSCC, head and neck squamous cell carcinoma; IFN-γ, interferon gamma; M1, proinflammatory phenotype; SP140, speckled protein 140.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Expressing, Activation Assay, Quantitative Proteomics

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: SP140 negatively regulates STAT1 transcription and phosphorylation in macrophages. (A–D) SP140 was downregulated using siRNA in naïve (undifferentiated) macrophages, and cells were collected for RNA expression analysis by qPCR for SP140, STAT1, IL-1Ra and arginase-1, after 24 hours. 18S was used as an endogenous normalizer. The delta–delta Ct method was used to identify the relative gene expression. Data are presented as mean and SD of fold change compared with the control. (E) ATAC-seq read coverage (average count over an arbitrary sliding window) of SP140 KO and non-KO macrophages treated with LPS for 4 hours (along with their respective 0-hour controls) and average H3K27me3 ChIP-seq coverage for IFN-γ stimulated and unstimulated macrophages in the neighborhood of the STAT1 gene. (F) SP140 was downregulated using siRNA in naïve (undifferentiated) macrophages, and cells were collected for protein isolation and western blot 48 hours post transfection. (I) Effect of SP140 overexpressed on Levels of pSTAT1 Tyr701 was quantified by flow cytometry in controls (shaded line indicates control macrophages; blue line indicates scramble control (CRISPR/dCAS9 control), and red line indicates test sample (SP140 overexpression, CRISPR/dCAS9)) after 48 hours. *P<0.05, **P<0.01, ***P<0.001. ATAC-seq, assay of transposase accessible chromatin sequencing; CHiP, chromatin immunoprecipitation; IFN-γ, interferon gamma; IL, interleukin; qPCR, quantitative PCR; siRNA, small interfering RNA; SP140, speckled protein 140.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Phospho-proteomics, RNA Expression, Gene Expression, Control, ChIP-sequencing, Isolation, Western Blot, Transfection, Flow Cytometry, CRISPR, Over Expression, Sequencing, Chromatin Immunoprecipitation, Real-time Polymerase Chain Reaction, Small Interfering RNA

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: Overexpression of SP140 induces the production of inflammatory cytokines and chemokines and reprogram macrophages to induce TAM-mediated tumor cytotoxicity. (A, B) Control CRISPR/dCAS9 (control) or SP140 CRISPR/dCAS9 (SP140 overexpression) was transfected in naïve macrophages, and the expression of CD80 and CD86 was quantified by flow cytometry. (C–E) SP140 CRISPR/dCAS9 (SP140 OE) or scramble control (control CRISPR/dCAS9) were introduced to the naïve macrophages, and a multiplex fluorescence BioLegend assay was used to identify the levels of CXCL10, IFN-γ, and IL-12 (P70) in the supernatant after 48 hours. LPS (100 nM) was administered after 24 hours. (F–I) Correlation of SP140 expression and IFNG, CXCL10, IL-12B, and IL-12A levels in over 500 HNSCCs in the TCGA dataset. (J) Control CRISPR/dCAS9 or SP140 CRISPR/dCAS9 was introduced in TAMs isolated from syngeneic HNSCC tumor and cocultured with murine SCC7 cells. Cell viability after 48 hours of coculture was quantified with a quantitative viability assay kit. Data are presented based on the fold change of non-treated control. *P<0.05, ***P<0.001, ****P<0.0001. HNSCC, head and neck squamous cell carcinoma; IFN-γ, interferon gamma; IL, interleukin; SP140, speckled protein 140; TAM, tumor-associated macrophage; TCGA, The Cancer Genome Atlas.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Over Expression, Control, CRISPR, Transfection, Expressing, Flow Cytometry, Multiplex Assay, Fluorescence, Isolation, Viability Assay

Journal: Journal for Immunotherapy of Cancer

Article Title: SP140 inhibits STAT1 signaling, induces IFN-γ in tumor-associated macrophages, and is a predictive biomarker of immunotherapy response

doi: 10.1136/jitc-2022-005088

Figure Lengend Snippet: High expression of SP140 in tumors is associated with a favorable response to immunotherapy. (A) RNA was isolated from pretreatment specimens of HNSCC and lung cancer responders and non-responders to anti-PD-1 immunotherapy. RNA was isolated and levels of SP140 were quantified by RT-qPCR (n=21). 18S was used for normalization. Student’s t-test was used for statistical analysis. (B, C) Receiver operating curves of SP140 and PD-L1 for discriminating responders or non-responder cases. Y-axis represents sensitivity (%) and X-axis represents 100% specificity (%). (D) Patients with metastatic melanoma were dichotomized to high expression and low expression groups based on expression of SP140 (the highest quartile vs the rest). Overall survival of patients with high levels of SP140 (n=12) and low levels of SP140 (n=28) was graphed and analyzed using the Kaplan-Meier estimate. (E, F) Tumors with high expression of SP140 (n=12) versus tumors with low levels of SP140 (n=28) showed higher infiltration of M1 macrophages, CD8 T cells, gamma delta T cells, and overall immune score. The Wilcoxon test was used for statistical analysis, and the p value was corrected for multiple comparisons. **P<0.01, ***P<0.001, ****P<0.0001. HNSCC, head and neck squamous cell carcinoma; M1, proinflammatory phenotype; PD-1, programmed cell death protein 1; PD-L1, programmed death-ligand 1; RT-qPCR, reverse transcription–quantitative PCR; SP140, speckled protein 140.

Article Snippet: The culture media was replaced with RPMI-1640 medium plus 10% fetal bovine serum (FBS) and 5 μg/mL polybrene (Thermo Fisher), then transduced with SP140 lentiviral activation transduction particles (Santa Cruz Biotechnology).

Techniques: Expressing, Isolation, Quantitative RT-PCR, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) PLA utilizes a pair of oligonucleotide-conjugated secondary antibodies that bind corresponding antibodies targeting each interacting protein partner. The proximity of these two protein partners allows circular DNA amplification of the oligonucleotides template upon in situ ligation. Fluorescent probes complementary to this amplicon engender a detectable signal via fluorescence microscopy. Plasma membrane permeabilization was used for intracellular protein interactions. (B and C) PLA indicating protein-protein interactions of PAI-1 and low-density LRP1 on the cell surface (B) or intracellular (C) due to ASC-CM exposure in the presence or absence of the LRP1 inhibitor RAP. Representative micrograph images showing positive signal (red) for PAI-1/LRP1 interactions. DAPI was used to stain nuclei (blue). (D) ChIP-qPCR analysis of LRP1-ICD binding to two promoter regions of the SMAD4 gene in control (Ctrl) cells and CM-exposed cells treated and untreated with RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; **p < 0.01, ***p < 0.001. (E) qRT-PCR analysis of mRNA expression levels of SMAD4 gene in control cells and CM-exposed cells treated and untreated with Tip (50 μM) or RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; *p < 0.05. (F) PLA demonstrating protein-protein interactions of SMAD4 and ubiquitin due to ASC-CM exposure in the presence or absence of the following inhibitors: Tip for PAI-1, RAP for LRP1, and protease inhibitor MG132. Micrograph images showing positive PLA signal (green) for SMAD4-ubiquitin interactions. DAPI was used to stain nuclei (blue). (G) Protein expression of LRP1 and SMAD4 by capillary western immunoassays. Endometrial epithelial EME6/7t cells were treated with control media, ASC-CM, and ASC-CM plus Tip, or with pre-treatment with LRP1 antagonist RAP or protease inhibitor MG132. The result shown is a representative of three independent experiments. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: DNA Amplification, In Situ, Ligation, Amplification, Fluorescence, Microscopy, Clinical Proteomics, Membrane, Protein-Protein interactions, Staining, ChIP-qPCR, Binding Assay, Control, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) Bar plots showing mRNA expression levels of selected PAI1-regulated JAC genes by qRT-PCR in the two sh SMAD4 knockdown clones, compared to shCtrl in the presence or absence of ASC-CM. (B) ChIP-qPCR analysis of SMAD4 binding to promoter regions of the JAC genes in control (Ctrl) cells and ASC-CM-exposed cells. Data represent mean ± SD, n = 3 technical replicates; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Clone Assay, ChIP-qPCR, Binding Assay, Control

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A and B) tSNE plots of mass cytometry (CyTOF) analysis visualizing 74,048 epithelial cells, which were divided into clusters (n = 27) based on their proteomic profiles (A). These cells included immortalized EEC line EME6/7t (exposed to ASC-CM ± Tip) and primary endometrial tumors of patients with known BMIs (B). (C) tSNE-based phenographs showing abundance and cellular distributions of JAC proteins. (D) tSNE-based phenographs showing EpCAM and CK8/18 as epithelial cell markers and TGFR-2, SMAD2, and SMAD4 as TGF-β factors. See also and and .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Mass Cytometry

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A–C) Expression profiles of SMAD4 and JAC based on CyTOF were classified into five categories: I, II, III, IV, and V. Violin plots show ordered SMAD4 expression level (low to high) and distribution in the individual subpopulations (A). Expression heatmaps of JAC were aligned according to SMAD4 expression levels (in descending order) in different cellular subpopulations (B). Area of circle denotes relative cell proportion of a subpopulation within each sample and ordered in the aforementioned five categories in cellular subpopulations of EME6/7t lines and primary tumors (C). Tumors were ordered from top to bottom based on increased patients’ BMIs and histology classes. (D) Balloon plots of subpopulation distributions of endometrial tumors in the five categories based on histology and BMI. Circle area indicates the size of each subpopulation. Categories IV and V were associated with relatively high expression levels of SMAD4 and JAC, whereas low levels of SMAD4 and JAC were noted in categories I, II, and III. (E) Violin plots showing the average expression levels of four JAC markers—ADAM15, ROCK1, Cx43, and Gas1—based on patients’ tumor histology: papillary serous and endometrioid. (F) Balloon plots of subpopulation distributions in the five categories stratified by BMI into three groups: normal weight (NW), obesity (OB), and morbid obesity (MOB). (G) Violin plots showing the average expression levels of ADAM15, ROCK1, Cx43, and Gas1, based on three BMI groups: NW, OB, and MOB. Lower JAC expressions corresponded with patients with higher BMIs. ****p < 0.0001. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Ubiquitin Proteomics, Recombinant, Multiplex Assay, Reverse Transcription, SYBR Green Assay, In Situ, shRNA, Software

Journal: Cell systems

Article Title: Proteogenomic Network Analysis of Context-Specific KRAS Signaling In Mouse-to-Human Cross-Species Translation

doi: 10.1016/j.cels.2019.07.006

Figure Lengend Snippet: A. CDK2 and EPHA2 kinase-pathway subnetworks in the humanized CRC KiPNA including ASL and CRAT, synthetic lethal partners in CRCs expressing KRASG12D. B. CDK2 Kinase-pathway subnetwork in the humanized PDAC KiPNA including JUN, MET, SMAD3, and SRSF1, synthetic lethal partners in PDACs expressing KRASG12D. Wilcoxon Mann-Whitney p-values (p < 0.05) and fold changes (FC) are shown for gene essentiality probabilities in colon cell lines (KRASG12D vs. and other KRAS alleles). C. Quantitative real-time PCR (qRT-PCR) analysis of knock-down efficiency with siRNA against indicated genes in colon (n = 4) and pancreas (n = 4) cell lines. Data presented as a mean fold-change relative to scrambled control ± SEM (n = 3 technical replicates in each of 2 biological replicates). D. Caspase 3/7 activity 96 hours after siRNA-mediated gene knock-down in KRASG12D and KRASWT or KRASG13D colon cell lines. Bar plot shows mean of caspase 3/7 activity (normalized to scrambled control) in colon cell lines with KRASG12D mutations (LS513, LS180) or other KRAS allele [HT115 (WT), DLD1 (G13D)]. Bars represent the average of means from cell lines in each group ± SEM (n = 3 technical replicates in each of 2 biological replicates). Data were compared by Welch’s t-test [p > 0.05 (n.s.); p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***); p ≤ 0.0001 (****)]. E. Caspase 3/7 activity 96 hours after siRNA-mediated gene knock-down in KRASG12D and KRASG12R pancreas cell lines. Bar plot shows mean of caspase 3/7 activity (normalized to scrambled control) in pancreas cell lines with G12D mutations (SUIT2, KP4) or with G12R mutations (PSN1, TCCPAN2). Bars represent the average of means from cell lines in each group ± SEM (n = 3 technical replicates in each of 2 biological replicates). Data were compared by Welch’s t-test [p > 0.05 (ns); p ≤ 0.05 (*); p ≤ 0.01 (**); p ≤ 0.001 (***); p ≤ 0.0001 (****)]. (See also Figure S4).

Article Snippet: Human: LS513 Cells , ATCC , CRL-2134; RRID:CVCL_1386.

Techniques: Expressing, MANN-WHITNEY, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, Knockdown, Control, Activity Assay

Journal: Cell systems

Article Title: Proteogenomic Network Analysis of Context-Specific KRAS Signaling In Mouse-to-Human Cross-Species Translation

doi: 10.1016/j.cels.2019.07.006

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Human: LS513 Cells , ATCC , CRL-2134; RRID:CVCL_1386.

Techniques: Protein Array, Mutagenesis, Recombinant, BIA-KA, Extraction, Multiplex Assay, Reverse Transcription, Transfection, Phospho-proteomics, Software