Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: CXCL14 silencing is associated with adverse prognosis of CRC. ( A ) CRC datasets show CXCL14 downregulation in cancer development and metastasis (mean + SEM). * p < 0.05, *** p < 0.001. ( B ) The ProteinAtlas database demonstrates that CXCL14 is highly expressed in normal colon (( i ), PID3753) and rectal (( iv ), PID:3243) gland cells (arrow), but significantly reduced in colonic adenocarcinoma tissue (( ii ), PID:2002; ( iii ), PID:1958) and rectal adenocarcinoma (( v ), PID:1424; ( vi ), PID:1416). The bar represents 50 μm. ( C ) CXCL14 is silenced in human colon cancer cell lines (data from GTEx database) compared to normal tissue (data from ProteinAtlas database). ( D ) Mouse colon tissue and cell line qPCR results show very low CXCL14 in MC38 and CT26 cells. ( E ) Clinical datasets reveal CXCL14 negatively correlates with EMT markers in colon cancer ( p < 0.01). ( F ) CXCL14 exhibits a general negative correlation with the tumor cell proliferation marker TK1 in the four CRC datasets. ( G ) Kaplan–Meier curve of post-progression survival (PPS) for colon cancer patients stratified by CXCL14 expression (probe 222484_s_at). ( H ) CXCL14 strongly correlates with T cell (0.4 < r < 0.7) and NK cell markers (r > 0.6) in GTEx colon tissue datasets, but weakly or not at all with Treg cells, neutrophils, and macrophages (r < 0.2). ( I ) Analysis of the correlation between CXCL14 expression levels and the expression levels of MMP family genes in the Sidra-LUMC dataset.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Marker, Expressing

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Overexpression of CXCL14 inhibits proliferation of colon cancer cells. ( A ) RNA-seq data ( n = 2), q-PCR ( n = 3) and WB result of CXCL14 expression in the non-induced and DOX-induced MC38-TetOn-CXCL14 cell line. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001, **** p < 0.0001, t -test. ( B ) Proliferation curves of control and 0.5 and 1 μg/mL DOX-induced MC38 cell ( n = 6). Data represent the mean ± SD. *** p < 0.001, one-way ANOVA. ( C ) RNA-seq data of TK1 mRNA of control and CXCL14 -overexpressed MC38 cell ( n = 2). Data represent the mean ± SD. * p < 0.05, t -test. ( D ) WB detection, qPCR data of CXCL14 expression (left) and proliferation curves of parental and CXCL14-overexpressed CT26 cells. Data represent the mean ± SD. *** p < 0.001, t -test and one-way ANOVA. ( E ) WB assay and qPCR data of CXCL14 expression (left) and proliferation curves of parental and CXCL14 -overexpressed HCT15 cells. Data represent the mean ± SD. ** p < 0.01, *** p < 0.001, t -test and one-way ANOVA. ( F ) The proliferation inhibition effect of recombinant human CXCL14 (rhCXCL14) on colon cancer cell line in vitro. **** p < 0.0001, one-way ANOVA. ( G ) The tumor growth curves of DOX-induced and non-induced groups of C57bl/6n mice with subcutaneous MC38-plvx-TetOn-CXCL14 cells. The tumor volume of the DOX-induced group was significantly lower than that of the non-induced group ( n = 8), * p < 0.05, ** p < 0.01, t -test. ( H ) Appearance of dissected subcutaneous tumor tissue ( n = 8 for control and DOX+ group). ( I ) The tumor tissue was excised from the subcutaneous tissue and weighed. The tumor weight of the DOX-induced group was significantly lower than that of the non-induced group ( n = 8), ** p < 0.01, t -test. ( J ) The body weight curves of non-induced and DOX-induced mice ( n = 8), no significant differences by t -test.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Over Expression, RNA Sequencing, Expressing, Control, Inhibition, Recombinant, In Vitro

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: RNA-seq analysis of the regulatory role of CXCL14 overexpression on the transcriptome of MC38 cells. ( A ) The volcanic plot displayed that the induction of CXCL14 expression led to a significant upregulation of 625 genes and downregulation of 1237 genes ( p -adj < 0.05) in MC38 cells, representing approximately 15% of the total genes. ( B ) The Gene Ontology (GO) analysis of differentially expressed genes (DEGs) between the non-induced and DOX-induced MC38-TetOn-CXCL14 cell line. ( C ) GSEA showed that CXCL14 significantly suppressed cell EMT signaling. Dot size indicates the number of genes counted in each term. ( D ) RNA-seq data of EMT-related genes expression of MC38-TetOn-CXCL14 cell line prior or post DOX induction (all p -values < 0.01). ( E ) The GSEA analysis of the impacts of CXCL14 overexpression on MC38 cell migration, locomotion and chemotaxis. ( F ) RNA-seq data of MMP family genes of non-induced and DOX-induced MC38-TetOn-CXCL14 cell line ( n = 2). Here only genes with non-zero values are displayed. family. * p < 0.05, ** p < 0.01, *** p < 0.001.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: RNA Sequencing, Over Expression, Expressing, Migration, Chemotaxis Assay

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Overexpression of CXCL14 inhibits invasion of colon cancer cells. ( A ) The invasion assay of non-induced and DOX-induced CXCL14-overexpressed MC38 cells ( n = 3). Data represent the mean ± SD. * p = 0.011, t -test. ( B ) The invasion assay demonstrates that overexpression of CXCL14 in CT26 cells impairs their invasion capability ( n = 3), * p < 0.05, t -test. ( C ) The invasion assay has revealed that the restored expression of CXCL14 in HCT15 cells effectively suppresses their invasion capacity. * p <0.01, t -test. ( D ) The wound repair assay of control and 1 μg/mL DOX-induced MC38 cell ( n = 10). The red lines indicate the leading edge of the migrating cells. Data represent the mean ± SD. *** p < 0.001, t -test. ( E ) The wound repair assay of parental and CXCL14-overexpressed-HCT15 cell lines ( n = 6). Data represent the mean ± SD. ** p < 0.01, one-way ANOVA. ( F ) WB assay of the effect of CXCL14-conditioned medium on the phosphorylation of Erk, Akt, and p38 proteins in MC38 cells. ( G ) The grayscale quantification results of the WB bands in ( F ). the data represents the consistent outcomes from multiple replicates of the experiment. ( H ) qPCR data of EMT-related genes in parental and CXCL14-overexpressed CT26 cells ( n = 2), * p < 0.05, ** p <0.01, t -test. ( I ) The appearance of the liver 21 days after spleen inoculation with MC38. ( J ) The liver weight data, n = 5, * p = 0.02. ( K ) Number of metastatic tumor foci on the liver surface. ** p < 0.01, n = 5.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Over Expression, Invasion Assay, Expressing, Control, Phospho-proteomics

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: Transcriptome analysis showed that CXCL14 regulates the tumor microenvironment. ( A ) Frozen sections from mice bearing subcutaneous tumors revealed the presence of the reporter gene EGFP, indicating the induction of CXCL14 expression within the tumor. ( B ) RNA-seq data of the expression level of CXCL14 in non-induced and DOX-induced CXCL14 overexpression tumor tissues ( n = 4), * p < 0.05, t -test. ( C ) RNA-seq results revealed that there were 720 significantly upregulated genes and 234 downregulated genes in CXCL14 OE MC38 subcutaneous tumors compared to MC38 non-induced tumors. ( D ) The GO analysis results indicate that the gene changes caused by CXCL14 overexpression are mainly concentrated in immune/inflammatory response, chemotaxis, and T cell-related signal transduction. ( E ) The KEGG enrichment analysis results indicate that the gene changes caused by CXCL14 overexpression are mainly concentrated in immune deficiency, Th cell differentiation, and antigen presentation. ( F ) The RNAseq data of T cell marker genes in the subcutaneous tumor with non-induced and DOX-induced CXCL14 overexpression ( n = 4), * p < 0.05, t -test. ( G ) The heat map of qPCR data of dendritic cell markers in the subcutaneous tumor with non-induced and DOX-induced CXCL14 overexpression ( n = 4), p < 0.05, t -test. ( H ) GSEA of hallmark inflammatory and IFNγ gene signatures was performed to compare CXCL14-overexpressed MC38 cells with non-induced MC38 cells.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Expressing, RNA Sequencing, Over Expression, Chemotaxis Assay, Transduction, Cell Differentiation, Immunopeptidomics, RNA sequencing, Marker

Journal: Cells

Article Title: CXCL14 Inhibits Colon Cancer Progression by Modulating Tumor Cell Invasion and Immune Microenvironment

doi: 10.3390/cells15100860

Figure Lengend Snippet: CXCL14 enhances tumor T cell infiltration. ( A ) The H-E staining of the tumor showed that there were more necrotic areas in the tumor overexpressing CXCL14. ( B ) The abundance of CD3 cells was significantly increased in the tumor tissues induced by DOX+ overexpression of CXCL14. Each treatment had 4 biological replicates, and 5 high-power fields were randomly selected from each tumor to count the positive cells. ( C ) The CD3 (PE-Cy5.5) and SSC scatter plot of the tumor cell suspension. The gate in the figure selects CD3-positive T lymphocytes. ( D ) The flow cytometry results of CD4 (FITC) vs. CD8 (APC). The Q1 quadrant represents CD4+CD8-T lymphocytes, and the Q3 quadrant represents CD4-CD8+ T lymphocytes. ( E ) The statistical results of the Q1 quadrant cells in ( D ) show that CD4+CD8- T lymphocytes were significantly increased in the tumor cells induced by DOX overexpression ( p < 0.001). ( F ) The statistical results of the Q3 quadrant cells in ( D ) showed that CD4-CD8+ T lymphocytes were significantly increased in the tumor cells induced by DOX overexpression ( n = 4), * p < 0.05, *** p < 0.001, t -test. ( G ) The scatter plot of FSC/SSC of the tumor cell suspension. The gate encloses live cells. ( H ) The scatter plot of CD40/APC and CD11b/PC7 of tumor cells. The Q2 quadrant represents double-positive cells, which are tumor macrophages. Colors indicate the relative cell density in each region of the plot. ( I ) There was no significant change in the content of macrophages in tumor-infiltrating cells ( n = 4), t -test.

Article Snippet: In the proliferation inhibition assay, recombinant human CXCL14 protein, purchased from MCE (Monmouth Junction, NJ, USA; Cat# HY- P72683 ), was used in the culture medium.

Techniques: Staining, Over Expression, Suspension, Flow Cytometry

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Structures of LF-268 and 268-TCO ( top ) and biochemical IC 50 values (bottom) of LF-268, 268-TCO, and LXH-254 for purified Y301D/Y302D ARaf, WT BRaf, and Y340D/Y341D CRaf (LF-268: n=3, 268-TCO and LXH-254: n=1). b, Schematic of the competitive 268-TCO pulldown experiment performed with HCT-116 cells using tetrazine-linked beads for enrichment ( left ) and a heatmap ( right ) showing the proteins that were significantly competed (log 2 ratio > 2 and p < 0.05) by 5 μM LF-268 compared to DMSO-treated cells (n=3). c, Schematic depicting the competitive kinobead-based inhibitor profiling workflow. Lysates were either incubated with 10 μM LF-268 or DMSO and only EPHA2 was significantly competed with a log 2 ratio > 2 for 10 μM LF-268 relative to DMSO (n=2). d, Representative western blots showing pErk levels in mutant KRas-expressing cells treated with a range of LF-268 doses for 4 h (n=2, **n=3). e, Quantification of normalized pErk percentages and determination of pErk IC 50 values from the western blots obtained in (d). The percent pErk value shown at each LF-268 concentration relative to DMSO is the mean of all replicates performed. IC 50 curves were fit to the percent pErk values obtained from all replicates. pErk IC 50 values and 95% confidence intervals (Cis) for each cell line are listed in Table S1 . f, Correlation plot of LF-268 and LXH-254 pErk IC 50 values for eleven mutant KRas-expressing cell lines. Pearson’s r value is shown. Values shown in blue indicate the six cell lines that comprise the comparative panel of mutant KRas-expressing cell lines.

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Purification, Incubation, Western Blot, Mutagenesis, Expressing, Concentration Assay

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Percent activity values for purified Y301D/Y302D ARaf, WT BRaf, and Y340D/Y341D CRaf in the presence of a range of LF-268, 268-TCO, or LXH-254 concentrations (LF-268 n=3, 268-TCO n=1, LXH-254 n=1). For LF-268, individual data points represent the average of n=3 replicates and error bars represent standard error of the mean (s.e.m.). Calculated IC 50 values are shown in . b, STRING protein:protein interaction network mapping proteins with at least one phospho-site that exhibits a >2-fold average decrease in HCT-116 cells treated for 4 h with 5 μM LF-268 relative to DMSO (n=3). The thickness of the line indicates the relative strength of data that indicates a protein:protein interaction. MAPK1 and MAPK3 (Erk1 and Erk2) are labeled in red. Phospho-peptide quantifications are in Table S3 . c, Histogram of log 2 ratios of quantified phospho-peptides for HCT-116 cells treated with 5 μM of LF-268 for 4 h relative to DMSO following kinobead enrichment (n=3). Log 2 ratios are plotted from lowest to highest and the dotted line indicates a log 2 ratio < −2. Phospho-peptide quantifications are in Table S4 .

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Activity Assay, Purification, Labeling

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blot ( top left ) of ARaf, BRaf, and CRaf expression levels across the comparative panel of mutant KRas-expressing cell lines. Correlation plots of GAPDH-normalized ARaf ( top right ), BRaf ( bottom left ), and CRaf ( bottom right ) levels as measured by western blot (n=1) versus LXH-254 pErk IC 50 values. b, Western blots of phosphorylation levels of BRaf and CRaf regulatory phospho-sites (n=1). All images are from individual western blots. c, The ratio of CRaf co-immunoprecipitated with BRaf (normalized to immunoprecipitated BRaf) from CIAR-293 cells pre-treated with A115 relative to DMSO for 1 h, followed by incubation with LXH-254 for 3 h. CRaf co-immunoprecipitation levels for DMSO and A115 pre-treated CIAR-293 cells were probed on the same western blot, allowing relative levels to be compared. The values shown are from the experiments described in . Bars represent the mean of n=3 replicates and error bars represent standard error of the mean (s.e.m.).

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Expressing, Mutagenesis, Phospho-proteomics, Immunoprecipitation, Incubation

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blots ( top ) of ARaf, BRaf, and CRaf levels in size exclusion chromatography fractions from the lysates of Mia PaCa-2 cells treated with 10 μM LXH-254 or DMSO (n=1). Quantification indicates the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions ( bottom ). b, Representative western blots ( top ) and quantification ( bottom ) of ARaf levels co-immunoprecipitated with CRaf from Mia PaCa-2 treated with a range of LXH-254 doses for 3 h. The percent CRaf levels (normalized to immunoprecipitated BRaf) shown at each LXH-254 concentration are represent singlicate data. DC 50 curves were fit to percent ARaf levels obtained from all three replicates and ARaf:CRaf dimerization DC 50 values and 95% CIs are listed in Table S1 . The Mia PaCa-2 pErk IC 50 and BRaf:CRaf dimerization DC 50 curves from are overlayed to demonstrate at which LXH-254 concentrations pErk IC 50 s and dimerization DC 50 values intersect. Error bars represent standard error of the mean (s.e.m.). c, Representative western blot ( top ) of pErk levels in HCT-116 cells treated with range of encorafenib doses for 1 h followed by a 1 h washout. Quantification (bottom) of normalized pErk percentages and determination of pErk PA 50 value. The percent pErk value shown at each LXH-254 concentration relative to DMSO is the mean of n=3 replicates. PA 50 curves were fit to percent pErk values obtained from all replicates. pErk PA 50 values and 95% confidence intervals (CIs) are listed in Table S1 . d, Representative western blot ( top ) and quantification (bottom) of pErk levels in HCT-116 cells treated with a range of LXH-254 doses for 4 h (same data as shown in ). e, In-cell western assay ( top ) of pErk levels in HCT-116 cells treated with a range of LXH-254 doses for various treatment times. Quantification ( bottom ) of normalized pErk percentages (relative to DMSO-treated cells) observed at each LXH-254 concentration from the in-cell western assay (n=1).

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Size-exclusion Chromatography, Immunoprecipitation, Concentration Assay, In-Cell ELISA

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a and b, Representative western blots ( a ) and quantification ( b ) of CRaf levels co-immunoprecipitated with BRaf from Mia PaCa-2 cells pre-treated with DMSO or a partial (∼85%) inhibitory dose of a Mek inhibitor for 1 h, followed by treatment with a range of LXH-254 doses for 3 h. The BRaf-normalized percent CRaf levels shown at each LXH-254 concentration relative to maximum CRaf levels are the mean of n=3 replicates and error bars represent standard error of the mean (s.e.m.). DC 50 curves were fit to percent CRaf levels obtained from all three replicates. BRaf:CRaf dimerization DC 50 values and 95% confidence intervals (CIs) for each cell line are listed in Table S1 . BRaf:CRaf DC 50 curves shown for LXH-254 only treated cells are from . Mek inhibitor pre-treatment concentrations used: cobimetinib = 200 nM, Ro 5126766 = 30 nM, binimetinib = 80 nM, trametinib = 10 nM, GDC-0623 = 10 nM. c, Correlation plot of LXH-254 BRaf:CRaf DC 50 s from (b) and pErk IC 50 s calculated from , d. Pearson’s r value = 0.595. d, Schematic depicting workflow for quantifying biotinylation of individual Raf isoforms by KRas-TurboID in CIAR-293 cells. e, Representative western blots ( top left ) showing levels of biotinylation of immunoprecipitated ARaf, BRaf, and CRaf in CIAR-293 cells transfected with a KRas-TurboID construct, treated with DMSO, LXH-254 (10 μM), cobimetinib (500 nM), or GDC-0623 (40 nM) for 4 h, followed by treatment with 100 μM biotin for 15 min. Quantified biotinylation levels of ARaf, BRaf, and CRaf (normalized to total immunoprecipitated ARaf, BRaf, and CRaf, respectively, and represented as a fraction relative to DMSO) were obtained from n=3 replicates. Bars equal the mean of all replicates and error bars represent standard error of the mean (s.e.m.). f, Representative western blots ( top ) of pErk levels in Mia PaCa-2 cells pre-treated with 40 nM cobimetinib or DMSO for 1 h, followed by incubation with a range of encorafenib doses for 3 h. The percent pErk values shown at each encorafenib concentration relative to DMSO are the mean of n=3 replicates. Error bars represent s.e.m. PA 50 curves were fit to percent pErk values obtained from all three replicates. pErk PA 50 values and 95% CIs for each cell line are listed in Table S1 .

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Immunoprecipitation, Concentration Assay, Transfection, Construct, Incubation

Journal: bioRxiv

Article Title: Uncovering the mechanistic basis of intracellular Raf inhibitor sensitivity reveals synergistic cotreatment strategies

doi: 10.1101/2024.09.18.613772

Figure Lengend Snippet: a, Western blots ( top ) of ARaf, BRaf, CRaf, and Mek levels in size exclusion chromatography fractions from the lysates of Mia PaCa-2 cells treated with cobimetinib or GDC-0623 (n=1). Quantification indicates the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions ( bottom ). b, Quantification shows the amount of each Raf isoform eluted in each fraction as a percentage of the total isoform in all fractions.

Article Snippet: Meanwhile, Protein G agarose beads (Cell Signaling Technology) were loaded with BRaf Antibody (F-7, Santa Cruz Biotechnology) or ARaf Antibody (A-5) Santa Cruz Biotechnology) and Protein A agarose beads were loaded with c-Raf (D4B3J) Rabbit mAb (Cell Signaling Technology) using the following protocol: beads were quickly washed by gentle centrifugation three times with modified RIPA buffer on ice.

Techniques: Western Blot, Size-exclusion Chromatography

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that ROCK1 mean intensities within circumferential rings (CRs) are similar between WT and LUZP1 KO cells (40.87 ± 9.95 arbitrary units [a.u.] [WT] vs. 39.48 ± 6.04 a.u. [LUZP1 KO]). n = 3. P = 0.54 (Mann–Whitney U test). Bars and error bars represent the mean ± standard deviation (SD). In vitro myosin light chain (MLC) phosphorylation assay using 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the ppMLC/MLC ratio relative to the control showed that LUZP1 did not change the ratio (1.00 [1 st lane, control] vs. 1.13 ± 0.24 [2 nd lane] vs. 1.01 ± 0.44 [3 rd lane] vs. 1.08 ± 0.73 [4 th lane]). n = 4. P = 0.49 (Kruskal–Wallis test). Bars and error bars represent the mean ± SD. IB, immunoblotting. Representative confocal micrographs of co‐cultures of Venus‐LUZP1‐expressing LUZP1 KO (REV) and LUZP1 KO Eph4 cells treated with 100 nM calyculin A for 30 min. Scale bar, 10 μm. Bar plots with dot density plots showing that calyculin A reversed the difference in ppMLC levels within CRs between REV and LUZP1 KO cells (control, 21.14 ± 16.80 a.u. [WT] vs. 3.10 ± 1.72 a.u. [LUZP1 KO]; calyculin A, 25.24 ± 10.54 a.u. [WT] vs. 20.65 ± 5.62 a.u. [LUZP1 KO]; washout, 22.09 ± 7.90 a.u. [WT] vs. 7.92 ± 4.01 a.u. [LUZP1 KO]). ** P < 0.01 (Mann–Whitney U test). Bars and error bars represent the mean ± SD. n = 3. Representative immunoblot of WT, LUZP1 KO, and Venus‐LUZP1‐expressing LUZP1 knockout (REV) Eph4 cells treated with 100 nM calyculin A for 30 min. Quantification of the ppMLC/MLC ratio relative to WT control, confirming the reversal of the difference in ppMLC levels within CRs between WT and LUZP1 KO cells by calyculin A (WT, 1.00 [control] vs. 1.40 ± 0.06 [calyculin A] vs. 1.14 ± 0.33 [washout]; KO, 0.09 ± 0.04 [control] vs. 1.49 ± 0.06 [calyculin A] vs. 0.81 ± 0.99 [washout]; REV, 2.06 ± 1.78 [control] vs. 1.82 ± 1.50 [calyculin A] vs. 1.80 ± 1.14 [washout]). n = 3. Bars and error bars represent the mean ± SD. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Knock-Out, MANN-WHITNEY, Standard Deviation, In Vitro, Phosphorylation Assay, Western Blot, Expressing

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: A schematic drawing of myosin phosphatase. Myosin phosphatase consists of PP1c β/δ, myosin phosphatase targeting subunit 1 (MYPT1), and a small 20‐kDa regulatory subunit (M20). PP1c β/δ represents a catalytic subunit responsible for dephosphorylating myosin light chain (MLC), whereas MYPT1 targets myosin phosphatase to MLC by binding both PP1c β/δ and MLC. Representative confocal micrographs of co‐cultures of wild‐type (WT) and LUZP1 knockout (LUZP1 KO) Eph4 cells in the apical plane. Scale bar, 10 μm. Bar plots with dot density plots showing that PP1c mean intensities within CRs are similar between WT and LUZP1 KO cells (28.68 ± 9.60 arbitrary units [a.u.] [WT] vs. 25.04 ± 9.47 a.u. [LUZP1 KO]). P = 0.09 [Mann–Whitney U test]. n = 3. Bars and error bars represent the mean ± standard deviation (SD). Co‐immunoprecipitation of HA‐PP1c β/δ and GFP‐LUZP1. LUZP1 binds to PP1c β/δ. IB, immunoblotting. In vitro MLC phosphorylation assay using 1 μg GST‐PP1c β/δ in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, and 0–5 μg GST‐LUZP1. Quantification of the di‐phosphorylated MLC (ppMLC)/MLC ratio relative to the control showed that LUZP1 upregulated ppMLC/MLC levels in a dose‐dependent manner (1.00 [1 st lane, control] vs. 1.27 ± 0.33 [2 nd lane] vs. 1.76 ± 0.68 [3 rd lane] vs. 2.53 ± 1.65 [4 th lane] vs. 2.93 ± 2.45 [5 th lane]). n = 3 or 6. ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. In vitro Merlin phosphorylation assay using 1 μg GST‐PP1c β/δ, 100 ng GST‐Merlin, 2 pg p21‐activated kinase 1 (PAK1), and 5 μg GST‐LUZP1. Quantification of the phosphorylated Merlin (pMerlin)/Merlin ratio relative to the control showed that LUZP1 upregulated pMerlin/Merlin levels (0.23 ± 0.15 [1 st lane] vs. 1.00 [2 nd lane, control] vs. 0.32 ± 0.17 [3 rd lane] vs. 0.97 ± 0.42 [4 th lane] vs. 1.25 ± 0.39 [5 th lane]). n = 4 or 9. * P < 0.05, ** P < 0.01 (Kruskal–Wallis test followed by Steel test [compared with 3 rd lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among ppMLC, LUZP1, and myosin phosphatase at tight junction (TJ)‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Binding Assay, Knock-Out, MANN-WHITNEY, Standard Deviation, Immunoprecipitation, Western Blot, In Vitro, Phosphorylation Assay

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet: Box plots with dot density plots showing the ratio of the apical area/basal area in co‐cultures of Venus‐LUZP1‐expressing LUZP1 knockout (REV) and LUZP1 knockout (LUZP1 KO) Eph4 cells; 2 μM nocodazole treatment for 30 min partially reversed apical constriction of REV cells (REV, 0.65 ± 0.16 [control] vs. 0.90 ± 0.18 [nocodazole] vs. 0.64 ± 0.16 [washout]; KO, 1.30 ± 0.17 [control] vs. 1.07 ± 0.13 [nocodazole] vs. 1.32 ± 0.19 [washout]). ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). The solid lines represent the medians, and the boxes represent the interquartile ranges. The error bars extending from the box represent the data within 1.5 times of the interquartile range. Representative confocal micrographs of co‐cultures of LUZP1‐expressing wild‐type (WT) and LUZP1 KO Eph4 cell treated with 2 μM nocodazole for 30 min. Nocodazole treatment partially reversed the difference in di‐phosphorylated MLC (ppMLC) levels within circumferential rings (CRs) between WT and LUZP1 KO cells. Scale bar, 10 μm. Bar plots with dot density plots showing that ppMLC levels within CRs were significantly downregulated in WT Eph4 cells after nocodazole treatment. Importantly, ppMLC levels in LUZP1 KO Eph4 cells were unchanged after nocodazole treatment (WT, 21.43 ± 6.96 arbitrary units [a.u.] [control] vs. 17.67 ± 5.40 a.u. [nocodazole] vs. 20.84 ± 7.19 a.u. [washout]; KO, 8.74 ± 1.71 a.u. [control] vs. 8.67 ± 1.89 a.u. [nocodazole] vs. 7.96 ± 2.35 a.u. [washout]). n = 3. ** P < 0.01 (Kruskal–Wallis test followed by Steel–Dwass test). Bars and error bars represent the mean ± standard deviation (SD). In vitro MLC phosphorylation assay using 1 μg MTs in addition to 25 ng GST‐MLC, 4 ng GST‐ROCK1 catalytic domain, 1 mM ATP, 1 μg GST‐protein phosphatase 1c β/δ (PP1c β/δ), and 0–5 μg GST‐LUZP1. Quantification of the relative ppMLC/MLC ratio to the control showed that MTs promote LUZP1‐mediated inhibition of PP1c β/δ (1.00 [1 st ‐lane, control] vs. 1.42 ± 0.59 [2 nd ‐lane] vs. 1.72 ± 0.76 [3 rd ‐lane] vs. 1.99 ± 0.56 [4 th ‐lane] vs. 1.14 ± 0.37 [5 th ‐lane] vs. 2.87 ± 1.51 [6 th ‐lane] vs. 2.74 ± 1.19 [7 th ‐lane] vs. 2.50 ± 0.88 [8 th ‐lane]). n = 6. * P < 0.05 (Kruskal–Wallis test followed by Steel test [compared with 1 st lane]). Bars and error bars represent the mean ± SD. A schematic drawing of the relationships among MTs, ppMLC, LUZP1, and myosin phosphatase at TJ‐associated CRs to promote apical constriction. Source data are available online for this figure.

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Expressing, Knock-Out, Standard Deviation, In Vitro, Phosphorylation Assay, Inhibition

Journal: The EMBO Journal

Article Title: A microtubule‐LUZP1 association around tight junction promotes epithelial cell apical constriction

doi: 10.15252/embj.2020104712

Figure Lengend Snippet:

Article Snippet: GST‐ROCK1‐catalytic domain , Carna biosciences , Cat#01‐109.

Techniques: Recombinant, Plasmid Preparation, Sequencing, Transfection, Protease Inhibitor, Purification, Western Blot, Blocking Assay, Software, Imaging, Modification

Journal: International journal of cancer

Article Title: The presence of Merkel cell polyomavirus is associated with deregulated expression of BRAF and Bcl-2 genes in non-small cell lung cancer.

doi: 10.1002/ijc.28062

Figure Lengend Snippet: Figure 2. Western blot analysis of phospho-BRAF (Ser445), BRAF and Bcl-2 in MCPyV-infected and noninfected NSCLC specimens. (a) Compared to MCPyV noninfected cells (lanes 1–3), both activa- tion of BRAF at Ser445 and overexpression of BRAF protein were evident in MCPyV-infected tissues (lanes 4–6). (b) Bcl-2 protein levels were significantly decreased in MCPyV-positive samples (lanes 4–6) compared to noninfected tissue specimens. Actin was used as a stable endogenous control. 2ve: negative samples; 1ve: positive samples.

Article Snippet: Primary antibodies were then added at the appropriate dilution [phosphor-BRAF (Ser445), BRAF and Bcl-2 1/ 1,000; beta-actin 1/2,000) with overnight incubation at 4 C. All primary antibodies were purchased from Cell Signaling Technology, MA.

Techniques: Western Blot, Infection, Over Expression, Control

Journal: bioRxiv

Article Title: MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

doi: 10.1101/252742

Figure Lengend Snippet: A. UM-SCC47 cells were treated for 72 hr with either control siRNA, or siRNA specific for human Sdc4, β4 integrin (ITGB4), EGFR, or α3 integrin (ITGA3), followed by addition of EdU for 45 min to monitor DNA synthesis prior to fixation and staining; Western blots showing individual receptor expression 72 hr after siRNA transfection are shown (inset); B . Either NOKs or UM-SCC47 cells were allowed to invade through LN332-coated filters for 16 hr in serum-free conditions following stimulation with 10 ng/ml EGF +/− 30 μM SSTN EGFR or vehicle; C. Quantification of NOK or UM-SCC47 cell invasion as in ( B ) in the presence or absence of the EGFR kinase inhibitors gefitinib (3 μM) or erlotinib (2 μM), SFK inhibitor PP2 or its inactive analogue PP3 (3 μM), MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-0777607 (3 μM), c-Abl inhibitor GNF5 (2 μM), pan-p38MAPK inhibitor BIRB-796 (100 nM) and α6β4 (3E1) or α3β1 (P1B5) integrin blocking antibody (10 μg/ml); D. DNA synthesis is detected by EdU incorporation in UM-SCC47 cells grown for 3 hr in medium containing SSTN EGFR , the EGFR kinase inhibitors gefitinib or erlotinib, or SFK inhibitor PP2 versus its inactive analogue PP3.

Article Snippet: Western blotting primary antibodies include: goat polyclonal human Sdc4 (AF2918, 0.5 μg/mL), EGFR (AF231, 1 μg/mL) and total MST1R/RON kinase (AF691, 1 μg/mL); rabbit polyclonal pY1238/1239 MST1R/RON (AF1947, 1 μg/mL) and mouse human ITGB4 mAb 422325 (1 μg/mL) from R&D Systems (Minneapolis, MN); rabbit polyclonal ITGA3 (1:250) from Novus Biologicals (Littleton, CO); rabbit mAbs 73E5 (c-Abl pY245, 1 μg/mL), 247C7 (c-Abl pY412, 1 μg/mL), D13E1 (total p38MAPK, 1:1000), 11H10 (tubulin, 1:1000), 133D3 (pS345-CHK1, 1:1000), rabbit polyclonal pT68-CHK2 (2661S, 1:1000) and pT183/Y185 p38MAPK mouse mAb 28B10 (1:2000) from Cell Signaling Technology (Danvers, MA); mouse mAbs 8E9 (total c-Abl, 1 μg/mL) and TU-01 (tubulin, 1:1000), and rabbit polyclonal Sdc2 (1 μg/mL) from Invitrogen/ThermoFisher Scientific (Rockford, IL); p53 mouse mAb DO-1 (1:750) from Santa Cruz Biotechnology (Dallas, TX) and mouse mAbs from Millipore-Sigma, JBW301 (pS139-γH2AX, 1:1000) and AC-74 (β-actin, 1:5000).

Techniques: DNA Synthesis, Staining, Western Blot, Expressing, Transfection, Blocking Assay

Journal: bioRxiv

Article Title: MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

doi: 10.1101/252742

Figure Lengend Snippet: A, B. Select neoplastic, preneoplastic and non-transformed epithelial cells were treated for 3 hr in DMSO (vehicle), the MST1R/RON inhibitors CAS 913376-84-8 (1 μM) or BMS-777607 (3 μM) or the Abl kinase inhibitors GNF5 (2 μM) or PPY-A (0.2 μM). EdU was added for the last 45 min. The cells were fixed and extracted to stain for EdU incorporation ( A ) or DNA-bound PCNA ( B ); C. UM-SCC47 cells treated with vehicle alone or 30 μM SSTN EGFR for 3 hr were lysed and subjected to immunoprecipitation with non-specific isotype control IgG or mAb 8G3 to Sdc4. Immunoprecipitates were probed for the presence of EGFR, α3 integrin (ITGA3), β4 integrin (ITGB4), total and active MST1R/RON (pY1238/1239), total and active c-Abl (pY412 and pY245), Sdc2 and Sdc4; D . UM-SCC47 cells were treated for 3 hr with vehicle or 30 μM SSTN EGFR , then extracted and analyzed on western blots for active pY245 or pY412 phosphorylated c-Abl.

Article Snippet: Western blotting primary antibodies include: goat polyclonal human Sdc4 (AF2918, 0.5 μg/mL), EGFR (AF231, 1 μg/mL) and total MST1R/RON kinase (AF691, 1 μg/mL); rabbit polyclonal pY1238/1239 MST1R/RON (AF1947, 1 μg/mL) and mouse human ITGB4 mAb 422325 (1 μg/mL) from R&D Systems (Minneapolis, MN); rabbit polyclonal ITGA3 (1:250) from Novus Biologicals (Littleton, CO); rabbit mAbs 73E5 (c-Abl pY245, 1 μg/mL), 247C7 (c-Abl pY412, 1 μg/mL), D13E1 (total p38MAPK, 1:1000), 11H10 (tubulin, 1:1000), 133D3 (pS345-CHK1, 1:1000), rabbit polyclonal pT68-CHK2 (2661S, 1:1000) and pT183/Y185 p38MAPK mouse mAb 28B10 (1:2000) from Cell Signaling Technology (Danvers, MA); mouse mAbs 8E9 (total c-Abl, 1 μg/mL) and TU-01 (tubulin, 1:1000), and rabbit polyclonal Sdc2 (1 μg/mL) from Invitrogen/ThermoFisher Scientific (Rockford, IL); p53 mouse mAb DO-1 (1:750) from Santa Cruz Biotechnology (Dallas, TX) and mouse mAbs from Millipore-Sigma, JBW301 (pS139-γH2AX, 1:1000) and AC-74 (β-actin, 1:5000).

Techniques: Transformation Assay, Staining, Immunoprecipitation, Western Blot

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits MAPK signaling pathway through suppressing CRAF and MEK1/2 but not BRAF kinase activity. a , b The inhibitory effect of erianin on the activity of MEK1 and MEK2 kinase. Active GST-MEK1 full length or GST-MEK2 full length (60 ng) and various doses of erianin were incubated with inactive GST-ERK1 or tag free ERK2 (400 ng) as substrate at 30 °C for 30 min. The phosphorylation of ERK1/2 (Thr202/Tyr204) was detected by western blotting. c The inhibitory effect of erianin on the activity of CRAF kinase. Active CRAF (306-end) (50 ng) and various doses of erianin were incubated with inactive GST-MEK1 (600 ng) as substrate at 30 °C for 30 min. d – f Quantifications of integrated density in ( a – c ) were performed. Data were shown as means ± S.D. of three independent experiments. The asterisks (* p < 0.05, ** p < 0.01, *** p < 0.001) indicate a significant difference in the expression of phosphorylation of ERK1 or ERK2 vs total ERK1 or ERK2 in control and erianin-treated group. g The luminescent ADP detection assay was developed to detect the luminescence signal of ATP-to-ADP using the same concentration kinases and substrates described in above kinase assay. Three independent repeats were conducted in this experiment. h Immunoprecipitation (IP)/WB of endogenous CRAF from lysates of SK-MEL-2 (NRAS mut) and A375 (BRAF V600E) cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for BRAF, CRAF, and MEK1. i IP/WB of endogenous MEK1 from lysates of SK-MEL-2 and A375 cells treated with DMSO or erianin at 12.5, 25, 50 nM for 24 h. Total lysates were immunoblotted for CRAF and MEK1. j , k Western blotting of phospho-CRAF, phospho-MEK1/2 and phospho-ERK1/2 by erianin, vemurafenib, cobimetinib or LY3009120 at indicated concentration for 24 h in NRAS mutant SK-MEL-2 and BRAF V600E mutant A375 cell lines. l Western blotting of MAPK signa l ing pathway by erianin, vemurafenib, cobimetinib, or LY3009120 at indicated concentrations for 24 h in KRAS mutant HCT116 cell line

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Activity Assay, Incubation, Phospho-proteomics, Western Blot, Expressing, Control, Detection Assay, Concentration Assay, Kinase Assay, Immunoprecipitation, Mutagenesis

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin inhibits proliferation in BRAF V600E or RAS mutant cell lines. a Chemical structure of erianin. b Cytotoxicity of erianin in normal NHEM and NHDF cell lines using MTT assay. c The left panel shows representative dose–response curves by MTT assay. The SK-MEL-2 (NRAS mut), HCT116 (KRAS mut), A375 (BRAF V600E), and SK-MEL-28 (BRAF V600E) cell lines were exposed to erianin for 72 h. The concentrations are transformed to Log10 values; the Y -axis shows the corresponding relative cell viability. The right panel shows IC50 values of erianin, three BRAF inhibitors (vemurafenib, dabrafenib and encorafenib) and three MEK inhibitors (cobimetinib, trametinib, and binimetinib) calculated in GraphPad Prism 7.0. d The effect of erianin on growth of SK-MEL-2, A375, SK-MEL-28, and HCT 116 cells was estimated by MTT assay at 24, 48, or 72 h. Data were shown as means ± S.D. e The effect of erianin on anchorage-independent growth in above cells was evaluated. Data were shown as means ± S.D. Scale bars: 400 μm. The colonies numbers were calculated in Image-Pro Plus software. * p < 0.05; ** p < 0.01; *** p < 0.001. f Synergism effect of erianin and vemurafenib in SK-MEL-2, A375, SK-MEL-28, and HCT 116. The synergism and antagonism (CI value) were determined and analyzed using CompuSyn 1.0. CI value > 1.1 indicates antagonism, 1.1 ≥ CI value > 0.9 shows addictive effect and CI value ≤ 0.9 indicates synergism

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, MTT Assay, Transformation Assay, Software

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin suppresses either BRAF V600E or RAS mutant cell growth in CDX model. a NOD-SCID mice were injected subcutaneously with SK-MEL-2 (NRAS mut, 5 × 10 6 cell/mouse), A375 (BRAF V600E, 1 × 10 7 cell/mouse), SK-MEL-28 (BRAF V600E, 5 × 10 6 cell/mouse) and HCT116 (KRAS mut, 1 × 10 7 cell/mouse) cells mixed with Matrigel (1:1); erianin (50 mg/kg), vemurafenib (50 mg/kg) or the combine was given through oral gavage and the size of the tumors was monitored twice per week. Tumor volume (mm 3 ) = (length × width × height) × 0.52. SK-MEL-2: n = 10; A375 and SK-MEL-28: n = 8; HCT116: n = 9. b The photographs show tumors from CDX mice treated with vehicle, erianin, vemurafenib, or the combination. c The weight of the tumors was quantified and expressed as the treatment groups compared with the vehicle-treated group. Data were presented as mean ± S.D. One-way ANOVA test. * p < 0.05; ** p < 0.01. d Western blotting shows the expression of phospho-MEK1/2 and phospho-ERK1/2 by erianin in SK-MEL-2, A375, and SK-MEL-28 CDX tumor tissues. The tissue lysates were prepared from CDX tumor tissues in each treatment group. Three samples were randomly prepared for each group and every blot shows one sample. e The quantization (IOD values) of IHC staining in the treatment groups compared with the vehicle-treated group. Each point represents the IOD values of four quantified data from one mouse. Scale bars: 50 μm. One-way ANOVA test. *** p < 0.001. f Kaplan–Meier curve depicting tumors less than 1000 mm 3 in the treatment groups compared with the vehicle-treated group

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: Mutagenesis, Injection, Western Blot, Expressing, Immunohistochemistry

Journal: Signal Transduction and Targeted Therapy

Article Title: Erianin suppresses constitutive activation of MAPK signaling pathway by inhibition of CRAF and MEK1/2

doi: 10.1038/s41392-023-01329-3

Figure Lengend Snippet: Erianin exerts antitumor efficacy in melanoma and colorectal cancer in vivo. a Tumor pharmacodynamic assay was performed in tumor-bearing NPG mice (tumor has been passaged from melanoma patient to mice for three generations). The photographs show tumors from melanoma PDX mice treated with vehicle or drugs. b The effect of erianin on the volume of PDX tumors over time (within 78 days) was plotted. Vehicle, erianin (50 mg/kg, once a day), vemurafenib (50 mg/kg, once a day), erianin and vemurafenib combination therapy, cobimetinib (5 mg/kg, twice a week) or vemurafenib and cobimetinib combination therapy (once a day and twice a week, respectively) were administered by oral gavage, n = 8 in each group. Tumor volume was measured once a week. One-way ANOVA test. * p < 0.05; ** p < 0.01. c Tumor weight was measured after treatment on the last day of the study. d The expression of phospho-MEK1/2 and phospho-ERK1/2 were examined by immunofluorescence analysis. Scale bars: 20 μm. One-way ANOVA test. *** p < 0.001. e Antitumor efficacy of erianin with or without immunity using B16F10 cell xenograft in C57BL-6J mouse. f , g Trend of tumor volume over time and tumor weight was measured after treatment on the last day of the study. One-way ANOVA test. * p < 0.05; ** p < 0.01. h The model depicts that erianin suppresses constitutive activation of MAPK signaling pathway in either BRAF V600E or RAS mutant cancers (Created with BioRender.com). Through inhibition of CRAF and MEK1/2 kinases, erianin suppresses phospho-MEK1/2 and phospho-ERK1/2 without paradoxical activation in vitro and in vivo

Article Snippet: Active BRAF (#B08-11BG), active BRAF V600E (#B08-12G), active RAF1 (EE) (#R01-13G), corresponding RAF substrate inactive MEK1 (#M02-14BG), as well as active MEK1 (#M02-10G), active MEK2 (#M03-10G), inactive ERK1 protein (#M29-14G), and inactive ERK2 protein (#M28-14U) were obtained from Signal Chem.

Techniques: In Vivo, Expressing, Immunofluorescence, Activation Assay, Mutagenesis, Inhibition, In Vitro

Journal: British Journal of Cancer

Article Title: The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

doi: 10.1038/bjc.2012.70

Figure Lengend Snippet: Growth inhibition induced by the PI3K inhibitors, NVP-BEZ235 or GDC-0941, and the MEK inhibitors, AZD6244 or PD0325901, alone and in combination. HCT116 and HT29 cells were treated with the indicated fractions of the GI 50 concentrations of the inhibitors, alone or in combination, derived from , for 72 h, and an SRB assay was subsequently performed. Data are presented as a percentage of the control, in which cells were treated with 0.5% (v/v) DMSO. Points represent the mean of three independent experiments±standard error. Lines were fitted using non-linear regression analysis.

Article Snippet: HCT116 (KRas mutant, PIK3CA mutant), DLD1 (KRas mutant, PIK3CA mutant) and HT29 (BRaf mutant, PIK3CA mutant) human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U ml −1 )–streptomycin (50 mg ml −1 ) and 2 m M L -glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert (Cambrex, Charles City, IA, USA).

Techniques: Inhibition, Derivative Assay, Sulforhodamine B Assay, Control

Journal: British Journal of Cancer

Article Title: The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

doi: 10.1038/bjc.2012.70

Figure Lengend Snippet: Effect of the MEK inhibitors, PD0325901 and AZD6244, as single agents on MAPK signal transduction and the PI3K inhibitors, NVP-BEZ235 and GDC-0941, as single agents on PI3K/AKT signal transduction. HCT116 and HT29 cells were treated with the indicated concentrations of the inhibitors, derived from , for 24 h. Cell lysates were subjected to electrophoresis, followed by western blotting using the indicated phospho-specific antibodies. Blots were then stripped and reprobed with the corresponding total antibody to confirm equal protein loading. Data shown are representative of ⩾3 independent experiments.

Article Snippet: HCT116 (KRas mutant, PIK3CA mutant), DLD1 (KRas mutant, PIK3CA mutant) and HT29 (BRaf mutant, PIK3CA mutant) human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U ml −1 )–streptomycin (50 mg ml −1 ) and 2 m M L -glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert (Cambrex, Charles City, IA, USA).

Techniques: Transduction, Derivative Assay, Electrophoresis, Western Blot

Journal: British Journal of Cancer

Article Title: The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

doi: 10.1038/bjc.2012.70

Figure Lengend Snippet: Summary of the effect of the PI3K inhibitors, NVP-BEZ235 or GDC-0941, in combination with the MEK inhibitor PD0325901 on protein phosphorylation

Article Snippet: HCT116 (KRas mutant, PIK3CA mutant), DLD1 (KRas mutant, PIK3CA mutant) and HT29 (BRaf mutant, PIK3CA mutant) human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U ml −1 )–streptomycin (50 mg ml −1 ) and 2 m M L -glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert (Cambrex, Charles City, IA, USA).

Techniques: Phospho-proteomics, Inhibition

Journal: British Journal of Cancer

Article Title: The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

doi: 10.1038/bjc.2012.70

Figure Lengend Snippet: Effect of the PI3K/mTOR inhibitor NVP-BEZ235 or the PI3K inhibitor GDC-0941, mixed at an equipotent concentration with the mTORC1/2 inhibitor KU0063794 in combination with the MEK inhibitor, PD0325901. ( A ) Growth inhibition. HCT116 and HT29 cells were treated with the indicated fractions of the GI 50 concentration of the mixed PI3K and mTOR inhibitors (data not shown) and the GI 50 concentration of the MEK inhibitor (calculated from ), alone or in combination for 72 h, and an SRB assay was subsequently performed. Data are presented as a percentage of the control, in which cells were treated with 0.5% (v/v) DMSO. Points represent the mean of three independent experiments±standard error and lines were fitted using non-linear regression analysis. ( B ) Interaction of the combinations. Median effect analysis (CalcuSyn software) was used to evaluate the interaction between the inhibitor combinations. Points represent the mean of three independent experiments±standard error. Horizontal dotted lines indicate the boundaries for each interaction classification.

Article Snippet: HCT116 (KRas mutant, PIK3CA mutant), DLD1 (KRas mutant, PIK3CA mutant) and HT29 (BRaf mutant, PIK3CA mutant) human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U ml −1 )–streptomycin (50 mg ml −1 ) and 2 m M L -glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert (Cambrex, Charles City, IA, USA).

Techniques: Concentration Assay, Inhibition, Sulforhodamine B Assay, Control, Software

Journal: British Journal of Cancer

Article Title: The synergistic interaction of MEK and PI3K inhibitors is modulated by mTOR inhibition

doi: 10.1038/bjc.2012.70

Figure Lengend Snippet: Relationship between the PI3K inhibitor (GDC-0941): mTORC1/2 inhibitor (KU0063794) concentration ratio and synergy with the MEK inhibitor PD0325901 in the HCT116, DLD1 and HT29 cell lines. Cells were treated with the PI3K inhibitor GDC-0941 alone, the mTORC1/2 inhibitor KU0063794 alone or the indicated ratios of the two inhibitors together, in combination with the MEK inhibitor PD0325901 for 72 h, and an SRB assay was subsequently performed. Median effect analysis (CalcuSyn software) was used to evaluate the interaction between the inhibitor combinations. Points represent the mean of the combination index at 1 × the GI 50 concentration for the HCT116 and HT29 cell lines, and 0.5 × the GI 50 concentration for the DLD1 cell line, of three independent experiments±standard error. Correlation ( r ) and significance ( P ) were calculated using two-tailed Spearman's Rank Correlation analysis.

Article Snippet: HCT116 (KRas mutant, PIK3CA mutant), DLD1 (KRas mutant, PIK3CA mutant) and HT29 (BRaf mutant, PIK3CA mutant) human colorectal cancer cells were obtained from the American Type Culture Collection, were grown in RPMI-1640 medium (supplemented with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin (50 U ml −1 )–streptomycin (50 mg ml −1 ) and 2 m M L -glutamine) and confirmed free of mycoplasma contamination by regular testing with Mycoalert (Cambrex, Charles City, IA, USA).

Techniques: Concentration Assay, Sulforhodamine B Assay, Software, Two Tailed Test