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: 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: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: EGF induces the ERK/AKT/mTOR signaling pathways and YB-1 phosphorylation but reduces CXCL14 mRNA expression. (a, b) LNCap cells were treated with or without rhEGF and the AKT inhibitor LY294002, the ERK pathway inhibitor PD98059 or the mTOR pathway inhibitor Rapamycin. AKT-mTOR pathway and ERK pathway related proteins, YB-1, phospho-YB-1, cyclinD1 and cleaved PARP were assessed using WB analysis. (c) CXCL14 and YB-1 mRNA expression levels were measured by qRT-PCR after treatment of LNCap cells with or without rhEGF. A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments: *P < 0.05, **P < 0.01

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques: Protein-Protein interactions, Phospho-proteomics, Expressing, Quantitative RT-PCR

Journal: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: Effects of YB-1 and CXCL14 silencing on cell cycle and apoptosis. (a) LNCAP cells were cultured for 48 h with scrambled siRNA and YB-1 or CXCL14 siRNA, stained with propidium iodide and analyzed for cell cycle by flow cytometry. (b) Cells transfected as in (a) were harvested after 48 h and analyzed for apoptosis by flow cytometry. Percentages correspond to the proportion of cells positive for each profile

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques: Cell Culture, Staining, Flow Cytometry, Transfection

Journal: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: CXCL14 expression is inversely correlated with YB-1. (a) YB-1 and CXCL14 mRNA expression levels were examined by qRT-PCR after knockdown of YB-1. (b) Western blot analysis was performed to assess the expression of YB-1 after knockdown of CXCL14 in LNCap cells; GAPDH served as the internal control. A representative image of at least three independent experiments with similar results is shown. Each value represents the mean ± SD (bars) of three independent experiments: *P < 0.05

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Western Blot, Control

Journal: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: Quantification of YB-1, EGFR and CXCL14 in human prostate cancer tissues. YB-1 was weakly expressed in low Gleason score samples, while CXCL14 and EGFR were positively expressed in the cytoplasm. In high Gleason score samples, the tumor cells were solid without an adenoid structure. YB-1 and EGFR were positively expressed in the nucleus and cytoplasm, respectively, while CXCL14 was negatively expressed in these tissues

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques:

Journal: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: Correlation of YB-1, EGFR and CXCL14 expression with clinicopathological features in prostate cancer cases

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques: Expressing

Journal: Bioengineered

Article Title: The roles of Y-box-binding protein (YB)-1 and C-X-C motif chemokine ligand 14 (CXCL14) in the progression of prostate cancer via extracellular-signal-regulated kinase (ERK) signaling

doi: 10.1080/21655979.2021.1993537

Figure Lengend Snippet: Correlation between overall survival of prostate cancer patients in YB-1, EGFR and CXCL14 expressing tissues. (a) The overall survival of prostate cancer patients with a high expression level of YB-1 was much lower than that of patients with a low level of YB-1(P = 0.0116); (b-d) There was no significant difference in the overall survival of prostate cancer patients with EGFR or CXCL14 expression levels or with Gleason score

Article Snippet: TaqMan probes for YB-1 (Hs00898625_g1), CXCL14 (Hs00171135_m1) and ACTB (Hs99999903_m1) were used for qRT-PCR.

Techniques: Expressing

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A) Dot plots comparing pre-therapy and on-therapy CD45 (PTPRC) mRNA levels in melanoma patients treated with BRAFi or BRAF/MEKi. Matched patient sample size of n = 6. Statistics were calculated using a two-tailed paired t-test. (B) Average tumor volume curves in response to daily BRAF/MEKi in YUMM1.7 melanoma-bearing NSG mice. Tumor volumes were measured daily. (n = 5-6 tumors, mean ± SEM) (C) Schematic illustrating the different phases of syngeneic melanoma C57BL6/J mouse models. BRAF/MEKi was administered by daily oral gavage when tumors reached ∼700mm3 in size. Growing tumor: 3-day vehicle control treatment. Regressing tumor: 3-day BRAF/MEKi treatment. Residual disease: 14-day BRAF/MEKi treatment. Resistant tumor: BRAF/MEKi treatment until the rebounding tumor reaches its initial size. Created with BioRender.com. (D) Average tumor volume curves in response to daily BRAF/MEKi in different melanoma C57BL6/J models. Tumor volumes were measured daily. (n = 3-5 tumors, mean ± SEM) (E) Representative flow cytometry plots of CD45+ cells gated on live cells in different phases in BRAF/MEKi-treated C57BL/6J mice bearing YUMM1.7 (growing tumors were treated with vehicle control). The numbers in the plots represent the percentage of cells within each gate. (F-H) Quantification of CD45+ cell infiltration in different phases in BRAF/MEKi-treated C57BL/6J mice bearing YUMM1.7 (D), YUMMUV1.7 (E), and YUMMUV3.3 (F) (growing tumors were treated with vehicle control). YUMMUV3.3 tumors did not exhibit profound regressing phase as YUMM1.7 and YUMMUV1.7 but the collection timepoint was after 3-day BRAF/MEKi, consistent with timeline defined in (C). (n = 3-9 tumors, one-way ANOVA with Tukey’ s multiple comparisons test, mean ± SEM)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Two Tailed Test, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A) Schematic showing immune cell types, including macrophages, CD8+ T cells, and NK cells, that may contribute to the BRAF/MEKi tumor response. Created with BioRender.com. (B and C) Depletion efficiencies of clodronate liposomes in tumors at the endpoint (full resistance). Quantification of F4/80-high macrophages (B) and F4/80-low macrophages (C). (n = 4-6 tumors, two-tailed unpaired t-test, mean ± SEM) (D) Kaplan-Meier curve for BRAF/MEKi-treated mice bearing YUMM1.7 receiving clodronate liposomes (CL) or PBS liposomes (PL). (n = 4-6 tumors, Log-rank Mantel-Cox test) (E and F) Depletion efficiencies of the anti-CD8b antibody. Quantification of circulating CD8+ T cells six days post anti-CD8b antibody injection (E) and tumor-infiltrating CD8+ T cells at the endpoint (full resistance) (F). (n = 4 tumors, two-tailed unpaired t-test, mean ± SEM) (G) Kaplan-Meier curve for BRAF/MEKi-treated mice bearing YUMM1.7 receiving anti-CD8b antibody or isotype control. (n = 4 tumors, Log-rank Mantel-Cox test) (H) Depletion efficiency of the anti-ASGM1 antibody in tumors at the endpoint (full resistance). Representative flow cytometry plots and quantification of tumor-infiltrating NK cells. (n = 4-5 tumors, two-tailed unpaired t-test, mean ± SEM) (I) Bar plot showing best response (%) of BRAF/MEKi-treated male mice bearing YUMM1.7 receiving the anti-ASGM1 antibody or rabbit serum. (n = 5 tumors, two-tailed unpaired t-test, mean ± SEM) (J and K) Kaplan-Meier curve for BRAF/MEKi-treated male and female mice bearing YUMM1.7 (J) or YUMMUV1.7 (K) tumors receiving the anti-ASGM1 antibody or rabbit serum. (n = 4-5 tumors, Log-rank Mantel-Cox test)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Liposomes, Two Tailed Test, Injection, Control, Flow Cytometry

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A) Bar plot showing tumor volume change (%) of different phases in BRAF/MEKi-treated mice bearing YUMM1.7 (growing tumors were treated with vehicle control). (n = 5-8 tumors, mean ± SEM) (B) Quantification of NK cell infiltration in different phases in BRAF/MEKi-treated mice bearing YUMM1.7 (growing tumors were treated with vehicle control). (n = 4-9 tumors, one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM) (C) Correlation between NK cell infiltrate in YUMM1.7 tumors and therapy response to BRAF/MEKi. (Simple linear regression) (D) Representative immunofluorescence images of NK1.1 (red), CD3 (green) and nuclear DAPI (blue) of BRAF/MEKi-treated tumors in the indicated treatment groups. NK cells are NK1.1+CD3-. Scale bar 100 μm. (E) Scatter plot showing the NK cell signature between regressing tumors and residual disease. NK cell activation and inhibitory marker genes are shown in orange and green, respectively. (F and G) Violin plot showing the expression of activation (F) and inhibitory (G) marker genes in NK cells from regressing tumors and residual disease. (H) Quantification of CD8+ T cell infiltration in different phases in BRAF/MEKi-treated mice bearing YUMM1.7 (growing tumors were treated with vehicle control). (n = 4-9 tumors, one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM) (I) Correlation between CD8+ T cell infiltration and therapeutic response in YUMM1.7 tumors treated with BRAF/MEKi. (Simple linear regression) (J) Schematic illustrating the NK cytotoxicity assay. Created with BioRender.com. (K) Bar plot showing % NK cytotoxicity to YUMM1.7-GFP enriched from regressing tumors and residual disease at effector-to-target (E: T) ratios of 3:1 and 1:1. (n = 3 tumors, conducted in two independent experiments, multiple unpaired t-tests, mean ± SEM)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Control, Immunofluorescence, Activation Assay, Marker, Expressing, Clinical Proteomics, Cytotoxicity Assay

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A and B) Representative immunofluorescence images of F4/80 (green), NK1.1 (red), and nuclear DAPI (blue) in regressing tumors and residual disease treated with BRAF/MEKi (A). Scale bar, 100 μm, 10 μm. Quantification of F4/80+ cells next to NK1.1+ cells (B). (n = 3 tumors with 4-11 total microscopy fields analyzed across all tissues, at least 1,600 counted NK1.1+ cells per tumor, two-tailed unpaired t-test, mean ± SEM) (C and D) Depletion efficiencies with diphtheria toxin (DT) (LysM-cre;iDTR) in tumors during tumor regression. Quantification of F4/80-high macrophages (C) and F4/80-low macrophages (D). (n = 5-6 tumors, two-tailed unpaired t-test, mean ± SEM) (E) Quantification of tumor-infiltrating NK cells in BRAF/MEKi-treated mice receiving diphtheria toxin (DT) (LysM-cre;iDTR) or control (NaCl in LysM-cre;iDTR or DT in WT mice) during tumor regression. (n = 6-7 tumors, two-tailed unpaired t-test, mean ± SEM) (F and G) Violin plot showing the expression of Lyz2 (F) and Adgre1 (G) in each macrophage cluster. Mo/Mϕ, monocyte/macrophage. (H—J) Quantification of MHC II+ (H), CCL5+ (I), or CD63+ (J) F4/80-high macrophages in BRAF/MEKi-treated mice receiving diphtheria toxin (DT) (LysM-cre;iDTR) or control (NaCl in LysM-cre;iDTR) during tumor regression. (n = 9 tumors, two-tailed unpaired t-test, mean ± SEM) (K) Heatmap depicting the CCL signaling network among different immune cell types during tumor regression, analyzed using CellChat. Mo/Mϕ, monocyte/macrophage. (L) Selected DEGs of multiple Ccl genes in macrophage Clusters 0 and 3. Mo/Mϕ, monocyte/macrophage. (M) Bubble plot showing ligand-receptor-based CellChat analysis during tumor regression, with macrophages as senders and NK cells as receivers. Mo/Mϕ, monocyte/macrophage. (N) Schematic illustrating experimental design for analysis of NK cells migrated toward macrophage-derived conditioned media. Mϕ, macrophage. Created with BioRender.com (O) Bar chart showing the migrated NK cell ratio in the transwell assay using conditioned media derived from macrophages isolated from regressing tumors or residual disease and primary splenocytes treated with a CCR2/5 inhibitor (CVC) or DMSO. CVC, cenicriviroc. (n = 5-8 tumors, conducted in two independent experiments, one-way ANOVA with Tukey’s multiple comparisons test, mean ± SEM)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Immunofluorescence, Microscopy, Two Tailed Test, Control, Expressing, Derivative Assay, Transwell Assay, Isolation

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A) Violin plot showing Ptpn22 expression in each immune cell type. Mo/Mϕ, monocyte/macrophage. (B) Schematic illustrating experimental design for targeting Ptpn22 in YUMM1.7-bearing mice. Created with BioRender.com. (C) Quantification of tumor-infiltrating NK cells in BRAF/MEKi-treated mice receiving L-1 or vehicle control on day 13. (n = 9 tumors, two-tailed unpaired t-test, mean ± SEM) (D) Representative immunofluorescence images of NK1.1 (red), CD3 (green) and nuclear DAPI (blue) of BRAF/MEKi-treated tumors in the indicated treatment groups on day 13. NK cells are NK1.1+CD3-. Scale bar 100 μm. (E and F) Quantification of tumor-infiltrating F4/80-high (E) or F4/80-low (F) macrophages in BRAF/MEKi-treated mice receiving L-1 or vehicle control on day 13. (n = 6-7 tumors, two-tailed unpaired t-test, mean ± SEM) (G—I) Quantification of tumor-infiltrating Ccl5+ (G), Cd63+ (H), or MHC II+ (I) F4/80-high macrophages in BRAF/MEKi-treated mice receiving L-1 or vehicle control on day 13. (n = 6-7 tumors, two-tailed unpaired t-test, mean ± SEM) (J—N) Violin plot showing the expression of Adgre1 (J), Ccl5 (K), Cd63 (L), and H2-Ab1/Eb1 (M and N) in the monocyte/macrophage population between regressing tumors and residual disease. REG, regressing; RD, residual disease. (O) Kaplan-Meier curve for BRAF/MEKi-treated mice bearing YUMM1.7 receiving L-1 or vehicle control. (n = 5-6 tumors, Log-rank Mantel-Cox test)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Expressing, Control, Two Tailed Test, Immunofluorescence

Journal: bioRxiv

Article Title: Innate Immune Remodeling Drives Therapy Resistance via Macrophage–NK Cell Crosstalk

doi: 10.1101/2025.07.22.666055

Figure Lengend Snippet: (A) Schematic illustrating the timeline and response to BRAF/MEKi for four melanoma patients in GSE229908. Each dot represents one FNA biopsy. Grey represents baseline (before BRAF/MEKi), blue represents responding tumors, and red represents BRAF/MEKi-resistant tumors. (B) UMAP plot showing the cell populations of the four melanoma patients. (C) Bar plots showing NK cell quantification at each FNA timepoint in four melanoma patients. (D) Quantification of NK cells in FNA biopsies grouped into Baseline (before BRAF/MEKi treatment), Responding, and Resistant groups. (mean ± SEM) (E—G) Violin plots showing the expression of exhaustion markers (E), activation markers (F), or RAB27A (G) in the NK cell population in Patient 2 (Resistant), 3 (Responding) and 4 (Partial Responding). (H) Bar plot showing NK cell fractional changes across three treatment states in PRJNA591860, containing 49 lung cancer biopsies before and during TKI treatment. (I) Schematic illustrating the timeline and response to TKI treatment in three lung cancer patients with sequential biopsies in PRJNA591860. Each dot represents one FNA biopsy. Grey represents naïve (before TKI treatment), blue represents responding tumors, and red represents resistant tumors. (J) Bar plots showing NK cell fractional changes at each FNA timepoint in three lung cancer patients. (K) NK fractional changes in FNA biopsies from the three patients grouped into Naïve, Responding, and Resistant groups. (mean ± SEM)

Article Snippet: Mice received BRAF/MEKi therapy at a dose of 25 mg/kg dabrafenib (Medchem Express, catalog no. HY-14660) and 0.15 mg/kg trametinib (Medchem Express, catalog no. HY-10999).

Techniques: Expressing, Activation Assay