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New England Biolabs point mutations
Point Mutations, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 97/100, based on 648 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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point mutations - by Bioz Stars, 2026-06

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Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Diagram depicting different MRTFA partners that can promote cortical stiffness in cancer cells. (B) Brightfield images of E0771 cells overexpressing MRTFA WT treated with DMSO (vehicle only control), TGFβ inhibitor (SB-505124; 2.5 µM) or YAP inhibitor (Verteporfin; 10 µM) for 24 hrs. Brightfield images of E0771 cells overexpressing MRTFA WT in the absence or presence of shRNA mediated SRF knockdown (shSRF). Scale bars: 50 µm. (C) Relative transcript levels of downstream MRTFA target genes, Acta2 and Myh11 . Data are shown as fold change calculated from 3 technical replicates per sample and normalized GAPDH. Each graph is representative of 3 independent experiments. (D) Confocal images of representative cells stained with phalloidin (F-actin). Scale bar = 10 µm. (E) Anisotropy measurements for cell lines in (D). Each graph is a compilation of 3 independent experiments; AT-3: pRetroX = 95 cells, MRTFA WT = 95 cells, MRTFA Y238A = 92 cells; E0771: Control (pRetroX) = 165 cells, MRTFA WT =112 cells, MRTFA Y238A =158 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (F) Raw AFM stiffness measurements comparing MRTFA WT and MRTFA Y238A compared to control cells. Each graph is a compilation of 2 independent experiments. AT-3: pRetroX = 19 cells, MRTFA WT = 19 cells, MRTFA Y238A = 19 cells; E0771: Control (pRetroX) = 15 cells, MRTFA WT = 16 cells, MRTFA Y238A = 15 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001. (G) Raw AFM stiffness measurements comparing SRF knockdown effect on MRTFA WT cell stiffness. Results are combined from 2 independent experiments. E0771: Control (pRetroX) = 10 cells, MRTFA WT + Control (pLKO) = 11 cells, MRTFA WT + shSRF=10 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; * P <0.05; ** P <0.01; *** P <0.001.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Control, shRNA, Knockdown, Staining

(A) Immunoblot of YAP in E0771 MRTFA WT cells treated with 10 µM verteporfin for 24 hours compared to DMSO control. GAPDH is used as loading control. (B) Immunoblot of SMAD phosphorylation in E0771 MRTFA WT cells treated with 2.5ng/mL TGFß2 for 1 hour in the presence or absence of 2.5 µM SB-505124 pre-treatment. GAPDH is used as loading control. (C) MRTFA and MRTFB amino acid alignment around the conserved Y238 and Y305 residues, respectively. (D) Immunoblot of MRTFA overexpression in MRTFA WT and MRTFA Y238A cells compared to control. GAPDH is used as loading control. * indicates endogenous MRTFA. (E) Relative transcript levels of actin-related established MRTFA target genes Actg2 and Myh11 in MRTFA WT and MRTFA Y238A mutant overexpressing cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Each graph is representative of 3 independent experiments. (F) Representative immunofluorescent images depicting MRTFA protein localization in MRTFA WT and MRTFA Y238A mutant overexpressing cell lines. Scale bars = 50 µm. (G) Quantitation of MRTFA protein localization in MRTFA WT and mutant overexpressing cell lines. AT-3: Control (pRetroX) = 298 cells, MRTFA WT = 140 cells, MRTFA Y238A = 169 cells; E0771: Control (pRetroX) = 185 cells, MRTFA WT = 315 cells, MRTFA Y238A = 232 cells. (H) Relative transcript levels of SRF in E0771 MRTFA WT cell lines with/without SRF knockdown (shSRF). Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Graph is representative of 3 independent experiments.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Immunoblot of YAP in E0771 MRTFA WT cells treated with 10 µM verteporfin for 24 hours compared to DMSO control. GAPDH is used as loading control. (B) Immunoblot of SMAD phosphorylation in E0771 MRTFA WT cells treated with 2.5ng/mL TGFß2 for 1 hour in the presence or absence of 2.5 µM SB-505124 pre-treatment. GAPDH is used as loading control. (C) MRTFA and MRTFB amino acid alignment around the conserved Y238 and Y305 residues, respectively. (D) Immunoblot of MRTFA overexpression in MRTFA WT and MRTFA Y238A cells compared to control. GAPDH is used as loading control. * indicates endogenous MRTFA. (E) Relative transcript levels of actin-related established MRTFA target genes Actg2 and Myh11 in MRTFA WT and MRTFA Y238A mutant overexpressing cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Each graph is representative of 3 independent experiments. (F) Representative immunofluorescent images depicting MRTFA protein localization in MRTFA WT and MRTFA Y238A mutant overexpressing cell lines. Scale bars = 50 µm. (G) Quantitation of MRTFA protein localization in MRTFA WT and mutant overexpressing cell lines. AT-3: Control (pRetroX) = 298 cells, MRTFA WT = 140 cells, MRTFA Y238A = 169 cells; E0771: Control (pRetroX) = 185 cells, MRTFA WT = 315 cells, MRTFA Y238A = 232 cells. (H) Relative transcript levels of SRF in E0771 MRTFA WT cell lines with/without SRF knockdown (shSRF). Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Graph is representative of 3 independent experiments.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Western Blot, Control, Phospho-proteomics, Over Expression, Mutagenesis, Quantitation Assay, Knockdown

(A) Venn diagram representing the location of the putative stiffness genes in AT-3 and E0771 cell lines where they are exclusively upregulated at least 2-fold by MRTFA WT expression. (B) Gene Ontology analysis of putative “stiffness genes” from A. See Table S1 for full GO term enrichment sets. (C) GSEA analysis performed on patient data from TCGA PanCancer Atlas of invasive breast cancer by using “monoatomic”, “channel” or “transport” keyword containing gene signatures. Patients’ samples were allocated to MRTFA high or MRTFA low categories based on the median MRTFA expression value. (D) Same GSEA analysis as (C), except patients’ samples were allocated to SRF high versus SRF low categories based on the median SRF expression value. (E) Heat map of bulk RNA-seq data showing top ion-associated genes upregulated in AT-3 or E0771 MRTFA WT cells compared to MRTFA Y238A . (F) mRNA expression values of all genes in (E) accessed through cBioportal in MRTFA high/low and SRF high/low patients in TCGA. Multiple hypothesis testing correction for statistical significance and the resulting q values were calculated by cBioportal. See Table S3 for all genes. (G) Relative transcript levels of Kcnmb1 in MRTFA WT and MRTFA Y238A cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Each graph is representative of 3 independent experiments. (H-I) Relative transcript levels of KCNMB1 in mouse and human shMRTFA/B and shSRF cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized GAPDH. Each graph is representative of 3 independent experiments.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Venn diagram representing the location of the putative stiffness genes in AT-3 and E0771 cell lines where they are exclusively upregulated at least 2-fold by MRTFA WT expression. (B) Gene Ontology analysis of putative “stiffness genes” from A. See Table S1 for full GO term enrichment sets. (C) GSEA analysis performed on patient data from TCGA PanCancer Atlas of invasive breast cancer by using “monoatomic”, “channel” or “transport” keyword containing gene signatures. Patients’ samples were allocated to MRTFA high or MRTFA low categories based on the median MRTFA expression value. (D) Same GSEA analysis as (C), except patients’ samples were allocated to SRF high versus SRF low categories based on the median SRF expression value. (E) Heat map of bulk RNA-seq data showing top ion-associated genes upregulated in AT-3 or E0771 MRTFA WT cells compared to MRTFA Y238A . (F) mRNA expression values of all genes in (E) accessed through cBioportal in MRTFA high/low and SRF high/low patients in TCGA. Multiple hypothesis testing correction for statistical significance and the resulting q values were calculated by cBioportal. See Table S3 for all genes. (G) Relative transcript levels of Kcnmb1 in MRTFA WT and MRTFA Y238A cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH. Each graph is representative of 3 independent experiments. (H-I) Relative transcript levels of KCNMB1 in mouse and human shMRTFA/B and shSRF cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized GAPDH. Each graph is representative of 3 independent experiments.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Expressing, RNA Sequencing

(A) Venn diagram and gene ontology analyses of the SRF independent genes that are exclusively upregulated by MRTFA Y238A in both AT-3 and E0771 cells to at least 2-fold or more. See Table S2 for full results. (B) Examples of statistically significant GSEA for MRTFA high/low breast cancer patient tumors from TCGA. (C) Examples of statistically significant GSEA for SRF high/low breast cancer patient tumors from TCGA. (D) Relative mRNA expression levels of MRTFA and MRTFB in shMRTFA/B and SRF in shSRF mouse cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH levels. Each graph is representative of 3 independent experiments. (E) Relative mRNA expression levels of MRTFA and MRTFB in shMRTFA/B and SRF in shSRF human cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH levels. Each graph is representative of 3 independent experiments. (F) Relative mRNA expression levels of canonical YAP target gene CCN2, which encodes connective tissue growth factor (CTGF), and KCNMB1 in AT-3 cells overexpressing wildtype YAP or hyperactive YAP 5SA mutant or treated for 24 hours with TGFβ inhibitor SB-505124. (G) KCNMB1 human and mouse genetic loci analyzed for SRF (purple tracks) binding by using University of California Santa Cruz Genome Browser data publicly available Chromatin immunoprecipitation sequencing data. Each purple solid line under the ReMap density map indicates an independent dataset where SRF was found to be associated with the locus. In the Encode cCREs track, red solid boxes indicate promoter regions, orange and yellow solid boxes indicate proximal and distal enhancers, respectively, and blue tracks indicate CTCF only binding regions. (H) Chromatin immunoprecipitation-PCR from AT-3 cells using KCNMB1 primers and the indicated antibodies against SRF, MRTFA, histone H3 (as a positive control) and IgG (negative control). Water was used as input material as a negative control for the PCR reaction. Image is representative of 3 independent experiments. (I) Raw counts from bulk RNA-seq data for Myocd , Mrtfa , and Mrtfb genes in AT-3 and E0771 cells.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Venn diagram and gene ontology analyses of the SRF independent genes that are exclusively upregulated by MRTFA Y238A in both AT-3 and E0771 cells to at least 2-fold or more. See Table S2 for full results. (B) Examples of statistically significant GSEA for MRTFA high/low breast cancer patient tumors from TCGA. (C) Examples of statistically significant GSEA for SRF high/low breast cancer patient tumors from TCGA. (D) Relative mRNA expression levels of MRTFA and MRTFB in shMRTFA/B and SRF in shSRF mouse cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH levels. Each graph is representative of 3 independent experiments. (E) Relative mRNA expression levels of MRTFA and MRTFB in shMRTFA/B and SRF in shSRF human cell lines. Data are shown as fold change calculated from 3 technical replicates per sample and normalized to GAPDH levels. Each graph is representative of 3 independent experiments. (F) Relative mRNA expression levels of canonical YAP target gene CCN2, which encodes connective tissue growth factor (CTGF), and KCNMB1 in AT-3 cells overexpressing wildtype YAP or hyperactive YAP 5SA mutant or treated for 24 hours with TGFβ inhibitor SB-505124. (G) KCNMB1 human and mouse genetic loci analyzed for SRF (purple tracks) binding by using University of California Santa Cruz Genome Browser data publicly available Chromatin immunoprecipitation sequencing data. Each purple solid line under the ReMap density map indicates an independent dataset where SRF was found to be associated with the locus. In the Encode cCREs track, red solid boxes indicate promoter regions, orange and yellow solid boxes indicate proximal and distal enhancers, respectively, and blue tracks indicate CTCF only binding regions. (H) Chromatin immunoprecipitation-PCR from AT-3 cells using KCNMB1 primers and the indicated antibodies against SRF, MRTFA, histone H3 (as a positive control) and IgG (negative control). Water was used as input material as a negative control for the PCR reaction. Image is representative of 3 independent experiments. (I) Raw counts from bulk RNA-seq data for Myocd , Mrtfa , and Mrtfb genes in AT-3 and E0771 cells.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Expressing, Mutagenesis, Binding Assay, ChIP-sequencing, Chromatin Immunoprecipitation, Positive Control, Negative Control, RNA Sequencing

(A and B) Top: Confocal images of representative cells loaded with IPG-1 AM K + binding dye. Scale bar = 10 µm. Bottom: Graphs showing mean fluorescence intensity of intracellular K + bound dye. Each graph is representative of 3 independent experiments; AT-3: Control (pRetroX) = 261 cells, MRTFA WT = 196 cells, MRTFA Y238A = 291 cells, Control (pLKO) = 125 cells, shMRTFA/B=134 cells, shSRF = 140 cells; E0771: Control (pRetroX) = 353 cells, MRTFA WT = 258 cells, MRTFA Y238A = 362 cells, Control (pLKO) = 147 cells, shMRTFA/B = 119 cells, shSRF = 132 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A and B) Top: Confocal images of representative cells loaded with IPG-1 AM K + binding dye. Scale bar = 10 µm. Bottom: Graphs showing mean fluorescence intensity of intracellular K + bound dye. Each graph is representative of 3 independent experiments; AT-3: Control (pRetroX) = 261 cells, MRTFA WT = 196 cells, MRTFA Y238A = 291 cells, Control (pLKO) = 125 cells, shMRTFA/B=134 cells, shSRF = 140 cells; E0771: Control (pRetroX) = 353 cells, MRTFA WT = 258 cells, MRTFA Y238A = 362 cells, Control (pLKO) = 147 cells, shMRTFA/B = 119 cells, shSRF = 132 cells; Kruskal-Wallace with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Binding Assay, Fluorescence, Control

(A-B) Left: Ratiometric images of representative cells loaded with Fluo-4 and FuraRed Ca 2+ binding dyes. FuraRed signal indicates Ca 2+ -free dye loading and Fluo-4 indicates Ca 2+ -bound dye. Scale bar = 10 µm. Right: Graphs showing mean fluorescence intensity ratio of Fluo-4 and Fura Red. Each graph is representative of 3 independent experiments; AT-3: Control (pRetroX) = 415 cells, MRTFA WT = 862 cells, MRTFA Y238A = 670 cells, Control (pLKO) = 77 cells, shMRTFA/B = 924 cells, shSRF = 776 cells; E0771: Control (pRetroX) = 367 cells, MRTFA WT = 303 cells, MRTFA Y238A = 238 cells, Control (pLKO) = 140 cells, shMRTFA/B = 108 cells, shSRF = 190 cells; Kruskal-Wallace test with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (C) Schematic of different Ca 2+ chelating agents their site of extra- and intra-cellular action. (D) AFM stiffness measurements of AT-3 and E0771 MRTFA WT cells treated with EDTA or BAPTA-AM compared to DMSO control groups. Results are from 3 independent experiments combined. AT-3: Control (water as a control for EDTA) = 24 cells, EDTA = 23 cells, DMSO (Control for BAPTA-AM) = 22 cells, BAPTA-AM = 20 cells; E0771: Control (water) = 15 cells, EDTA=15 cells, DMSO (Control for BAPTA-AM) = 8 cells, BAPTA-AM = 9 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (E) Confocal images of representative AT-3 and E0771 MRTFA WT overexpressing cells treated with EDTA or BAPTA-AM compared to controls and stained with phalloidin (F-actin). Scale bar=10 µm. (F) Anisotropy measurements of cells in (E). Each graph is representative of 2 independent experiments per cell type and treatment. AT-3: Control (water as a control for EDTA) = 31 cells, EDTA=31 cells, DMSO (Control for BAPTA-AM) = 32 cells, BAPTA-AM = 35 cells; E0771: Control (water) = 46 cells, EDTA = 73 cells, DMSO (Control for BAPTA-AM) = 40 cells, BAPTA-AM = 43 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (G) Anisotropy measurements of unmodified AT-3 and E0771 cells treated with EDTA or BAPTA, compared to respective controls. Each graph is representative of 2 independent experiments per cell type and treatment. AT-3: Control (water as a control for EDTA) = 77 cells, EDTA=74 cells, DMSO (Control for BAPTA-AM) = 76 cells, BAPTA-AM=89 cells; E0771: Control (water) = 93 cells, EDTA = 104 cells, DMSO (Control for BAPTA-AM) = 84 cells, BAPTA-AM = 95 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (H) Representative images of AT-3 and E0771 cells treated with EDTA or BAPTA-AM and stained with cell death marker propidium iodide (PI, red) during live culture. Arrow heads point to PI + cells that are never in high abundance under any condition. Images are representative of 3 independent experiments. Scale bar = 600 µm. (I) Left: Representative immunofluorescent images of MRTFA protein localization in cells treated with EDTA or BAPTA-AM, compared to respective controls. Scale bars= 50 µm. Right: Quantitation of MRTFA protein localization in cells as seen at left. Graphs are a compilation of 3 independent experiments. AT-3: Control (water as a control for EDTA) = 316 cells, EDTA = 69 cells, DMSO (Control for BAPTA-AM) = 357 cells, BAPTA-AM = 187 cells; E0771: Control (water) = 178 cells, EDTA = 72 cells, DMSO (Control for BAPTA-AM) = 234 cells, BAPTA-AM = 128 cells.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A-B) Left: Ratiometric images of representative cells loaded with Fluo-4 and FuraRed Ca 2+ binding dyes. FuraRed signal indicates Ca 2+ -free dye loading and Fluo-4 indicates Ca 2+ -bound dye. Scale bar = 10 µm. Right: Graphs showing mean fluorescence intensity ratio of Fluo-4 and Fura Red. Each graph is representative of 3 independent experiments; AT-3: Control (pRetroX) = 415 cells, MRTFA WT = 862 cells, MRTFA Y238A = 670 cells, Control (pLKO) = 77 cells, shMRTFA/B = 924 cells, shSRF = 776 cells; E0771: Control (pRetroX) = 367 cells, MRTFA WT = 303 cells, MRTFA Y238A = 238 cells, Control (pLKO) = 140 cells, shMRTFA/B = 108 cells, shSRF = 190 cells; Kruskal-Wallace test with multiple comparisons. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (C) Schematic of different Ca 2+ chelating agents their site of extra- and intra-cellular action. (D) AFM stiffness measurements of AT-3 and E0771 MRTFA WT cells treated with EDTA or BAPTA-AM compared to DMSO control groups. Results are from 3 independent experiments combined. AT-3: Control (water as a control for EDTA) = 24 cells, EDTA = 23 cells, DMSO (Control for BAPTA-AM) = 22 cells, BAPTA-AM = 20 cells; E0771: Control (water) = 15 cells, EDTA=15 cells, DMSO (Control for BAPTA-AM) = 8 cells, BAPTA-AM = 9 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (E) Confocal images of representative AT-3 and E0771 MRTFA WT overexpressing cells treated with EDTA or BAPTA-AM compared to controls and stained with phalloidin (F-actin). Scale bar=10 µm. (F) Anisotropy measurements of cells in (E). Each graph is representative of 2 independent experiments per cell type and treatment. AT-3: Control (water as a control for EDTA) = 31 cells, EDTA=31 cells, DMSO (Control for BAPTA-AM) = 32 cells, BAPTA-AM = 35 cells; E0771: Control (water) = 46 cells, EDTA = 73 cells, DMSO (Control for BAPTA-AM) = 40 cells, BAPTA-AM = 43 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (G) Anisotropy measurements of unmodified AT-3 and E0771 cells treated with EDTA or BAPTA, compared to respective controls. Each graph is representative of 2 independent experiments per cell type and treatment. AT-3: Control (water as a control for EDTA) = 77 cells, EDTA=74 cells, DMSO (Control for BAPTA-AM) = 76 cells, BAPTA-AM=89 cells; E0771: Control (water) = 93 cells, EDTA = 104 cells, DMSO (Control for BAPTA-AM) = 84 cells, BAPTA-AM = 95 cells; Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (H) Representative images of AT-3 and E0771 cells treated with EDTA or BAPTA-AM and stained with cell death marker propidium iodide (PI, red) during live culture. Arrow heads point to PI + cells that are never in high abundance under any condition. Images are representative of 3 independent experiments. Scale bar = 600 µm. (I) Left: Representative immunofluorescent images of MRTFA protein localization in cells treated with EDTA or BAPTA-AM, compared to respective controls. Scale bars= 50 µm. Right: Quantitation of MRTFA protein localization in cells as seen at left. Graphs are a compilation of 3 independent experiments. AT-3: Control (water as a control for EDTA) = 316 cells, EDTA = 69 cells, DMSO (Control for BAPTA-AM) = 357 cells, BAPTA-AM = 187 cells; E0771: Control (water) = 178 cells, EDTA = 72 cells, DMSO (Control for BAPTA-AM) = 234 cells, BAPTA-AM = 128 cells.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Binding Assay, Fluorescence, Control, MANN-WHITNEY, Staining, Marker, Quantitation Assay

(A) Schematic of the experimental approach to qualitatively assess relative amounts of extracellular potassium in the tumor microenvironment. (B) Representative image from a lung section showing accumulated K + in a metastatic lesion. Green triangle points to accumulated [K + ] e . Scale: 50µm. (C) Schematic showing elevated Lamp1+ granules at the immune synapse during antigen mediated cancer cell targeting by OT-I+ CD8+ Cytotoxic T lymphocytes. Graph on the right shows OVA dependent increase in Lamp1+ positivity as a measure of T-lymphocyte granulation under different conditions. Results are representative of 3 independent experiments. (D) Measurements of time to death of a cancer cell after it has been contacted 1:1 by a CD8 + T lymphocyte as determined by PI accumulation in the cancer cell. Data are compiled from 4 independent experiments with. P-values are calculated by Kruskal-Wallis test with multiple comparisons. Mean values are shown. ***:P<0.001, ****:P<0.0001. Control = 119 cells, high [K + ] e = 117 cells, BMS = 107 cells, high [K + ] e +BMS = 109 cells. (E) AFM stiffness measurements of representative cancer cells treated with high [K + ] e (45 mM KCl) compared to control. Results are from 2 combined experiments. AT-3: Control = 30 cells, high [K + ] e = 33 cells; E0771: Control = 34 cells, high [K + ] e = 37 cells. Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (F) AFM stiffness measurements of representative cancer cells treated with high [K + ] e and with/without 10 µM BMS-204352. Results are from 2 combined experiments. AT-3: high [K + ] e =25 cells, high [K + ] e +BMS = 21 cells; E0771: high [K + ] e = 20 cells, high [K + ] e +BMS = 20 cells. Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (G) Schematic depiction of how cortical F-actin influences surface roughness and the surface roughness measurements performed by AFM. Data is combined from 4 independent experiments. AT-3: Control = 28 cells, high [K + ] e = 28 cells, BMS = 24 cells, high [K + ] e +BMS = 24 cells, E0771: Control = 23 cells, high [K + ] e = 25 cells, BMS = 24 cells, high [K + ] e +BMS = 23 cells. P-values calculated by Mann-Whitney rank sum test. Mean+/-s.e.m are shown. RMS: root-mean square. (H) Cell height of cells used in (G) measured by AFM. (I) Venn diagrams showing the number of differentially regulated genes in high [K + ] e . The intersections represent the genes “rescued” which means the BMS-204352 treatment caused differential expression in the opposite direction. “Commonly rescued” genes are conserved across two different cell types. (J) Gene ontology analyses of “Commonly rescued” genes in (F). See Table S6 for a full list of GO terms that are enriched. (K) Working model: MRTFA/SRF upregulates the expression of F-actin bundling proteins and BK channel subunit, KCNMB1, for promoting cellular stiffness. Elevated cancer cell stiffness triggers mechanosurveillance by cytotoxic lymphocytes for inhibiting metastatic colonization.

Journal: bioRxiv

Article Title: Ionic Regulation of Mechanosurveillance and Metastasis via the MRTFA/KCNMB1 Axis

doi: 10.64898/2026.01.13.699089

Figure Lengend Snippet: (A) Schematic of the experimental approach to qualitatively assess relative amounts of extracellular potassium in the tumor microenvironment. (B) Representative image from a lung section showing accumulated K + in a metastatic lesion. Green triangle points to accumulated [K + ] e . Scale: 50µm. (C) Schematic showing elevated Lamp1+ granules at the immune synapse during antigen mediated cancer cell targeting by OT-I+ CD8+ Cytotoxic T lymphocytes. Graph on the right shows OVA dependent increase in Lamp1+ positivity as a measure of T-lymphocyte granulation under different conditions. Results are representative of 3 independent experiments. (D) Measurements of time to death of a cancer cell after it has been contacted 1:1 by a CD8 + T lymphocyte as determined by PI accumulation in the cancer cell. Data are compiled from 4 independent experiments with. P-values are calculated by Kruskal-Wallis test with multiple comparisons. Mean values are shown. ***:P<0.001, ****:P<0.0001. Control = 119 cells, high [K + ] e = 117 cells, BMS = 107 cells, high [K + ] e +BMS = 109 cells. (E) AFM stiffness measurements of representative cancer cells treated with high [K + ] e (45 mM KCl) compared to control. Results are from 2 combined experiments. AT-3: Control = 30 cells, high [K + ] e = 33 cells; E0771: Control = 34 cells, high [K + ] e = 37 cells. Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (F) AFM stiffness measurements of representative cancer cells treated with high [K + ] e and with/without 10 µM BMS-204352. Results are from 2 combined experiments. AT-3: high [K + ] e =25 cells, high [K + ] e +BMS = 21 cells; E0771: high [K + ] e = 20 cells, high [K + ] e +BMS = 20 cells. Mann-Whitney test. All error bars are mean±s.e.m. ns, not significant; *P<0.05; **P<0.01; ***P<0.001. (G) Schematic depiction of how cortical F-actin influences surface roughness and the surface roughness measurements performed by AFM. Data is combined from 4 independent experiments. AT-3: Control = 28 cells, high [K + ] e = 28 cells, BMS = 24 cells, high [K + ] e +BMS = 24 cells, E0771: Control = 23 cells, high [K + ] e = 25 cells, BMS = 24 cells, high [K + ] e +BMS = 23 cells. P-values calculated by Mann-Whitney rank sum test. Mean+/-s.e.m are shown. RMS: root-mean square. (H) Cell height of cells used in (G) measured by AFM. (I) Venn diagrams showing the number of differentially regulated genes in high [K + ] e . The intersections represent the genes “rescued” which means the BMS-204352 treatment caused differential expression in the opposite direction. “Commonly rescued” genes are conserved across two different cell types. (J) Gene ontology analyses of “Commonly rescued” genes in (F). See Table S6 for a full list of GO terms that are enriched. (K) Working model: MRTFA/SRF upregulates the expression of F-actin bundling proteins and BK channel subunit, KCNMB1, for promoting cellular stiffness. Elevated cancer cell stiffness triggers mechanosurveillance by cytotoxic lymphocytes for inhibiting metastatic colonization.

Article Snippet: The MRTFA Y238A and KCNMB1 E65K mutants were created by single point mutations in MRTFA (Addgene #19846) and Kcnmb1 (Addgene #113565) and were verified by Sanger sequencing.

Techniques: Control, MANN-WHITNEY, Quantitative Proteomics, Expressing

a Schematic diagram of gRNA target sequences and partial donor oligo sequences in CRISPR/Cas-mediated genome engineering for generating the C57BL/6 mouse model with a point mutation (T23A or T23D) at the PA28γ gene locus. The gRNA specifically targets the exon 2 region of the PA28γ gene, which encodes T23. The donor oligo introduced the T23A (ACA to GCA) or T23D (ACA to GAC) mutation into exon 2 by homology-directed repair. In addition, a synonymous mutation R21 (CGG to CGC) was introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. b Sanger sequencing results derived from genomic DNA of PA28γ T23A and PA28γ T23D mice. c Experimental schematic illustrating the strategy for inducing HNSCC in WT, PA28γ T23A , and PA28γ T23D mice ( n = 16/group) using the carcinogen 4NQO. d Representative images of tongue lesions in 4NQO-induced HNSCC mouse models at week 24. The dotted circles indicate macroscopically prominent lesions. e The number (Left) and volume (Right) of macroscopic prominent lesions in the tongues were quantified in week 24 of the 4NQO-induced HNSCC experiment. Data shown represent the mean ± SD for n = 16 mice; statistical significance was assessed by two-sided unpaired t -test. f Survival analysis using the Kaplan-Meier method on over 10% body weight loss in WT, PA28γ T23A , and PA28γ T23D mice during weeks 16–24 of the 4NQO-induced HNSCC experiment. Statistical significance was assessed by the log-rank (Mantel-Cox) test. g Representative H&E staining of severe dysplasia or carcinoma in-situ, microinvasive carcinoma, and invasive carcinoma in tongue lesions (Left). Scale bars, 100 μm. The tongue lesions of three mouse groups were evaluated and compared (Right). Statistical significance was assessed by two-sided Fisher’s exact test. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: Phosphorylation of PA28γ by CK2 kinase facilitates HNSCC tumor formation and progression

doi: 10.1038/s41467-025-67131-7

Figure Lengend Snippet: a Schematic diagram of gRNA target sequences and partial donor oligo sequences in CRISPR/Cas-mediated genome engineering for generating the C57BL/6 mouse model with a point mutation (T23A or T23D) at the PA28γ gene locus. The gRNA specifically targets the exon 2 region of the PA28γ gene, which encodes T23. The donor oligo introduced the T23A (ACA to GCA) or T23D (ACA to GAC) mutation into exon 2 by homology-directed repair. In addition, a synonymous mutation R21 (CGG to CGC) was introduced to prevent the binding and re-cutting of the sequence by gRNA after homology-directed repair. b Sanger sequencing results derived from genomic DNA of PA28γ T23A and PA28γ T23D mice. c Experimental schematic illustrating the strategy for inducing HNSCC in WT, PA28γ T23A , and PA28γ T23D mice ( n = 16/group) using the carcinogen 4NQO. d Representative images of tongue lesions in 4NQO-induced HNSCC mouse models at week 24. The dotted circles indicate macroscopically prominent lesions. e The number (Left) and volume (Right) of macroscopic prominent lesions in the tongues were quantified in week 24 of the 4NQO-induced HNSCC experiment. Data shown represent the mean ± SD for n = 16 mice; statistical significance was assessed by two-sided unpaired t -test. f Survival analysis using the Kaplan-Meier method on over 10% body weight loss in WT, PA28γ T23A , and PA28γ T23D mice during weeks 16–24 of the 4NQO-induced HNSCC experiment. Statistical significance was assessed by the log-rank (Mantel-Cox) test. g Representative H&E staining of severe dysplasia or carcinoma in-situ, microinvasive carcinoma, and invasive carcinoma in tongue lesions (Left). Scale bars, 100 μm. The tongue lesions of three mouse groups were evaluated and compared (Right). Statistical significance was assessed by two-sided Fisher’s exact test. Source data are provided as a Source Data file.

Article Snippet: The C57BL/6 J mouse models with the point mutation (PA28γ T23A or PA28γ T23D ) at the PA28γ gene locus created by CRISPR/Cas-mediated genome engineering were obtained from Cyagen Biosciences.

Techniques: CRISPR, Mutagenesis, Binding Assay, Sequencing, Derivative Assay, Staining, In Situ

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