Journal: Genes & Diseases

Article Title: MFAP2 promotes metastasis and drug resistance by regulating epithelial-to-mesenchymal transition through EGFR signaling pathway in colorectal cancer cells

doi: 10.1016/j.gendis.2025.101800

Figure Lengend Snippet: MFAP2 promotes epithelial–mesenchymal transition (EMT) through the EGFR-AKT-STAT3 signaling pathway in colorectal cancer (CRC) cells. (A, B) The top 20 enrichment signaling pathways regulated by MFAP2 knockdown. (C, D) VEGFR2 signaling pathway was enriched in the MFAP2 knockdown cells, shown by Gene Set Enrichment Analysis (GSEA) and essential genes in this enrichment. (E) MFAP2 knockdown affected the EGFR-AKT-STAT3 axis.

Article Snippet: Following blocking with 5% non-fat milk in PBS with 0.02% Tween 20 detergent (PBST) at room temperature for 2 h, the membranes were incubated with primary antibodies, including MFAP2 (Solarbio, China), GAPDH (BBI Co., Ltd., China), epidermal growth factor receptor (EGFR; Proteintech, China), protein kinase B (AKT) (Proteintech), signal transducer and activator of transcription 3 (STAT3) (Proteintech), and vascular endothelial growth factor A (VEGFA) (Proteintech), p-EGFR (Cell Signaling Technology, USA), p-STAT3 (Cell Signaling Technology), and p-AKT ser473 (Cell Signaling Technology) antibodies, at 4 °C overnight.

Techniques: Protein-Protein interactions, Knockdown

Journal: International Journal of Oncology

Article Title: Hypoxia-induced exosomal CAMTA1 promotes radio-resistance in MDA-MB-231 cells by regulating NRG1 to mediate M2 macrophage polarization

doi: 10.3892/ijo.2026.5875

Figure Lengend Snippet: Exosomal CAMTA1 promotes tumor growth in vivo . (A) The transfection efficacy of Ov-CAMTA1 was detected using RT-qPCR and immunoblotting analysis. (B) The appearance of tumor. The tumor (C) volume and (D) weight. (E) The mRNA expression of CAMTA1 was detected using RT-qPCR. (F) The level of IL-10 was detected using ELISA-related IL-10 assay kits. (G) The level of CD163 was detected using immunohistochemistry analysis. (H) H&E staining. (I) The expression of Caspase 3 was detected using immunohistochemistry analysis. (J) The Spearman correlation analysis of CAMTA1 and NRG1. (K) The expression of NRG1 was detected using immunohistochemistry analysis. * P<0.05, ** P<0.01 and *** P<0.001. CAMTA1, Calmodulin-binding Transcription Activator 1; Ov, overexpression; RT-qPCR, reverse transcription-quantitative PCR; H&E, hematoxylin and eosin; NRG1, neuregulin 1.

Article Snippet: The slices were then incubated with primary antibody against NRG1 (cat. no. 83323-6-RR; 1:500; Proteintech Group, Inc.), CD163 (cat. no. 83285-4-RR; 1:2,000; Proteintech Group, Inc.) and Caspase-3 (cat. no. 19677-1-AP; 1:500; Proteintech Group, Inc.) overnight at 4°C.

Techniques: In Vivo, Transfection, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Immunohistochemistry, Staining, Binding Assay, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: The Journal of Cell Biology

Article Title: Spectrin coordinates cell shape and signaling essential for epidermal differentiation

doi: 10.1083/jcb.202502071

Figure Lengend Snippet: αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin from shCtr and shSptan1 0595 -transduced E17.5 embryos immunolabeled for the basal layer marker K14, the differentiation marker K10, and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (b) Quantification of cell area and shape of suprabasal shSptan1 K14 + and K14 - cells in E17.5 embryos. Values of basal and suprabasal shCtr and basal shSptan1 are equal to Lines: Mean values; dots: single cells pooled from 3 embryos with >100 cells for each condition. ****P < 0.0001 with Kruskal–Wallis, Dunn’s multiple comparison test. (c) Immunofluorescence analysis for the TJ marker occludin in Ctr and Sptan1 −/− primary keratinocytes differentiated for 48 h in high Ca 2+ . Representative example of three biological replicates each. (d) Transepithelial resistance (TER) measurements in Ctr and αII-spectrin knockdown keratinocytes after switching to high Ca 2+ . Line represent means over time of three biological replicates each. Representative experiment of n > 10 biological replicates. (e) Newborn epidermal whole-mount immunofluorescence analysis for the TJ marker ZO-1, revealing impaired alignment of the upper old (red arrowheads) and the lower new TJ rings (blue arrowheads) in the granular layer 2 (SG2). Maximum projection of the granular layer. (f) Illustration of cell shapes and TJ organization in the SG2 of Ctr and Sptan1 epi−/− epidermis. (g) Dorsal skin sections from shCtr and shSptan1 0595- transduced E17.5 embryos. Sections were processed for transglutaminase 1 (TGM1) activity assay. Upper Insets show the transduced cells (H2B−RFP+). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean ± SD of 30 ROI from n = 3 embryos per condition. Bars: mean normalized intensity; dots individual microscopy fields. *P = 0.0471 by unpaired t- test. (i) Quantification of TGM1 activity cortical enrichment from the data shown in g. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: TGM1 activity cortex/cytoplasm intensity ratio mean; dots: individual cells. ***P = 0.0003 with Kolmogorov–Smirnov. (j) Dye exclusion assay: shCtr and shSptan1 0595 -transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (k) Dye exclusion assay: Ctr and Sptan1 epi−/− E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (l) Dorsal skin section from Ctr and E-cadherin epi−/− newborn mice. Sections were processed for TGM1 activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (m) Quantification of TGM1 intensity from data shown in l. (n) Transepidermal water loss (TEWL) measurements on Ctr and Sptan1 epi −/− newborn mice. Dots represent individual mice. Data are the mean of 27 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. ROI, region of interest.

Article Snippet: Ankyrin3 (IF 1:500, WB 1:1,000, #27766-1-AP; Proteintech), E-cadherin (IF 1:200, #610182; BD Transduction Laboratories, clone number 36 or IF 1:500, #3195; Cell Signaling), EGFR (IF 1:500, #ab52894; Abcam), pEGFR (Y1068) (IF 1:500, #ab40815; Abcam), GAPDH (WB 1:10,000, #AM4300; Ambion or WB 1:1,000, #5174; Cell Signaling), GFP (1:3,000, #ab13970; Abcam), Keratin10 (IF 1:1,000, #PRB-159P; BioLegend), Keratin14 (IF 1:2,000, #PRB 155P; Covance), Loricrin (1:1,000, #Poly19051; BioLegend), Myosin heavy chain IIa (IF 1:500, PRB-440; BioLegend), phospho-Myosin Light Chain 2 (Thr18/Ser19) (IF 1:100, #3674; Cell Signaling), occludin (IF 1:400, #33-1,500; Invitrogen), TRPV3 (IF 1:1,000, #b94582; Abcam), αII-Spectrin (IF 1:500, WB 1:1,000, ab11755; Abcam), TGM1 (IF 1:500, #12912-3-AP; Proteintech), α-catenin (IF 1:2,000, #C2081; Sigma-Aldrich), β-catenin (IF 1:1,000, #ab32572; Abcam), and YAP (IF 1:500, #14074; Cell Signaling).

Techniques: Immunolabeling, Marker, Comparison, Immunofluorescence, Knockdown, Activity Assay, Microscopy, Exclusion Assay

Journal: The Journal of Cell Biology

Article Title: Spectrin coordinates cell shape and signaling essential for epidermal differentiation

doi: 10.1083/jcb.202502071

Figure Lengend Snippet: αII-spectrin regulates epidermal differentiation and barrier function. (a) Dorsal skin sections from shSptan1 0595 -transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the suprabasal marker K10. (b) Dorsal skin sections from shSptan1 9753 -transduced E17.5 embryos immunolabeled for the basal layer marker K14 and the granular layer marker loricrin. Insets show the transduced cells (H2B−GFP+). (c) Dorsal skin sections from shSptan1 0595 -transduced E17.5 embryos immunolabeled for the cleavage furrow marker survivin. Yellow lines show representative axes of division. Graph: Quantification of spindle orientation plotted as a cumulative frequency distribution. NS: P = 0.3485 with Mann–Whitney. (d) Dorsal skin sections from shSptan1 0595 -transduced E17.5 embryos immunolabeled for EdU. (e) Quantification of EdU + basal and suprabasal layer cells from the data shown in d. Bars: mean ± SD from n = 3 embryos per condition. Dots: average EdU + basal and suprabasal layer cells from each embryo with Mann–Whitney. (f) Quantification of cell (nuclei) numbers from primary Ctr and Sptan1 −/− or Sptan1 siRNA-treated keratinocytes differentiated for 48 h in high Ca 2+ . Dots: Mean values from biological replicates. >360 cells counted for Ctr/ Sptan1 −/− each and >20,000 cells for siCtr/ siSptan1 each with Mann–Whitney. (g) Dorsal skin section from Ctr and Sptan1 epi−/− newborn mice. Sections were processed for transglutaminase 1 (TGM1) activity assay or negative Ctr (mutated TGM substrate, pepQNK5). (h) Quantification of TGM1 intensity from data shown in g. Data are the mean of 30 fields of view from n = 3 newborn mice per condition. Bars: Mean intensity; dots individual microscopy fields. ****P < 0.0001 with Kolmogorov–Smirnov. (i) Dorsal skin sections from Ctr, Ecad epi−/− and Sptan1 epi−/− newborn mice immunolabeled for total TGM1 protein. (j and k) Quantification of TGM1 intensity in Ctr, Ecad epi−/− and Sptan1 epi−/− . Lines: Mean values/biological replicate. Nonsignificant with Mann–Whitney. (l) Dye exclusion assay: shCtr and shSptan1 9753 -transduced E17.5 embryos were treated with toluidine blue dye to evaluate the skin barrier. (m) Dye exclusion assay: shCtr and shSptan1 0595 -transduced E18.5 embryos were treated with toluidine blue.

Article Snippet: Ankyrin3 (IF 1:500, WB 1:1,000, #27766-1-AP; Proteintech), E-cadherin (IF 1:200, #610182; BD Transduction Laboratories, clone number 36 or IF 1:500, #3195; Cell Signaling), EGFR (IF 1:500, #ab52894; Abcam), pEGFR (Y1068) (IF 1:500, #ab40815; Abcam), GAPDH (WB 1:10,000, #AM4300; Ambion or WB 1:1,000, #5174; Cell Signaling), GFP (1:3,000, #ab13970; Abcam), Keratin10 (IF 1:1,000, #PRB-159P; BioLegend), Keratin14 (IF 1:2,000, #PRB 155P; Covance), Loricrin (1:1,000, #Poly19051; BioLegend), Myosin heavy chain IIa (IF 1:500, PRB-440; BioLegend), phospho-Myosin Light Chain 2 (Thr18/Ser19) (IF 1:100, #3674; Cell Signaling), occludin (IF 1:400, #33-1,500; Invitrogen), TRPV3 (IF 1:1,000, #b94582; Abcam), αII-Spectrin (IF 1:500, WB 1:1,000, ab11755; Abcam), TGM1 (IF 1:500, #12912-3-AP; Proteintech), α-catenin (IF 1:2,000, #C2081; Sigma-Aldrich), β-catenin (IF 1:1,000, #ab32572; Abcam), and YAP (IF 1:500, #14074; Cell Signaling).

Techniques: Immunolabeling, Marker, MANN-WHITNEY, Activity Assay, Microscopy, Exclusion Assay

Journal: The Journal of Cell Biology

Article Title: Spectrin coordinates cell shape and signaling essential for epidermal differentiation

doi: 10.1083/jcb.202502071

Figure Lengend Snippet: EGFR activity regulates cortical TRPV3 localization. (a) Dorsal skin sections from shCtr; TRPV3-GFP–transduced E17.5 embryos treated with DMSO or gefitinib immunolabeled for αII-spectrin and pEGFR. (b and c) Quantification of TRPV3-GFP and αII-spectrin cortical enrichment from the data shown in a. Mean ± SD from 30 individual cells from n = 3 embryos per condition. Bars: TRPV3-GFP cortex/cytoplasm ratio; dots: individual cells. **P = 0.0029 for TRPV3-GFP and ***P = 0.0001 for αII-spectrin with Kolmogorov-Smirnov. (d and f) Primary mouse keratinocytes cultured in high-calcium (1.5 mM) medium treated with DMSO, gefitinib, or TGF-α and immunolabelled for p-EGFR, TRPV3, and αII-spectrin. Boxes indicate the location of the magnified area. (e and g) Quantification of TRPV3 and αII-spectrin intensity from the data shown in d and f. Mean ± SD from ∼200 mature junctions from n = 3 experiment per condition. Bars: mean normalized intensity; dots: individual junctions. Nuclei were stained with DAPI. **P = 0.001 and ****P > 0.0001 for TRPV3 intensity. ****P > 0.0001 and ***P = 0.0001 for αII-spectrin intensity with Kolmogorov–Smirnov. (h) Dorsal skin sections from E17.5 wild-type embryos treated with DMSO and gefitinib. Sections were processed for transglutaminase 1 (TGM1) activity assay. (i) Quantification of crosslinked TGM substrate intensity. Mean ± SD of 30 ROIs from n = 3 embryos per condition. Bars: mean normalized intensity; dots: individual microscopy fields. NS: P = 0.3876 with Kolmogorov–Smirnov. (j) Quantification of cortical enrichment of crosslinked TGM substrate. Mean ± SD from 60 individual cells from n = 3 embryos per condition. Bars: means of cortex/cytoplasm ratio; dots: individual cells. ****P < 0.0001 with Kolmogorov–Smirnov. Nuclei were stained with DAPI; dashed lines indicate the dermal-epidermal border. (k) Model - High-tension spectrin-actomyosin cortices regulate TGM activity. Illustration of junction and cytoskeleton distribution across epidermal layers, with spectrin most enriched in SG3 and F-actin most enriched in SG1. Lower left: illustration of spectrin and myosin-dependent organization of cortical F-actin. Upper left: Working model of how lattice organization and myosin tension regulate EGFR/TRPV3 signaling complexes, resulting in Ca 2+ influx and cortical TGM activation. ROI, region of interest.

Article Snippet: Ankyrin3 (IF 1:500, WB 1:1,000, #27766-1-AP; Proteintech), E-cadherin (IF 1:200, #610182; BD Transduction Laboratories, clone number 36 or IF 1:500, #3195; Cell Signaling), EGFR (IF 1:500, #ab52894; Abcam), pEGFR (Y1068) (IF 1:500, #ab40815; Abcam), GAPDH (WB 1:10,000, #AM4300; Ambion or WB 1:1,000, #5174; Cell Signaling), GFP (1:3,000, #ab13970; Abcam), Keratin10 (IF 1:1,000, #PRB-159P; BioLegend), Keratin14 (IF 1:2,000, #PRB 155P; Covance), Loricrin (1:1,000, #Poly19051; BioLegend), Myosin heavy chain IIa (IF 1:500, PRB-440; BioLegend), phospho-Myosin Light Chain 2 (Thr18/Ser19) (IF 1:100, #3674; Cell Signaling), occludin (IF 1:400, #33-1,500; Invitrogen), TRPV3 (IF 1:1,000, #b94582; Abcam), αII-Spectrin (IF 1:500, WB 1:1,000, ab11755; Abcam), TGM1 (IF 1:500, #12912-3-AP; Proteintech), α-catenin (IF 1:2,000, #C2081; Sigma-Aldrich), β-catenin (IF 1:1,000, #ab32572; Abcam), and YAP (IF 1:500, #14074; Cell Signaling).

Techniques: Activity Assay, Immunolabeling, Cell Culture, Staining, Microscopy, Activation Assay