Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Selective RNAi silencing of Schwann cell Piezo1 alleviates mechanical hypersensitization following peripheral nerve injury

doi: 10.1016/j.omtm.2025.101433

Figure Lengend Snippet: GFP expression after intrasciatic AAVolig001-CBA-GFP (A–D) GFP expression in the nerve fascicles (dashed circles) of cross-sectioned sciatic nerves (A and B). Detection of GFP signals in longitudinal sections of tibial and sural serves (C and D). (E–J) Representative IHC images illustrate selective GFP expression in both mSCs and nmSCs, colabeled with SCs markers MPZ, MBP, GAP43, and P75NTR (E–H). Empty and white arrowheads in (F) and (H) point to mSCs and nmSCs, respectively; no GFP signals in the afferent axons labeled by Tubb3 and NF200 (I and J). Scale bars (μm): (A) and (B), 100; (C), 500; (D)–(F), 100. Antibodies for double labeling are indicated in each montage IHC image. Scales: 50 μm for all. GAP43, growth-associated protein 43; MBP, myelin basic protein; MPZ, myelin protein zero; NF200, neurofilament 200; P75NTR, p75 neurotrophin receptor; and Tubb3, β3-tubulin.

Article Snippet: Cells and tissue sections were immunolabeled with the selected primary antibodies mouse Piezo1 (1:400, Novus Biologicals, NBP2-75617), rabbit GFP (1:500, Cell Signaling Technology [Danvers, MA], 2555S), rabbit mCherry (1:400, Cell Signaling Technology, 43590), rabbit GAP43 (1:400, Cell Signaling Technology, 8945), rabbit p75NTR (1:400, Cell Signaling Technology, 8238), rabbit MPZ (1:1,000, Cell Signaling Technology, 57518s), rabbit S100 (1:1,000, Cell Signaling Technology, 13018), goat MBP (1:1,000, Santa Cruz Biotechnology [SCB], sc13912), goat Sox10 (1:200, SCB, sc17342), rabbit NF200 (1:1,000, Cell Signaling Technology, 30564), rabbit Tubb3 (1:1,000, Cell Signaling Technology, 5586), mouse cytokeratin14 (CK14, 1:200, SCB, sc53253), and rabbit glial fibrillary acidic protein (1:1,000, Dako [Glostrup, Denmark, Z0334) in a humid atmosphere overnight at 4°C.

Techniques: Expressing, Labeling

Journal: Cellular and Molecular Life Sciences

Article Title: Met–HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1007/s00018-022-04149-w

Figure Lengend Snippet: Gene expression analysis identifies MPZL3 as a HER3-regulated transcript in MET -amplified cells. a Western blot analysis of the phosphorylation of Akt (Ser 473 ), ERK1/2 (Thr 202 /Tyr 204 ) and STAT3 (Tyr 705 ) in the indicated cell lines stably transduced with control (pLKO) or shRNAs targeting HER3. b Volcano plot of RNA sequencing data. Genes with significant increase or decrease in expression between control (pLKO) and HER3-depleted KatoII cell lines are labeled. c – d Measurement of the relative amounts of ERBB3 , MPZL3 , NYNRIN , BHLHE41 and LGR6 genes in the indicated cell lines by RT-qPCR. c Genes downregulated under steady-state conditions upon stable HER3 depletion in EBC1 cells. d Genes downregulated under steady-state conditions upon stable HER3 depletion in H1993 cells. Data are means (bars) of three individual replicates. Two-way ANOVA with Dunnett’s test for multiple comparisons; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Error bars indicate SEM

Article Snippet: Antibodies against V5 peptide were purchased from Abcam, while antibodies against MPZL3 were purchased from ProteinTech.

Techniques: Gene Expression, Amplification, Western Blot, Phospho-proteomics, Stable Transfection, Transduction, Control, RNA Sequencing, Expressing, Labeling, Quantitative RT-PCR

Journal: Cellular and Molecular Life Sciences

Article Title: Met–HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1007/s00018-022-04149-w

Figure Lengend Snippet: MPZL3 is required for MET -amplified cell expansion downstream of HER3. a Western blot analysis of total and phosphorylated levels of HER3 and MPZL3 in EBC1 cells overexpressing MPZL3, or an empty-vector control (pLKO) and transfected with control or HER3 - targeting siRNAs. b Live-cell imaging to measure cell confluence of EBC1 cell lines stably transduced with control (pLKO) or MPZL3 and transfected with control or HER3 - targeting siRNAs. Representative growth curves are shown; Error bars indicate SEM. Confluence relative to relevant siCtl-treated cells is quantified on the right panel ( n = 3); error bars show SEM. c , g Representative images of colony-forming assay are presented. c , h Colony formation of KatoII cells in soft agar ( c – d ) or H1993 cells in adherent conditions ( g – h ) transduced with shRNA targeting MPZL3 or controls (pLKO and shCtl). Quantification of the colony number ( n = 4). e , i MPZL3 transcript levels in KatoII ( e ) and H1993 ( i ) cells transduced with shRNA targeting MPZL3 or controls (pLKO and shCtl). f , j Western blot analysis of total and phosphorylated levels of HER3 in KatoII f and H1993 j cells transduced with shRNA targeting MPZL3 or controls (pLKO and shCtl). b : One-way ANOVA with the Sidak–Holm test for multiple comparisons; **** p ≤ 0.0001. d – e and h – i : One-way ANOVA with Dunnett’s test for multiple comparisons; * p ≤ 0.05; ** p ≤ 0.01

Article Snippet: Antibodies against V5 peptide were purchased from Abcam, while antibodies against MPZL3 were purchased from ProteinTech.

Techniques: Amplification, Western Blot, Plasmid Preparation, Control, Transfection, Live Cell Imaging, Stable Transfection, Transduction, shRNA

Journal: Cellular and Molecular Life Sciences

Article Title: Met–HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1007/s00018-022-04149-w

Figure Lengend Snippet: HER3 interacts with MPZL3. a Co-immuno-precipitation analysis of KatoII cells stably transduced with MPZL3- or GFP-V5 expression vectors, or control (pLKO). Cells were treated with PHA-665752 (0.5 mM, 1 h) or with vehicle control (DMSO). Immunoprecipitation (IP), Whole cell lysate (WCL). b HER3 and MPZL3 were analyzed by split intein-mediated protein ligation (SIMPL) assay to measure direct protein–protein interaction. c Schematic of MPZL3 mutant constructs. d FLAG transfer by point mutants and the ΔECD mutant were compared to wild-type MPZL3 using the SIMPL assay. e Co-immunoprecipitation of MPZL3 and HER3 was quantified, comparing HER3 recovered by the full-length or ΔECD MPZL3 mutants. Data are means (bars) of three individual replicates. Student’s t test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001. Error bars indicate SEM

Article Snippet: Antibodies against V5 peptide were purchased from Abcam, while antibodies against MPZL3 were purchased from ProteinTech.

Techniques: Immunoprecipitation, Stable Transfection, Transduction, Expressing, Control, Ligation, Mutagenesis, Construct

Journal: Cellular and Molecular Life Sciences

Article Title: Met–HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1007/s00018-022-04149-w

Figure Lengend Snippet: Amplification of genes encoding MET , EGFR and E RBB2 correlate with elevated levels of MPZL3 in the Cancer Cell Line Encyclopedia. a Met-amplified cells were identified in the CCLE by relative gene copy number (Log 2 relative to ploidy + 1 ≥ 2.3), and MPZL3 expression was observed to be higher than that in cells with no RTK amplified (median TPM: 2.8 [ MET -amplified] vs. 1.7 [control], Mann–Whitney p = 0.03). b Cell lines in CCLE were profiled for RTK amplification (Log 2 relative to ploidy + 1 ≥ 2.3), and MPZL3 expression was compared in cells with an amplified RTK to cells with no RTK amplification. ANOVA p < 0.0001

Article Snippet: Antibodies against V5 peptide were purchased from Abcam, while antibodies against MPZL3 were purchased from ProteinTech.

Techniques: Amplification, Expressing, Control, MANN-WHITNEY

Journal: Cellular and Molecular Life Sciences

Article Title: Met–HER3 crosstalk supports proliferation via MPZL3 in MET -amplified cancer cells

doi: 10.1007/s00018-022-04149-w

Figure Lengend Snippet: MPZL3 levels correlate with MET and ERBB3 gene expression and are associated with poor clinical outcome in gastric cancers. a – c Correlation between the indicated genes in gastric tumors, estimated using Pearson’s correlation ( n = 300). d Expression of MPZL3 in normal gastric tissues ( n = 100) and gastric tumors ( n = 300). e Expression of MPZL3 across gastric cancer subtypes. EMT, Epithelial-mesenchymal transition ( n = 46); MSI, microsatellite instability ( n = 68); MSS, microsatellite stable; TP53 – , TP53 loss ( n = 107); TP53 + , TP53 intact ( n = 79). f Recurrence-free survival across gastric intestinal adenocarcinoma according to Lauren classification ( n = 107). Data from GSE66229. Student’s t test; * p ≤ 0.05; ** p ≤ 0.01; *** p ≤ 0.001; **** p ≤ 0.0001

Article Snippet: Antibodies against V5 peptide were purchased from Abcam, while antibodies against MPZL3 were purchased from ProteinTech.

Techniques: Gene Expression, Expressing