Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Primers used for quantitative PCR analyses.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Real-time Polymerase Chain Reaction, Sequencing

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Narrowing down of the domain involved in the induction of COL1A1 expression in LX-2 cells. (A) Overexpression of both full-length fibrinogen domain of TNX [TNX-FG] and ITGA11 failed to induce the expression of fibrosis marker genes, including ACTA2, COL1A1 and TGFB1 . LX-2 cells were transfected with expression vectors for hTNX-FG (lane 2), ITGA11 (lane 3) and hTNX-FG and ITGA11 (lane 4) in DMEM/0.5% FBS. (B) Induction of COL1A1 expression by overexpression of both hTNX-FGF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGF (lane 2), hTNX-FGL (lane 3), ITGA11 (lane 4), hTNX-FGF and ITGA11 (lane 5) and hTNX-FGL and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Induction of COL1A1 expression by overexpression of both hTNX-FGFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFF (lane 2), hTNX-FGFL (lane 3), ITGA11 (lane 4), hTNX-FGFF and ITGA11 (lane 5) and hTNX-FGFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (D) Induction of COL1A1 expression by overexpression of both hTNX-FGFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFF (lane 2), hTNX-FGFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFF and ITGA11 (lane 5) and hTNX-FGFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (A-D) As a control, RNA from the cells without transfection (lane 1) was used. The cell lysate was prepared 48 h after transfection and then RNA was purified. Subsequently, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. The expression level of each gene in the control was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FG, fibrinogen-related domain of human tenascin-X; hTNX-FGF, first half of hTNX-FG; hTNX-FGL, latter half of hTNX-FG; hTNX-FGFF, first half of hTNX-FGF; hTNX-FGFL, latter half of hTNX-FGF; hTNX-FGFFF, first half of hTNX-FGFF; hTNX-FGFFL, latter half of hTNX-FGFF.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Marker, Transfection, Control, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Identification of the minimal sequence responsible for induction of COL1A1 expression in LX-2 cells. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFL (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFL and ITGA11 (lane 6) in DMEM/0.5% FBS. (B) Overexpression of both hTNX-FGpeptide2-5 and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGpeptide2-5 (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGpeptide2-5 and ITGA11 (lane 6) in DMEM/0.5% FBS. (C) Overexpression of both hTNX-FGFFFM and ITGA11 did not cause induction of COL1A1 expression. LX-2 cells were transfected with expression vectors for hTNX-FGFFFF (lane 2), hTNX-FGFFFM (lane 3), ITGA11 (lane 4), hTNX-FGFFFF and ITGA11 (lane 5) and hTNX-FGFFFM and ITGA11 (lane 6) in DMEM/0.5% FBS. After transfection followed by cell culture, cell lysate extraction and RNA purification, the expression levels of ACTA2, COL1A1 and TGFB1 were examined by reverse transcription-quantitative PCR. (A-C) The expression level of each gene in the control (lane 1, RNA from cells without transfection) was set to 1.0, and the relative expression level of each gene compared with that of the control is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1), one-way ANOVA with Dunnett's post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG); hTNX-FGFFFM, PRDCGEEMQNGAGAS peptide from hTNX-FG; hTNX-FGFFFL, QNGAGASRTSTIFL peptide from hTNX-FG; hTNX-FGpeptide2-5, GGLRIPF peptide from hTNX-FG.

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Sequencing, Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 in addition to inhibitors. (A) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a TGFBRI inhibitor (SB525334). DMSO (lanes 1, 2 and 3) and SB525334 (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). (B) Induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and ITGA11 with a YAP inhibitor (verteporfin). DMSO (lanes 1, 2 and 3) and vertepofin (lane 3) were added to the culture medium (DMEM/0.5% FBS) after the transfection of expression plasmids for both hTNX-FGFFFF and ITGA11 in LX-2 cells (lanes 2 and 3). Subsequently, the cells were cultured followed by cell lysate extraction, RNA purification and reverse transcription-quantitative PCR. (A and B) The expression level of each gene [ ACTA2, COL1A1 and TGFB1 for (A) and ACTA2, COL1A1 and YAP1 for (B)] in the control (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (lane 1) is shown (n=3). Data are presented as the mean ± SD. *P<0.05, **P<0.01 vs. control (lane 1); ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP, Yes-associated protein; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Expressing, Over Expression, Transfection, Cell Culture, Extraction, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction, Control

Journal: Molecular Medicine Reports

Article Title: COL1A1 expression induced by overexpression of both a 15-amino acid peptide from the fibrinogen domain of tenascin-X and integrin α11 in LX-2 cells

doi: 10.3892/mmr.2022.12846

Figure Lengend Snippet: Effect of YAP1 knockdown on induction of COL1A1 expression. YAP1 was knocked down with YAP1 siRNA prior to overexpression of both hTNX-FGFFFF and ITGA11, and then the expression levels of ACTA2 and COL1A1 were analyzed by reverse transcription-quantitative PCR in LX-2 cells. RNA from cells treated with transfection of control siRNA only (lane 1), transfection of control siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 2) and transfection of YAP1 siRNA followed by co-transfection with both hTNX-FGFFFF and ITGA11 expression plasmids (lane 3) was used. The expression level of each gene ( ACTA2, COL1A1 and YAP1 ) in the control (control siRNA only) (lane 1) was set to 1.0, and the relative expression level of each gene compared with that of the control (control siRNA) is shown (n=3). Data are presented as the mean ± SD. **P<0.01 vs. control (lane 1); # P<0.05, ## P<0.01 vs. lane 2, one-way ANOVA with the Bonferroni post hoc test. At the bottom of the figure, the relative expression levels of YAP1, hTNX-FGFFFF and ITGA11 are also shown, setting lane 2 to 1.0, since hTNX-FGFFFF expression was not detected in lane 1. COL1A1 , type I collagen α1 chain; TNX, tenascin-X; ITGA11, integrin α11; ACTA2 , α-smooth muscle actin; YAP1, Yes-associated protein 1; siRNA, small interfering RNA; hTNX-FGFFFF, GGLRIPFPRDCGEEM peptide from fibrinogen-related domain of human tenascin-X (hTNX-FG).

Article Snippet: To investigate the possible signaling pathway involved in the induction of COL1A1 expression by overexpression of both hTNX-FGFFFF and integrin α11 in LX-2 cells, a TGF-β receptor type 1 (TGFBRI) inhibitor (SB525334) (MedChemExpress) and a YAP inhibitor (verteporfin) (Cayman Chemical) were used.

Techniques: Knockdown, Expressing, Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Transfection, Control, Cotransfection, Small Interfering RNA

Journal: Nature Communications

Article Title: Tenascin C + myofibroblasts exacerbate vascular neointimal hyperplasia by propagation of nerve-macrophage interactions in mice

doi: 10.1038/s41467-026-69062-3

Figure Lengend Snippet: a Western blot analysis and quantification of TNC expression in the carotid arteries from ligated and sham-operated groups ( n = 6 biological replicates; males). b ELIZA quantification of serum TNC levels in ligated ( n = 9 biological replicates; males) and sham-operated ( n = 8 biological replicates; males) male mice. Values were normalized by subtracting baseline levels of untouched mice. c Immunofluorescence images of TNC (green) in the adventitia and neointima of carotid arteries from Col1a2 CreER Tnc fl/fl mice (cKO, n = 6 biological replicates; 4 males and 2 females) or control Tnc fl/fl mice (flox, n = 6 biological replicates; 3 males and 3 females). d Representative H&E-stained sections of carotid arteries from cKO ( n = 6 biological replicates; 4 males and 2 females) or control flox mice ( n = 6 biological replicates; 3males and 3 females), with quantification of the neointimal area and lumen-to-media ratios. e Sirus red-stained sections of carotid arteries from cKO ( n = 6 biological replicates; 4 males and 2 females) or control flox mice ( n = 6 biological replicates; 3 males and 3 females), with quantification of fibrotic area. f Immunofluorescence images of α-SMA (red) in the adventitia and neointima of carotid arteries from cKO or control flox mice. Cell nuclei are counterstained with DAPI (blue). g Immunofluorescence images of CD68 (magenta) and CD11c (cyan) in the adventitia of ligated carotid arteries from cKO ( n = 6 biological replicates; 4 males and 2 females) and control flox mice ( n = 6; males). h Maximal projection images of 3D whole-mount immunofluorescence staining of β Ⅲ-tubulin in ligated carotid arteries from cKO and flox mice. Scale bars: 100 μm in ( c – h ), enlargement scale bars: c 20 μm, f 50 μm g 20 μm. Data are shown as mean ± SD, and significance is determined by a two-tailed Mann-Whitney test ( d ) and an unpaired two-tailed Student’s t -test( a – c , e – g ). Source data are provided as a Source Data file.

Article Snippet: PDGFRα DreER -tdTomato mice were generated by crossing the PDGFRα DreER with the R26R-rox-tdTomato reporter strain; Col1a2 CreER Tnc fl/fl mice were generated by crossing the Col1a2 CreER with the Tnc fl/fl mouse strain; Cre-negative littermates Col1a2 CreER(−/−) ;Tnc fl/fl mice were generated by crossing the Col1a2 CreER (Cyagen, stock No. C001248) with the C57BL/6 J mouse strain; Na v 1.8-tdTomato mice were generated by crossing the Na v 1.8 Cre with the R26R-tdTomato reporter strain; Tamoxifen (MedChemExpress, HY-13757A) was dissolved in corn oil and injected intraperitoneally with 75 mg/kg dose.

Techniques: Western Blot, Expressing, Immunofluorescence, Control, Staining, Two Tailed Test, MANN-WHITNEY

Journal: Nature Communications

Article Title: Tenascin C + myofibroblasts exacerbate vascular neointimal hyperplasia by propagation of nerve-macrophage interactions in mice

doi: 10.1038/s41467-026-69062-3

Figure Lengend Snippet: a Whole-mount fluorescence images of ligated and sham-operated carotid arteries from sensory nerve-tracing Na v 1.8-tdTomato mice, showing Na v 1.8 + sensory neurons in red. 3 times each experiment was repeated independently with similar results. b Representative images of H&E-stained sections of ligated and sham-operated carotid arteries from fibroblast-specific conditional Tnc knockout (Col1a2 CreER Tnc fl/fl , cKO) mice or control (Tnc fl/fl , flox) mice treated with resiniferatoxin (RTX) or vehicle control (VEC). Quantification of neointimal area and lumen-to-media ratios is shown ( n = 5 biological replicates). c Sirus red-stained sections of ligated and sham-operated carotid arteries from cKO or flox mice treated with RTX or VEC, with quantification of fibrotic area ( n = 5 biological replicates). d – f Representative immunofluorescence images of d Ki67 (red), e α-SMA (magenta), and f Fn1 (Red) in ligated carotid arteries from cKO or flox mice treated with RTX or VEC ( n = 5 biological replicates). Scale bars: a 100 μm, 50 μm (enlargement), 10 μm (right); 100 μm in ( b , c ); 50 μm in ( d , e ); 25 μm in ( f ). Data are presented as mean ± SD, and significance is determined by two-way ANOVA with Šídák’s multiple comparisons test ( b – f ). Source data are provided as a Source Data file.

Article Snippet: PDGFRα DreER -tdTomato mice were generated by crossing the PDGFRα DreER with the R26R-rox-tdTomato reporter strain; Col1a2 CreER Tnc fl/fl mice were generated by crossing the Col1a2 CreER with the Tnc fl/fl mouse strain; Cre-negative littermates Col1a2 CreER(−/−) ;Tnc fl/fl mice were generated by crossing the Col1a2 CreER (Cyagen, stock No. C001248) with the C57BL/6 J mouse strain; Na v 1.8-tdTomato mice were generated by crossing the Na v 1.8 Cre with the R26R-tdTomato reporter strain; Tamoxifen (MedChemExpress, HY-13757A) was dissolved in corn oil and injected intraperitoneally with 75 mg/kg dose.

Techniques: Fluorescence, Staining, Knock-Out, Control, Immunofluorescence

Journal: Nature Communications

Article Title: Tenascin C + myofibroblasts exacerbate vascular neointimal hyperplasia by propagation of nerve-macrophage interactions in mice

doi: 10.1038/s41467-026-69062-3

Figure Lengend Snippet: a Time-lapse fluorescent images showing dynamic interactions between DRG neurons (red) from Na v 1.8-tdTomato mice with macrophages (green) from EGFP mice at the indicated times, showing macrophage migration and contact with neurons. b Whole-mount immunofluorescence of F4/80 (cyan) in ligation or sham carotid arteries from Na v 1.8-tdTomato mice, where sensory nerves are in red. Cell nuclei are counterstained with DAPI (blue). c RT-qPCR of ccl2 expression level (2 −ΔCT ) in DRG cell bodies from ligated ( n = 7 biological replicates) and sham-operated ( n = 5 biological replicates) mice. d Western blot and quantification of CCL2 expression in C1-T1 DRGs from ligated and sham-operated mice ( n = 6 biological replicates). e Western blot and quantification of CCL2 expression in C1-T1 DRGs from ligated fibroblast-specific Tnc knockout (Col1a2 CreER Tnc fl/fl , cKO)or control (Tnc fl/fl , flox) mice treated with resiniferatoxin (RTX) or vehicle (VEC) ( n = 8 biological replicates). f Immunofluorescence and quantification of βⅢ-tubulin (green) and CCL2 (red) in ligated carotid arteries from cKO or flox mice treated with RTX or VEC ( n = 5 biological replicates). g Immunofluorescence and quantification of F4/80 (green) and TGF-β1 (red) or IL-1β (cyan) in ligated carotid arteries from cKO or flox mice treated with RTX or VEC ( n = 5 biological replicates). h Representative images of Masson’s trichrome and H&E staining of human diseased (diseased, n = 10 technical replicates) and non-diseased controls (control, n = 10 technical replicates per specimen) arteries, with immunofluorescence of CD68 (yellow), β Ⅲ-tubulin (cyan), and CCL2(magenta). Dash lines indicate the adventitia (A), Media (M), neointima (N), and intima (I). Quantification includesβ Ⅲ-tubulin⁺ area, β Ⅲ-tubulin⁺CCL2⁺ area, and CD68⁺CCL2⁺ area. Data are shown as mean ± SD; significance is determined by Mann-Whitney test ( c ), two-tailed unpaired Student’s t -test ( d , h ), one-way ANOVA with Šídák’s multiple comparisons test ( e , g ), two-way ANOVA with Šídák’s multiple comparisons test ( f ). Scale bars: a 25 μm; b 100 μm; f – g 50 μm. h MS and H&E,1000 μm; enlargement, 100 μm. Experiments were independently repeated three ( b ) or five times ( a ) with similar results. Source data are provided as a Source Data file.

Article Snippet: PDGFRα DreER -tdTomato mice were generated by crossing the PDGFRα DreER with the R26R-rox-tdTomato reporter strain; Col1a2 CreER Tnc fl/fl mice were generated by crossing the Col1a2 CreER with the Tnc fl/fl mouse strain; Cre-negative littermates Col1a2 CreER(−/−) ;Tnc fl/fl mice were generated by crossing the Col1a2 CreER (Cyagen, stock No. C001248) with the C57BL/6 J mouse strain; Na v 1.8-tdTomato mice were generated by crossing the Na v 1.8 Cre with the R26R-tdTomato reporter strain; Tamoxifen (MedChemExpress, HY-13757A) was dissolved in corn oil and injected intraperitoneally with 75 mg/kg dose.

Techniques: Migration, Immunofluorescence, Ligation, Quantitative RT-PCR, Expressing, Western Blot, Knock-Out, Control, Staining, MANN-WHITNEY, Two Tailed Test

Journal: Nature Communications

Article Title: Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

doi: 10.1038/s41467-022-30466-6

Figure Lengend Snippet: A , B Nf1 +/neo , but not Nf1 +/1809 , DRG neuron AP firing rates are elevated relative to WT DRG neurons, as measured by ( A ) multi-electrode array (WT, n = 24, Nf1 +/neo , n = 10; P = 0.0005, Nf1 +/1809 n = 10, ns), or ( B ) calcium imaging recordings (WT n = 8, Nf1 +/neo n = 5, P < 0.0001, Nf1 +/1809 n = 14, ns). C , D TTX (1 µM) and lamotrigine (LTR; 200 µM) reduce Nf1 +/neo DRG neuron AP firing rate as measured by multi-electrode array (vehicle n = 4, TTX n = 7, P < 0.0001; LTR n = 6, P < 0.0001) and calcium imaging (vehicle n = 23, TTX n = 9, P < 0.0001, LTR n = 14, P < 0.0001). The right panels show representative ( A , C ) spike plots of entire multi-electrode array well recordings over 30 s, and ( B , D ) traces of neuronal activity over 3 min. E Schematic illustrating treatment of human sh NF1 Schwann cells with hiPSC-sensory neuron conditioned media (CM). NF1 -deficient Schwann cell proliferation is increased after treatment with NF1 C383X , NF1 R681X , and NF1 E2207X mutant neuron CM ( P < 0.0001), but not NF1 R1809C neuron CM relative to controls (CTL). n = 6 for all groups. F Analytical comparison of 2D gel electrophoresis (top-to-bottom: decreasing molecular weight; left-to-right: decreasing acidity) of NF1 R681X (left) and NF1 R1809C (right) CM relative to CTL hiPSC-sensory neuron CM. Red dots indicate proteins with increased expression, green dots indicate proteins with decreased expression, and yellow dots indicate unaltered proteins in NF1- mutant sensory neuron CM relative to CTL neuron CM. The six proteins uniquely increased more than 1.5-fold in NF1 R681X hiPSC-sensory neuron CM relative to CTL, but not in NF1 R1809C CM, relative to CTL are circled in blue and are listed in the lower panel. Representative CM from CTL, NF1 R1809C , and NF1 R681X sensory neurons was analyzed by 2D gel electrophoresis ( n = 1). G , H COL1A2 levels are increased in ( G ) NF1 C383X , NF1 R681X , and NF1 E2207X mutant neuron CM ( P < 0.0001), but not in NF1 R1809C neuron CM ( n = 4 for all groups), as well as in ( H ) Nf1 +/neo mouse DRG neuron CM ( P < 0.0001), but not in Nf1 +/1809 mouse DRG neuron CM ( n = 6 for all groups). I Nf1 -deficient DRG-NSC proliferation is increased after treatment with Nf1 +/neo DRG neuron CM ( P < 0.0001), but not Nf1 +/1809 DRG neuron CM, relative to WT controls. n = 6 for all groups. Data are presented as the mean ± SEM. A – E , G – I One-way ANOVA with ( A – D , G – I ) Dunnett’s, or ( E ) Tukey’s multiple comparisons test. P values are indicated within each panel. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: NF1 +/R681X or Nf1 +/neo sensory neurons were infected with three independent sh COL1A2 lentiviral particles or shRNA scrambled control particles (sc-108080; Santa Cruz Biotechnology) for 24 h. Neuronal media was refreshed and conditioned media was collected for subsequent assays 48–72 h post infection.

Techniques: Imaging, Activity Assay, Mutagenesis, Comparison, Two-Dimensional Gel Electrophoresis, Electrophoresis, Molecular Weight, Expressing

Journal: Nature Communications

Article Title: Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

doi: 10.1038/s41467-022-30466-6

Figure Lengend Snippet: A Immunofluorescent staining and corresponding quantitation of Ki67 + human sh NF1 Schwann cells (left) and Nf1 − /− mouse DRG–NSCs (right) following incubation with hiPSC-sensory neuron conditioned media (CM), with (h P = 0.0007; m P < 0.0001) and without ( P < 0.0001) collagenase ( n = 6 for all groups), COL1A2 alone with (h P = 0.0036; m P < 0.0001) and without ( P < 0.0001) collagenase ( n = 6 for all groups), as well as with and without control or short hairpins against COL1A2 ( n = 3 for all groups, P < 0.0001) or Col1a2 (vehicle n = 4, control short hairpin n = 7, sh Col1a2 -1 n = 4, sh Col1a2 -2 n = 4, sh Col1a2 -3 n = 3, P < 0.0001). B – C ( B ) Human and ( C ) mouse cutaneous (cNF) and plexiform neurofibromas (pNF) express COL1A2. Normal brain, lymph node and normal sural (human) or normal sciatic (mouse) nerves were negative for COL1A2 expression. Neurofilament was used as positive control for normal mouse nerve tissue. These data derive from a single-tissue microarray. D COL1A2 RNA expression is increased in human sh NF1 Schwann cells (left; P = 0.0014) and mouse Nf1 −/− DRG–NSCs (right; P = 0.0012) following COL1A2 treatment. n = 3 for all groups. E COL1A2 RNA expression is increased in human Schwann cells isolated from human cNF ( P = 0.0039) and pNF tumors ( P = 0.0022) relative to controls. Normal n = 10, cNF n = 11, pNF n = 11. Data are presented as the mean ± SEM. A , E One-way ANOVA with ( A ) Tukey’s or ( E ) Dunnett’s multiple comparisons test, or ( D ) paired two-tailed Student t test. Scale bars, 50 µm. Source data are provided as a Source Data file.

Article Snippet: NF1 +/R681X or Nf1 +/neo sensory neurons were infected with three independent sh COL1A2 lentiviral particles or shRNA scrambled control particles (sc-108080; Santa Cruz Biotechnology) for 24 h. Neuronal media was refreshed and conditioned media was collected for subsequent assays 48–72 h post infection.

Techniques: Staining, Quantitation Assay, Incubation, Control, Expressing, Positive Control, Microarray, RNA Expression, Isolation, Two Tailed Test

Journal: Nature Communications

Article Title: Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

doi: 10.1038/s41467-022-30466-6

Figure Lengend Snippet: A , B TTX (1 µM; A ; vehicle n = 6, TTX n = 6; P < 0.0001) and lamotrigine (LTR; 200 µM; B ; vehicle n = 9, LTR n = 9; P = 0.0001) reduce Nf1 +/neo DRG neuron Col1a2 secretion by 73 and 47% relative to vehicle-treated controls. C ZD7288 (ZD; 30 µM) increases Col1a2 secretion in WT ( n = 10 in both groups; P < 0.0001) and Nf1 +/1809 ( n = 4 in both groups; P = 0.0103) DRG neurons. D RAS activity is increased in both Nf1 +/neo and Nf1 +/1809 DRG neurons relative to controls ( n = 5 in all groups; P < 0.0001), ( E ) and is inhibited following TTX and LTR treatment ( n = 6 in all groups; P < 0.0001). F , G IN-1 has no effect on DRG neuronal activity, as measured by ( F ) multi-electrode array (vehicle n = 6, IN-1 n = 3, ns not significant), or ( G ) calcium-imaging recordings (vehicle n = 18, IN-1 n = 18; ns, not significant). Right: representative ( F ) spike plots of entire multi-electrode array well recordings over 30 s, and ( G ) traces of neuronal activity over 3 min. H IN-1 reduces Col1a2 secretion by 77.9% in Nf1 +/neo DRG neurons. n = 6 for both groups, P = 0.0001. I IN-1 reduces proliferation by 50% in Nf1 −/ − DRG–NSCs. n = 6 for both groups, P < 0.0001. J Lamotrigine treatment decreases pNF progression in vivo. Gross images and representative immunostaining of mouse pNFs demonstrate that LTR treatment reduces pNF size, partly restores neuronal histology (H&E), reduces proliferation (Ki67 + cells) and decreases Col1a2 production. Scale bars: gross anatomy images, 1 mm; sections, 100 µm. n = 5 for both groups. Data are represented as means ± SEM ( A – C , H , I ) using two-tailed paired Student’s t tests, ( F , G ) two-tailed unpaired t tests, or ( D , E ) one-way ANOVA with Dunnett’s post-test correction. P values are indicated within each panel. ns, not significant. Source data are provided as a Source Data file.

Article Snippet: NF1 +/R681X or Nf1 +/neo sensory neurons were infected with three independent sh COL1A2 lentiviral particles or shRNA scrambled control particles (sc-108080; Santa Cruz Biotechnology) for 24 h. Neuronal media was refreshed and conditioned media was collected for subsequent assays 48–72 h post infection.

Techniques: Activity Assay, Imaging, In Vivo, Immunostaining, Two Tailed Test

Journal: Nature Communications

Article Title: Neuronal hyperexcitability drives central and peripheral nervous system tumor progression in models of neurofibromatosis-1

doi: 10.1038/s41467-022-30466-6

Figure Lengend Snippet: A Tumor-associated NF1 -mutant sensory neurons have increased baseline neuron excitability and deregulated HCN channel function, leading to elevated COL1A2 secretion. COL1A2, in turn, increases NF1 −/ − Schwann cell proliferation to stimulate pNF growth. B Tumor-associated NF1- mutant retinal ganglion cell (RGC) activity is governed by two distinct mechanisms. First, visual experience (light)-induced activity enhances RGC production of soluble-Nlgn3 (s-Nlgn3), which drives OPG initiation and cell growth. Second, tumor-associated NF1- mutant RGCs have increased intrinsic baseline neuronal hyperexcitability, which is controlled by HCN channel function. Increased baseline HCN channel-regulated RGC excitability triggers increased midkine production to induce a T-cell (Ccl4) and microglial (Ccl5) signaling cascade that governs OPG progression and growth. PNS, peripheral nervous system, CNS, central nervous system, pNF, plexiform neurofibroma, OPG, optic pathway glioma. Small elements of this schematic were designed on BioRender.com. Source data are provided as a Source Data file.

Article Snippet: NF1 +/R681X or Nf1 +/neo sensory neurons were infected with three independent sh COL1A2 lentiviral particles or shRNA scrambled control particles (sc-108080; Santa Cruz Biotechnology) for 24 h. Neuronal media was refreshed and conditioned media was collected for subsequent assays 48–72 h post infection.

Techniques: Mutagenesis, Activity Assay

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification and Analysis of Hub Genes in Diabetic Cardiomyopathy: Potential Role of Cytochrome P450 1A1 in Mitochondrial Metabolism and STZ-Induced Myocardial Dysfunction

doi: 10.3389/fcvm.2022.835244

Figure Lengend Snippet: Primer sequence for RT-qPCR.

Article Snippet: Col1a2 , 5′-TTCTGTGGGTCCTGCTGGGAAA-3′ , Forward , Refer to OriGene's website.

Techniques: Sequencing

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification and Analysis of Hub Genes in Diabetic Cardiomyopathy: Potential Role of Cytochrome P450 1A1 in Mitochondrial Metabolism and STZ-Induced Myocardial Dysfunction

doi: 10.3389/fcvm.2022.835244

Figure Lengend Snippet: Common differentially expressed genes in GSE4745 and GSE688.

Article Snippet: Col1a2 , 5′-TTCTGTGGGTCCTGCTGGGAAA-3′ , Forward , Refer to OriGene's website.

Techniques:

Journal: Frontiers in Cardiovascular Medicine

Article Title: Identification and Analysis of Hub Genes in Diabetic Cardiomyopathy: Potential Role of Cytochrome P450 1A1 in Mitochondrial Metabolism and STZ-Induced Myocardial Dysfunction

doi: 10.3389/fcvm.2022.835244

Figure Lengend Snippet: Hub genes and Ctsk expression in the hearts of non-diabetic and STZ-induced diabetic mice. (A) Cyp1a1 mRNA fold change; (B) Cyp2e1 mRNA fold change; (C) Ctsk mRNA fold change; (D) Col1a1 mRNA fold change; (E) Col3a1 mRNA fold change; (F) Col1a2 mRNA fold change. Mean ± SEM, n = 4–7 mice per group. * p < 0.05 and ** p < 0.01 vs. Non diabetic control group. ns, no significance.

Article Snippet: Col1a2 , 5′-TTCTGTGGGTCCTGCTGGGAAA-3′ , Forward , Refer to OriGene's website.

Techniques: Expressing, Control