Journal: Aging (Albany NY)

Article Title: MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma

doi: 10.18632/aging.202424

Figure Lengend Snippet: miR-29a-3p inhibited migration and VM formation in glioma cells. ( A ) The effect of miR-29a-3p on cell movement was assessed using transwell migration assays (scale bar, 100 μm; n=3). ( B ) Quantification of transwell migration assays in ( A ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( C ) Effect of miR-29a-3p on VM formation ability (scale bar, 200 μm; n=3). ( D ) Quantification of relative VM number in ( C ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( E ) Western blot analysis of protein levels of N-cadherin, MMP9 and LAMB2 in A172 and U87 cells. GAPDH was used as a whole-cell protein loading control. Results are from three independent experiments.

Article Snippet: The blots were incubated with primary antibodies against ROBO1 (rabbit anti-ROBO1 polyclonal antibody, 1:500 dilution, 20219-1-AP; Proteintech; China), LAMB2 (rabbit anti-LAMB2 polyclonal antibody, 1:1000 dilution, 10895-1-AP; Proteintech; China), N-cadherin, MMP9, and GAPDH (1:1000; Cell Signaling Technology; USA).

Techniques: Migration, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: MicroRNA-29a-3p delivery via exosomes derived from engineered human mesenchymal stem cells exerts tumour suppressive effects by inhibiting migration and vasculogenic mimicry in glioma

doi: 10.18632/aging.202424

Figure Lengend Snippet: miR-29a-3p inhibited migration and VM formation by directly targeting ROBO1. ( A ) Schematic representation of the predicted binding sites for miR-29a-3p in the ROBO1 3′-UTR (wild type; WT) and the designed mutant versions (mutant; MUT) of the ROBO1 3’-UTR (left panel). Relative luciferase activity of HEK293T cells in the presence of the indicated treatments (middle and right plots). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( B ) Western blot analysis of the protein level of ROBO1,N-cadherin, MMP9 and LAMB2 after miR-29a-3p transfection. Results are from three independent experiments. ( C ) Western blot analysis of the expression of ROBO1,N-cadherin, MMP9 and LAMB2 after ROBO1 and miR-29a-3p overexpression. Results are from three independent experiments. ( D ) Representative images for the transwell assay (scale bar, 100 μm; n=3). ( E ) Quantification of transwell migration assays in ( D ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05). ( F ) Representative images for the VM formation assay (scale bar, 200 μm; n=3). ( G ) Quantification of relative VM numbers in ( F ). Data are shown as the mean±SD, n=3, one-way ANOVA (*, P < 0.05).

Article Snippet: The blots were incubated with primary antibodies against ROBO1 (rabbit anti-ROBO1 polyclonal antibody, 1:500 dilution, 20219-1-AP; Proteintech; China), LAMB2 (rabbit anti-LAMB2 polyclonal antibody, 1:1000 dilution, 10895-1-AP; Proteintech; China), N-cadherin, MMP9, and GAPDH (1:1000; Cell Signaling Technology; USA).

Techniques: Migration, Binding Assay, Mutagenesis, Luciferase, Activity Assay, Western Blot, Transfection, Expressing, Over Expression, Transwell Assay, Tube Formation Assay

Journal: Molecular Oncology

Article Title: LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

doi: 10.1002/1878-0261.13053

Figure Lengend Snippet: Laminin subunit gamma 1 (LAMC1) expression was upregulated by transforming growth factor β (TGFβ1) via SMAD family member 4 (SMAD4) and SP1 synergistic activation and was associated with a poor prognosis in patients with esophageal squamous cell carcinoma (ESCC). (A) Heatmap of the RNA‐seq data listing genes positively correlated with TGFβ1 in ESCC of GSE53625 ( r > 0.15, P < 0.05). (B) A total of 3084 genes of KYSE30 and KYSE180 were upregulated after TGFβ1 treatment using RNA‐seq data (fold change > 1). (C) Venn diagram of 652 overlapping genes based on positive regulation by TGFβ1 in ESCC. (D) The 31 genes of 652 genes were enriched in 11 pathways involved in the tumor microenvironment. (E,F) LAMC1 was more highly expressed in cancer than in para‐cancer at the RNA and protein levels. Representative immunohistochemical (IHC) images of LAMC1 staining in ESCC tumor tissues and nontumor tissues (original magnification: 200x). (G) Kaplan–Meier survival analysis of OS based on high ( n = 89) and low ( n = 90) LAMC1 expression in GSE53625 (left) and based on high ( n = 35) and low ( n = 20) LAMC1 expression (by IHC, right). (H,I) As verified by western blot and RT‐qPCR, TGFβ1 upregulated LAMC1 expression in a concentration‐ (at different concentrations for 24 h) and time‐dependent manner (at 5 ng·mL −1 for different times) in KYSE30 and KYSE450 cells (H), which could be reversed by the TGFβR1 inhibitor SB505124 (10 μ m ) at the protein (I) and RNA (J) levels. (K,L) After KYSE30 cells were treated with TGFβ1 (5 ng·mL −1 ) for 30 min, the localization of SMAD4 or SP1 to the LAMC1 promoter was detected by ChIP, and the expression of SMAD4 or SP1 was detected in anti‐SP1 or anti‐SMAD4 chromatin fractions separately by western blot (L). (M,N) Knockdown efficiency of SP1 combined with or without SMAD4 knockdown in ESCC cells was verified by western blotting. (O–Q) The expression levels of LAMC1 decreased in ESCC cells with SP1 knockdown combined with (P) or without (O) knockdown, but this effect could not be rescued by TGFβ1 treatment (Q). Three biological replicates were performed for in vitro assays. The data in bar charts are presented as the mean ± SD. ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t ‐test).

Article Snippet: Lenti ORF clone of human LAMC1 and vector was purchased form OriGene (RC216928L4, PS100093) for overexpression of LAMC1.

Techniques: Expressing, Activation Assay, RNA Sequencing Assay, Immunohistochemical staining, Staining, Western Blot, Quantitative RT-PCR, Concentration Assay, In Vitro

Journal: Molecular Oncology

Article Title: LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

doi: 10.1002/1878-0261.13053

Figure Lengend Snippet: LAMC1 promoted the proliferation and metastasis of ESCC cells in vivo and in vitro . (A,B) Knockdown or overexpression efficiency of LAMC1 in ESCC cells was verified by western blot and RT‐qPCR. (C) The rate of cell growth of KYSE30 and KYSE450 cells treated with shLAMC1 or overexpressing LAMC1 and vector control cells was measured by CCK‐8 assay. (D) Apoptosis of shLAMC1‐1, ‐2 and sh‐vec KYSE30 and KYSE450 cells was analyzed using the Annexin V APC Apoptosis Detection Kit. (E,F) The migration ability of shLAMC1‐1, ‐2, sh‐vec, LAMC1 and vector KYSE30 and KYSE450 cells was detected by chamber assay. The numbers of migrating cells were compared between the groups. (G–J) Representative images of tumor formation in nude mice subcutaneously inoculated with KYSE30 cells stably expressing LAMC1 shRNA or negative control and expressing LAMC1 or mock‐vehicle control (G, I), and tumor weights and volumes for groups (H, J). (K,L) Representative images of lung tissues isolated from mice injected with 1 × 10 6 shLAMC1, sh‐vec, LAMC1 or vector KYSE30 cells via the tail vein and hematoxylin and eosin stained images (200×) of such tissues, and quantification of lung metastasis (scale bars: 1 mm and 200 µm for the top and bottom rows of photomicrographs, respectively). Three biological replicates were performed for in vitro assays. The data in bar charts are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t ‐test).

Article Snippet: Lenti ORF clone of human LAMC1 and vector was purchased form OriGene (RC216928L4, PS100093) for overexpression of LAMC1.

Techniques: In Vivo, In Vitro, Over Expression, Western Blot, Quantitative RT-PCR, Plasmid Preparation, CCK-8 Assay, Migration, Boyden Chamber Assay, Stable Transfection, Expressing, shRNA, Negative Control, Isolation, Injection, Staining

Journal: Molecular Oncology

Article Title: LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

doi: 10.1002/1878-0261.13053

Figure Lengend Snippet: The positive effect of LAMC1 on ESCC cell migration occurred mainly via the Akt/IKKα/NF‐Κb/MMP9/14 pathway. (A) Antiapoptosis, NF‐κB and cytokine‐related pathways enriched by sh‐vec using GSEA. (B,C) Expression levels of phosphorylated Akt, NF‐κB, IKKα, MMP9 and MMP14 in KYSE30 and KYSE450 cells stably expressing LAMC1 shRNA or negative control and expressing LAMC1 or mock‐vehicle control. (D) Immunofluorescence staining of p65 in shLAMC1‐1, shLAMC1‐2, sh‐vec KYSE30 and KYSE450 cells. Nuclei were visualized with DAPI staining (blue). Representative images are shown. Scale bar: 34 μm. (E,F) Phosphorylation of IKKα, p65, MMP9 and MMP14 was also detected in shLAMC1 KYSE30 and KYSE450 cells treated with or without TNFα stimulation (E), and overexpressing LAMC1 with or without 5 μ m Akt phosphorylation selective inhibitor MK‐2206 2HCI and 10 μ m NF‐κB JSH‐23 stimulation (F). (G,H) Representative images of migration of these subgroups, shLAMC1 with or without TNFα (10 ng·mL −1 ) stimulation, overexpression of LAMC1 with or without JSH‐23 stimulation, measured by chamber assay. Three biological replicates were performed for in vitro assays.

Article Snippet: Lenti ORF clone of human LAMC1 and vector was purchased form OriGene (RC216928L4, PS100093) for overexpression of LAMC1.

Techniques: Migration, Expressing, Stable Transfection, shRNA, Negative Control, Immunofluorescence, Staining, Over Expression, Boyden Chamber Assay, In Vitro

Journal: Molecular Oncology

Article Title: LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

doi: 10.1002/1878-0261.13053

Figure Lengend Snippet: LAMC1 inhibited apoptosis possibly through the Akt/NF‐κB/caspase9‐caspase3‐PARP cascade. (A,B) Western blotting was conducted to detect the protein levels of cleaved caspase‐3, caspase‐9, and PARP in KYSE30 and KYSE450 cells stably expressing LAMC1 shRNA or negative control and expressing LAMC1 or mock‐vehicle control, which were induced by 10 µ m cisplatin for 24 h. (C,D) After cisplatin induction (10 m m for 24 h), cleaved caspase‐3, caspase‐9 and PARP were detected in KYSE30 and KYSE450 cells treated with shLAMC1 with or without TNFα stimulation (C) or overexpressing LAMC1 with or without 5 μ m Akt phosphorylation selective inhibitor MK‐2206 2HCI and 10 μ m NF‐κB nuclear translocation inhibitor JSH‐23 stimulation (D). (E–G) Proliferation and apoptosis of KYSE30 and KYSE450 cells stably expressing LAMC1 shRNA or negative control and expressing LAMC1 or mock‐vehicle control with or without TNFα or JSH‐38 (JSH), as measured by a CCK8 assay. Three biological replicates were performed for in vitro assays. The data in bar charts are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t ‐test).

Article Snippet: Lenti ORF clone of human LAMC1 and vector was purchased form OriGene (RC216928L4, PS100093) for overexpression of LAMC1.

Techniques: Western Blot, Stable Transfection, Expressing, shRNA, Negative Control, Translocation Assay, CCK-8 Assay, In Vitro

Journal: Molecular Oncology

Article Title: LAMC1 upregulation via TGFβ induces inflammatory cancer‐associated fibroblasts in esophageal squamous cell carcinoma via NF‐κB–CXCL1–STAT3

doi: 10.1002/1878-0261.13053

Figure Lengend Snippet: LAMC1 promoted CXCL1 secretion mainly by activating NF‐κB. (A) LAMC1 affected the cytokine and chemokine signaling pathways according to GSE53625 data. (B) A 48‐cytokine panel was used to detect cytokines in the CM of shLAMC1 and sh‐vec KYSE450 cells. (C) Expression of three cytokines in the CM of KYSE30 and KYSE450 cells stably expressing LAMC1 shRNA or negative control and expressing LAMC1 or mock‐vehicle control by western blot. (D) CXCL1 expression in the CM of shLAMC1 and LAMC1‐overexpressing KYSE30 and KYSE450 cells, as measured by ELISA. (E) Top 20 predicted transcription factors of CXCL1 in the Cistome network. (F,G) ELISA (F) and western blotting (G) were conducted to detect CXCL1 expression in the CM of shLAMC1 and sh‐vec cells with or without TNFα treatment, and of LAMC1‐overexpressing cells with or without 5 μ m Akt phosphorylation selective inhibitor MK‐2206 2HCI and 10 μ m NF‐κB nuclear translocation inhibitor JSH‐23 stimulation. (H) RT‐qPCR was conducted to detect CXCL1 expression in shLAMC1 and sh‐vec cells with or without TNFα treatment. (I) LAMC1 was positively associated with CXCL1 based on GSE53625 data. (J) CXCL1 was upregulated in tumor tissue compared with that in adjunct tissue in the GSE53625 dataset. Three biological replicates were performed for in vitro assays. The data in bar charts are presented as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001 (Student’s t ‐test).

Article Snippet: Lenti ORF clone of human LAMC1 and vector was purchased form OriGene (RC216928L4, PS100093) for overexpression of LAMC1.

Techniques: Expressing, Stable Transfection, shRNA, Negative Control, Western Blot, Enzyme-linked Immunosorbent Assay, Translocation Assay, Quantitative RT-PCR, In Vitro

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: Bright-field microscopy of hPSCs on (A) various laminin isoforms (y-axis) and (B) FDA-approved polymers over time (x-axis). Alg = alginate, Col = collagen-1, HA = hyaluronic acid, MC = methylcellulose, MTG (+) = Geltrex (positive control), and () = Tissue culture treated glass bottom plate (negative control). Scale bar = 100μm. (C) Example of OCT4 MFI with stem cell numbers, and (D) the calculated stemness index (SI) and growth rates on various hydrogel-forming FDA-approved proteins after 7 days.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Microscopy, Positive Control, Negative Control

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) Cell confluence of hPSC cultured in 2D on growth factor reduced Matrigel (Geltrex), Laminin 521, fibrin-laminin hydrogel, and fibrin-only gel after four days. Scale bar = 100μm. (B) hPSCs cultured in 3D hydrogels. Top panel: “High” concentration fibrin (5mg/ml) and “low” Laminin 521 (5μg/ml) concentration. Bottom panel: Alphagel containing structures resembling embryoid bodies, scale bars = 200μm. (C) SEM of fibrin-only hydrogel, scale bar = 1 μm, magnification 20K X, iProbe = 20pA, 6.00kV, WD = 11.0mm. (D) SEM of Alphagel, scale bar = 1 μm, magnification 20K X, iProbe = 13pA, 2.00kV, WD = 4.4mm. (E) Measurements of Young’s moduli in hydrogels with varying fibrin and laminin concentrations. One-way ANOVA; ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001. (F) Cell viability of hPSCs cultured in Alphagel, fibrin-only hydrogels, and 2D standard substrates. Mean ± S.E.M displayed. t-test; * = p < 0.05. (G) ELISA of Laminin 521 in culture media used with acellular Alphagel and (H) the calculated amount of fibrin-bound laminin over time.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Cell Culture, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: Stem cell marker expression (MFI) presented as mean ± standard error of the mean (S.E) and stemness index (SI) in various culture conditions after 3-4 months. Bar chart x-axis label key: (i) cell type, (ii) fibrin concentration (2mg/ml or 4mg/ml), (iii) gel-type = fibrin-laminin hydrogel (FLG) or fibrin only (F), and (iv) media type = mTeSRs/TeSR2 (M), TeSR-E8 (TE8), and E8 (E8). 2FLG is representative of Alphagel. HESCs cultured in 2mg/ml fibrin gel with FLG in E8 media. The absence of bars and/or S.E. was because cells died or differentiated in other repeats and no longer expressed stem cell markers. p values derived using the Mann Whitney U test (MFI), Chi-square test (SI) or Fisher test (when n < 5 for any cell).

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Marker, Expressing, Concentration Assay, Cell Culture, Derivative Assay, MANN-WHITNEY

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) MFI in varying thrombin concentrations. Thrombin 0 (control) = Laminin 521 and fibrinogen without thrombin; effectively a 2D Laminin 521 coating as fibrinogen was un-crosslinked. MFI was slightly lower in 3D fibrin-laminin with 0.01% thrombin because cross-linking of fibrin with Laminin 521 was sub-optimal. Statistical comparison using One-way ANOVA, * = p<0.05. (B) Cost-analysis to synthesize a 50μl hydrogel dome for 3D cell culture; Alphagel vs Matrigel.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Control, Comparison, Cell Culture

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) Macroscopic and microscopic appearances of cells in Alphagel. Contractile cardiomyocyte colonies (*) are visible by Day 6. By Day 14 contractile tissue resembling trabeculae carnae is observed. Scale bar: full line = 100μm, dotted line = 1cm. (B) Troponin T (TnT), alpha-smooth muscle actin (α-SMA), and DAPI in cardiomyocytes cultured with Alphagel. Scale bar = 100μm (fibres), 50μm (colonies), and 20μm (cells). (C) Neural progenitors showing Nestin, β catenin, and DAPI staining. Scale bar = 50μm. (D) Average lumen area of neuroepithelia, controls (neurospheres, fibrin-only gel, 2D laminin) vs. Alphagel. (E) Key hepatocyte markers in Alphagel-derived iHeps. ALB = albumin, HNF = Hepatocyte Nuclear Factor, CYP2A6 = Cytochrome 2A6, CD147 = Cluster of Differentiation protein 147, and E-CAD = E-cadherin. Scale bar = 25μm. (F) Key hepatocyte markers by gene expression (qPCR). (G) Albumin secretion (ELISA) and (H) CYP3A4 activity (P450-GloTM) in PHH vs iHeps cultured in various gels (Day 22). HCM = Hepatocyte Culture Media (Lonza). (I) LDL uptake (red) in iHeps cultured in Alphagel. Scale bar = 100μm. (J) CDFDA secretion (green) in iHeps derived in Alphagel. Top panel scale bar = 20μm, bottom panel scale bar = 50μm. For (D), the Kruskal-Wallis test was used for non-parametric data. For all other comparisons, the one-way ANOVA was used. * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Cell Culture, Staining, Derivative Assay, Gene Expression, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) H&E stains of sites injected with 1% Croton Oil, Alphagel, and 0.9% Saline at 1 and 2 weeks. Black arrow denotes areas of granulation and inflammation, * denotes necrotic and degenerating muscle fibres, and # denotes supportive changes around affected muscle fibres. Scale bar = 200μm. (B) Histological grade and scores of injection sites over 6 weeks. (C) Highlighted changes in gene expression in iHeps derived with Hepatologel versus, Alphagel, Matrigel, and adult primary human hepatocytes (PHH). (D) Albumin ELISA of culture media collected from iHeps 2 days after completion of hepatic differentiation and from PHH 2 days after plating. (E) Luciferin-based measure of CYP3A4 activity in iHeps 2 days after completion of hepatic differentiation and PHH 2 days after plating. One-way ANOVA; * = p < 0.05, ** = p < 0.01, *** = p < 0.001, and **** = p < 0.0001.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Injection, Saline, Gene Expression, Derivative Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) ELISA of culture medium for Apolipoprotein B (APOB), taken from iHeps 2 days after the end of the hepatic differentiation protocol, and from PHH culture media 2 days after plating. (B) Brightfield microscopy of 3D tubular branching strictures observed when Laminin 411 concentrations are increased. Scale bar = 100μm. (C) Alkaline Phsophatase (ALP) activity measured by colourimetric assay on culture medium taken 2 days the end of the hepatic differentiation protocol.

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Enzyme-linked Immunosorbent Assay, Microscopy, Activity Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: (A) Mouse livers excised 3 days after intra-hepatic injection with H-iHeps in Hepatogel and 0.9% saline. Red box = area magnified. * = site of injection, scale bar = 1mm. (B) Liver section and IF staining of human albumin (red) in transplanted iHeps engrafted in mouse liver 3 days after intra-hepatic injection. Scale bar = 100μm. (C) Cell counts for H-iHeps identified by albumin staining on liver histology; samples harvested 3 days after intra-hepatic injection. (D) ELISA of mouse serum for human albumin after injection with H-iHeps in Hepatogel and 0.9% saline over time. Day 0 = serum levels before injection. T-test; *** = p < 0.001, and **** = p < 0.0001

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Injection, Saline, Staining, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: A chemically defined and xeno-free hydrogel system for regenerative medicine

doi: 10.1101/2024.05.28.596179

Figure Lengend Snippet: Tube-like formations in hESCs on cultured on Laminin 111 with mTESR media on 2 nd passage, x40 magnification. Scale bar = 250μm

Article Snippet: Laminin concentrations in supernatants were measured using a laminin ELISA kit (Insight Biotechnology, #Boster Pikokine TM EK0434).

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: The Role of Sensory Nerves in Dental Pulp Homeostasis: Histological Changes and Cellular Consequences after Sensory Denervation

doi: 10.3390/ijms25021126

Figure Lengend Snippet: Antibodies used in this study.

Article Snippet: Laminin , Rabbit , Boster , PA1581.

Techniques:

Journal: Stem Cell Research & Therapy

Article Title: Pax7 as molecular switch regulating early and advanced stages of myogenic mouse ESC differentiation in teratomas

doi: 10.1186/s13287-020-01742-3

Figure Lengend Snippet: Analysis of basal lamina and NMJ functionality in Pax7+/+ and Pax7−/− teratomas. a – c Expression of mRNAs encoding Lama4 , Lama5 , and Lamb2 (for each genotype n = 8 to 6). d , e Expression of mRNAs encoding neuronal markers RbFox3 and Otx2 (for each genotype n = 6 to n = 7). f Immunolocalization of neurofilament (green, left column, TL transmitted light), synaptophysin (red, middle column; BTX—green; nuclei—blue), and Fasciculin (red, right column; BTX—green; nuclei—blue) in teratoma muscles. Scale bar 10 or 100 μm, as indicated. White bars—values for teratomas obtained from Pax7+/+ ESCs, gray bars—values for teratomas obtained from Pax7−/− ESCs. Data are presented as mean ± SD. Stars symbolize results of Student’s unpaired two-tailed t test: * p < 0.05; ** p < 0.01

Article Snippet: mRNA was isolated from teratomas, using mirVana Isolation Kit (Thermo Fisher Scientific). mRNA analysis reverse transcription was performed using 1 μg total RNA and RevertAid First Strand cDNA Synthesis Kit (Thermo Fisher Scientific) according to the manufacturer’s instruction. qPCR was performed using specific TaqMan® probes: Mm02019550_s1 ( Nanog ), Mm00435493_m1 ( Pax3 ), Mm00435125_m1 ( Myf5 ), Mm00440387_m1 ( Myod1 ), Mm00446194_m1 ( MyoG ), Mm00483191_m1 ( Cdh15 ), Mm01318252_m1 ( T encoding Brachyury), Mm00477791_m1 ( Nfix ), Mm00468267_m1 ( Eno3 ), Mm01321487_m1 ( Mck ), Mm00434400_m1 ( Itga7 ), Mm01318991_m1 ( Mef2a ), Mm00452375_m1 ( Nfatc4 ), Mm01332463_m1 ( Myh3 ), Mm01332564_m1 ( Myh2 ), Mm01319006_g1 ( Myh7 ), Mm01332541_m1 ( Myh4 ), Mm01226102_m1 ( Lama1 ), Mm00493080_m1 ( Lamb2 ), Mm01193660_m1 ( Lama4 ), Mm01222010_g1 ( Lama5 ), Mm01248771_m1 ( RbFox3 ), Mm00446859_m1 ( Otx2 ), Mm01205647_g1 ( Actb ), and the TaqMan Gene Expression Master Mix (Thermo Fisher Scientific).

Techniques: Expressing, Muscles, Two Tailed Test

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: Correlation between the density of LAMC1 at gastric cancer and clinicopathologic parameters

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Expressing

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: Gastric cancer with high expression of LAMC1 have higher migration and invasion ability in primary site. (A) Expression of LAMC1 in gastric cancer cell lines was analyzed by ELISA and Western blots. (B) Western blots were used for E-cadherin and vimentin expression in gastric cancer cells with LAMC1 knockout. (C) Transwell assay in gastric cancer cells with LAMC1 knockout. (D) The morphological characteristics of tumor xenograft model in AGS NC/si group, and tumor weight and volume were shown. (E) The HE staining and immunohistochemical results of xenograft tumor in AGS NC/si group.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Expressing, Migration, Enzyme-linked Immunosorbent Assay, Western Blot, Knock-Out, Transwell Assay, Staining, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: LAMC1-mediated preadipocytes differentiation promotes pre-metastatic niche formation and gastric cancer cell colonization in peritoneal microenvironment. (A) The Oil Red O staining and Western bolts for analyzing effect of different concentrations of LAMC1 (0, 50ng/ml and 100ng/ml) on 3T3-L1 and human omental preadipocytes differentiation for 5 days. (B) Cytokines secreted by 3T3-L1 after induction of different concentrations of LAMC1 for 24h were measured by RT-qPCR . ( C ) HSL expression in induced 3T3-L1 detected by RT-qPCR and western blots. (D) ELISA was used for analyzed FFAs, lipid droplets, adiponectin and leptin in 3T3-L1 CM after stimulation by different concentrations of LAMC1 for 4 days. (E) ELISA was used for analyzed FFAs in human omental preadipocytes CM after stimulation by different concentrations of LAMC1 for 4 days. (F) The flow chart of coculture. (G) ELISA for analyzing LAMC1 levels in supernatant of gastric cancer cell lines transfected with siLAMC1. (H) The lipid droplet formation ability of 3T3-L1 was measured by Oil Red O staining and adipocyte differentiation related protein after coculture for 5 days with gastric cancer cell supernatant transfected siLAMC1 for 48h. (I) Flowchart of reverse co-culture of 3T3-L1 supernatant and gastric cancer cells. (J) Western blots were used for E-cadherin and vimentin expression in gastric cells after coculture for 48h with 3T3-L1 supernatant induced by LAMC1 (0, 50ng/ml and 100ng/ml) for 4 days. (K) CCK8 assay for AGS cell proliferation detection . (L) The morphological characteristics of tumor xenograft model in AGS NC/si and 3T3-L1 coculture group, and tumor weight were shown. (M) The HE staining and immunohistochemical results of xenograft tumor in AGS NC/si and 3T3-L1 coculture group. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Staining, Western Blot, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Transfection, Co-Culture Assay, CCK-8 Assay, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: Differentiated preadipocytes remodel metabolic programming of gastric cancer cells. (A) Electron microscope was used to observe the content of lipid droplets in AGS cells after coculture for 48h with 3T3-L1 supernatant induced by LAMC1 (0, 50ng/ml and 100ng/ml) for 4 days. Blank group meant AGS cells weren't cocultured with 3T3-L1 supernatant. (B-D) The results of extracellular acidification, fatty acid oxidation and extracellular O 2 consumption in AGS cells after reverse coculture for 48h was shown. (E) The ratio of NADPH / NADP+ in AGS cells after reverse coculture for 48h was assessed as materials and method. (F) The ATP content in AGS cells after reverse coculture for 48h was shown. (G) AGS cells treated with 0.5mM palmitic acid for 48h were analyzed by mass spectrometry. The Heat map of differential genes was shown. (H) AGS cells treated with 0.5mM palmitic acid for 48h were analyzed by mass spectrometry. The Gene function classification of differential genes was shown. (I) The RT- qPCR was for analyzing expression of metabolism-related genes in AGS cells after treatment with 0.5mM palmitic acid or reverse coculture with 3T3-L1 CM for 24h. 10uM DC260126 was used to inhibit the effect of palmitic acid. (J) The immunohistochemical results of xenograft tumor in AGS NC/si and 3T3-L1 coculture group. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Microscopy, Mass Spectrometry, Quantitative RT-PCR, Expressing, Immunohistochemical staining

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: Palmitic acid phosphorylates STAT3 and promotes LAMC1 secretion through miR-193a-3p. (A) AGS cells treated with 0.5mM palmitic acid for 48h was used for mass spectrometry analysis and the three most abundant pathway proteins were shown. (B) The western blots were for analyzing pathway protein expression in AGS cells after treatment with 0.5mM palmitic acid for 48h or reverse coculture with 3T3-L1 supernatant induced by 0 and 100ng/ml LAMC1 for 48h. 10uM DC260126 was used to inhibit the effect of palmitic acid. (C) AGS cells transfected with siSTAT3 had a low LAMC1 expression determined by RT-qPCR, Western blots. (D) LAMC1 expression in supernatant of AGS cells transfected with siSTAT3. (E) Flowchart of co-culture of gastric cancer cell supernatant and 3T3-L1. (F) The gastric cancer cell supernatant transfected with siSTAT3 for 48h was collected to coculture with 3T3-L1 for 5 days. Western blots and the Oil Red O staining were used for lipid formation ability. (G) Three independent miRNA target databases were used to predict the potential miRNAs. (H) RT-qPCR was for analyzing miR193a-3p, miR384 and miR448 expression levels in AGS cells with STAT3 overexpression. (I) The gastric cancer cell supernatant transfected with miR-193a-3p mimic, inhibitor or NC for 48h was collected to coculture with 3T3-L1 for 5 days, and the Oil Red O staining and Western blots were used for lipid formation ability. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Mass Spectrometry, Western Blot, Expressing, Transfection, Quantitative RT-PCR, Co-Culture Assay, Staining, Over Expression

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: The miR-193a-3p inhibits LAMC1 expression in a post-transcriptional manner. (A-B) AGS cells were transfected with miR-193a-3p mimic, inhibitor or NC, and agarose gel electrophoresis and RT-qPCR were used to measure RNA level change, Western blots and ELISA for protein level. (C) The AGS cells were transfected with siLAMC1, RT-qPCR for miR-193a-3p expression. (D) A structure diagram of the pmirGLO dual-luciferase reporter vector with 3′-UTR of LAMC1 mRNA harbors miR-193a-3p binding sites. (E) The relative luciferase activity of reporter plasmids carrying wild-type or mutant LAMC1 3′-UTR. (F) The LAMC1 mRNA decay curves of AGS cells carrying miR-193a-3p mimic or mimic NC. (G) The AGS cells were transfected with miR-193a-3p mimic or mimic NC for 48h, and AGO2-RNA immunoprecipitation assay (RIP) was used for analyzed the combination method of miR-193a-3p and LAMC1. Error bars, SD. *P < 0.05; **P < 0.01; ***P < 0.001.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Expressing, Transfection, Agarose Gel Electrophoresis, Quantitative RT-PCR, Western Blot, Enzyme-linked Immunosorbent Assay, Luciferase, Plasmid Preparation, Binding Assay, Activity Assay, Mutagenesis, RNA Immunoprecipitation

Journal: International Journal of Biological Sciences

Article Title: LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis

doi: 10.7150/ijbs.70524

Figure Lengend Snippet: Gastric cancer with high LAMC1 expression has high risk of peritoneal metastasis. (A) Gross anatomy and nodule numbers of abdominal cavity in nude mice and the red arrows indicated micrometastases in the gastric cancer peritoneal metastasis model. (B) ROC curve. (C) The mechanism diagram of LAMC1-mediated preadipocytes differentiation promoted peritoneum pre-metastatic niche formation and gastric cancer metastasis.

Article Snippet: In order to confirm the effect of LAMC1 on the differentiation of adipocytes, different concentrations of LAMC1 recombinant protein (0, 50ng/ml, 100ng/ml) (Ag14674; Proteintech, USA) dissolved in phosphate-buffered saline (PBS, pH 7.4) were added to each fresh medium used for replacement.

Techniques: Expressing