Journal: Cancer letters

Article Title: KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer

doi: 10.1016/j.canlet.2021.10.019

Figure Lengend Snippet: A, KDM1A expression was examined in normal (n = 35) and tumor tissues (n = 546) from Uterine corpus endometrial carcinoma cases in the publicly available TCGA database using UALCAN portal. B, HEC1A and RL95–2 cells were transduced with control shRNA or KDM1A shRNA lentivirus and KDM1A knockdown was confirmed by Western blot analysis. C, Relative cell viability rates of control or KDM1A shRNA stably expressing HEC1A and RL95–2 cells were determined by MTT assay. HEC1A (D) and primary patient derived EC (E) cells were treated with different KDM1A inhibitors for 72 h and cell viability was examined using MTT assay. F, HEC1A control and KDM1A knockdown cells were treated with 119 FDA approved drugs and the drugs that showed significant inhibitory activity on the cell viability of KDM1A knockdown cells are shown. G, Control and KDM1A knockdown cells were treated with either vehicle or sirolimus (rapamycin), everolimus, or temsirolimus and the cell viability was examined using MTT assay. RL95–2 (H), primary patient derived EC (I, J) cells that stably express control shRNA or KDM1A shRNA were treated with rapamycin and the cell viability was determined using MTT assay. K, HEC1A cells were treated with NCD38 and rapamycin alone or in combination for 96 h and the cell viability was determined using MTT assay. Data are represented as mean ± SE. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Article Snippet: Human EC cell lines, HEC1A and RL95–2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as per ATCC guidelines.

Techniques: Expressing, Transduction, Control, shRNA, Knockdown, Western Blot, Stable Transfection, MTT Assay, Derivative Assay, Activity Assay

Journal: Cancer letters

Article Title: KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer

doi: 10.1016/j.canlet.2021.10.019

Figure Lengend Snippet: A, B, HEC1A and RL95–2 cells stably expressing control or KDM1A shRNA were serum starved for 24 h followed by stimulation with 10% FBS for 0, 10, 30, and 60 min. C, D, HEC1A and RL95–2 cells were serum starved for 24 h and pretreated with NCD38 (2 μM) for 1 h following stimulation with 10% FBS for 0, 10, 30, and 60 min. The activation of Akt/mTOR signaling components was profiled using western blotting. HEC1A (E) and RL95–2 (F) cells stably expressing control or KDM1A shRNA were treated with vehicle or rapamycin and the phosphorylation of Akt was determined using western blotting.

Article Snippet: Human EC cell lines, HEC1A and RL95–2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as per ATCC guidelines.

Techniques: Stable Transfection, Expressing, Control, shRNA, Activation Assay, Western Blot, Phospho-proteomics

Journal: Cancer letters

Article Title: KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer

doi: 10.1016/j.canlet.2021.10.019

Figure Lengend Snippet: A, B, HEC1A and RL95–2 cells stably expressing control or KDM1A shRNA were treated with either vehicle or rapamycin, and cell survival was determined by colony formation assays. Quantitation of colony area is shown (C, D). E, HEC1A cells were treated with NCD38 and rapamycin alone or in combination and survival was determined using colony formation assays. The colony area in each group was quantitated (F). G, H, Migratory ability of HEC1A cells stably expressing control shRNA or KDM1A shRNA following rapamycin treatment was determined using scratch wound healing assay. I, J, The effect on cell migration of HEC1A cells treated with NCD38 and rapamycin alone or in combination was determined. Relative migration of cells was quantified by ImageJ software. Data are represented as mean ± SE. **p < 0.01; ****p < 0.0001.

Article Snippet: Human EC cell lines, HEC1A and RL95–2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as per ATCC guidelines.

Techniques: Stable Transfection, Expressing, Control, shRNA, Quantitation Assay, Wound Healing Assay, Migration, Software

Journal: Cancer letters

Article Title: KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer

doi: 10.1016/j.canlet.2021.10.019

Figure Lengend Snippet: Total RNA was isolated from HEC1A cells stably expressing control or KDM1A shRNA after treatment with vehicle or rapamycin for 24 h and subjected to RNA sequencing. A, Heat map showing the hierarchical clustering of all samples and genes with log2 fold change>0.5, adjusted p-val<0.05. B, Volcano plots comparing the gene expression levels for the HEC1A KDM1A knockdown cells vs. control cells. Counts values were normalized with DESeq2, X axis showed the Log2 (fold change) of gene expression levels between groups, and Y axis indicated the -log10(adjusted p value). Significantly upregulated or down-regulated genes were marked in red and blue, respectively, with the criteria of padj<0.05 and abs(log2(fold change)) >0.25. Essential differentially expressed PI3K/AKT/mTOR pathway genes (MTOR, RICTOR, MAPKAP1, IRS1, IRS2, NRG1, PDPK1, PIK3CA, and PRKCB) were labeled in the plot. C-D, GSEA testing correlation of KDM1A shRNA modulated genes with signatures of PI3K events and phosphatidylinositol gene sets. E-F, the genes involved in mTORC2, PI3K and IGF signaling were validated in HEC1A and RL95–2 cells using RT-qPCR. G, HEC1A cells were subjected to chromatin immunoprecipitation (ChIP) and enrichment of KDM1A at the gene promoter regions was determined. H, HEC1A cells were treated with vehicle or NCD38 (3 μM) for 6 h and the enrichment of repressive histone methylation mark H3K9me2 at the gene promoter regions was analyzed by ChIP.

Article Snippet: Human EC cell lines, HEC1A and RL95–2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as per ATCC guidelines.

Techniques: Isolation, Stable Transfection, Expressing, Control, shRNA, RNA Sequencing, Gene Expression, Knockdown, Labeling, Quantitative RT-PCR, Chromatin Immunoprecipitation, Methylation

Journal: Cancer letters

Article Title: KDM1A inhibition augments the efficacy of rapamycin for the treatment of endometrial cancer

doi: 10.1016/j.canlet.2021.10.019

Figure Lengend Snippet: A, Athymic nude mice were implanted subcutaneously with HEC1A cells. After tumor establishment, mice were randomized to receive either vehicle or NCD38 (10 mg/kg body weight) or rapamycin (5 mg/kg body weight) or combination of NCD38+rapamycin daily for 5 days a week. Tumor volumes are shown in the graph. B, Body weights of the mice are shown. C, EC PDX (EC14) tumor bearing mice were treated with vehicle or SP2509 (30 mg/kg body weight) or rapamycin (5 mg/kg body weight) or combination of SP2509+rapamycin daily for 5 days a week. Tumor volumes are shown in the graph. D, Body weights of the mice are shown. E-G, Tumor sections collected from vehicle and treatment groups were processed and subjected to immunohistochemical staining for Ki67 and pAkt. Data represented as mean ± SE. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. H, Scatter plots from TIMER2.0 database illustrating the expression level correlations between KDM1A and PI3K/Akt/mTOR pathway genes (MTOR, MAPKAP1, RICTOR, PIK3CA, PDPK1, and IRS1) in Uterine Corpus Endometrial Carcinoma (UCEC). TPM values of each gene in TCGA-UCEC dataset (n = 545) were utilized for Spearman’s correlation coefficient calculation and then log2-transformed for visualization. P < 0.05 was considered as significant.

Article Snippet: Human EC cell lines, HEC1A and RL95–2, were obtained from the American Type Culture Collection (ATCC, Manassas, VA) and maintained as per ATCC guidelines.

Techniques: Immunohistochemical staining, Staining, Expressing, Transformation Assay

Journal: eLife

Article Title: Siglec1-expressing subcapsular sinus macrophages provide soil for melanoma lymph node metastasis

doi: 10.7554/eLife.48916

Figure Lengend Snippet:

Article Snippet: Antibody , Cy3 AffiniPure Goat Anti-Rat IgG (H+L) , Jackson ImmunoResearch , Cat# 112-165-167, RRID: AB_2338251 , (1:500).

Techniques: Control, Adsorption, Plasmid Preparation, Software, Recombinant, Expressing, Transfection, Construct, Negative Control, Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot

Journal: iScience

Article Title: Mice carrying the full-length human immunoglobulin loci produce antigen-specific human antibodies with the lambda light chain

doi: 10.1016/j.isci.2024.111258

Figure Lengend Snippet:

Article Snippet: Anti-Igλ, Human, Goat-Poly, HRP , Bethyl Laboratories , Cat# A80-116P; RRID: AB_67591.

Techniques: Activation Assay, Marker, Virus, Recombinant, Transfection, Adjuvant, Enzyme-linked Immunosorbent Assay, Expressing, Knock-Out, cDNA Synthesis, Plasmid Preparation, Software, Fluorescence, Imaging

Journal: Regenerative Therapy

Article Title: Preparation of iPS cell-derived CD31 + endothelial cells using three-dimensional suspension culture

doi: 10.1016/j.reth.2018.06.004

Figure Lengend Snippet: Characterisation of hiPS cell-derived CD31 + cells. (A) Endothelial marker gene expression in differentiated hiPS cells. hiPS cell differentiation was performed in 30- or 100-ml culture vessels. (B) Mean percentage of cells expressing CD31 on day 9 of differentiation from KDR + or KDR − cells. After 7 days of differentiation, both KDR + and KDR − cells were isolated by MACS, re-cultured onto collagen IV-coated tissue culture dishes, and immunostained with CD31 antibodies. Scale bars = 400 pm. CD31 + cell number was analysed using MetaXpress software. (C) Phase-contrast image of hiPS cell-derived CD31 + cells. Magnification, ×40. (D) Network formation by induced CD31 + cells after 24 h of culture on top of Matrigel. Magnification, ×40. (E) Comparison of endothelial marker gene expression between hiPS cell-derived CD31 + cells and tissue-derived vascular endothelial cells. The expression levels of genes were analysed by Taqman gene expression assay and gene expression was normalised to endogenous β-actin. (F) Immunostaining of CD31 + cells derived from hiPS cells on day 13 of differentiation. CD31 (left panel, red) and vascular endothelial cadherin (right panel, green). (B-F) hiPS cell differentiation from day 0 to day 7 was performed in 30-ml culture vessels. Nuclei were stained with Hoechst 33342 (blue) in (B) and (F). Scale bars = 400 pm in (B) and (F). Values are shown as mean ± standard deviation for at least three separate experiments in (A), (B), (E) and (F). Asterisk indicates statistically significant difference ( p < 0.05). NS, not significant.

Article Snippet: Phycoerythrin-conjugated monoclonal antibodies for human vascular endothelial (VE)-cadherin (R&D Systems) and monoclonal antibodies for human CD31 conjugated with phycoerythrin were used for immunocytochemistry.

Techniques: Derivative Assay, Marker, Gene Expression, Cell Differentiation, Expressing, Isolation, Cell Culture, Software, Comparison, Immunostaining, Staining, Standard Deviation

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 expression is upregulated in LUAD, and its increased expression is associated with unfavorable OS and FP. (A) METTL1 expression profile in LUAD tissues (n=483) and normal lung tissues (n=59) from TCGA database was analyzed using Gene Expression Profiling Interactive Analysis software. METTL1 expression was significantly upregulated in LUAD tissues. (B) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different tumor stages. The METTL1 expression profile in different LUAD stages (stage I, n=277; stage II, n=125; stage III, n=85 and stage IV, n=28) and normal lung tissues (n=59) from TCGA database was analyzed using UALCAN software. (C) The METTL1 expression profile in LUAD tissues (n=58) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. METTL1 expression was significantly upregulated in LUAD tissues. (D) METTL1 expression was significantly higher in different LUAD stages compared with normal tissues. No significant differences were observed among the different stages. The METTL1 expression profile in different stages (stage I, n=22; stage II, n=21; stage III, n=12 and stage IV, n=3) and normal lung tissues (n=49) from the GSE10072 dataset in the GEO database was analyzed using GraphPad Prism 5.0 software. (E) METTL1 expression was upregulated in LUAD tissues. Representative images of immunohistochemistry staining from the Human Protein Atlas database. Scale bar at low magnification, 200 µM; high magnification, 50 µM. HPA Patients’ ID, negative: 2268, medium: 3052, low: 2403. (F) High METTL1 expression was associated with unfavorable OS. The OS curve of 719 patients was plotted [cut-off value, 347; HR=1.7 (1.35–2.15), 95% CI; log-rank P=6×10 −6 −05]. (G) High METTL1 expression was associated with unfavorable FP. The FP curve of 461 patients was plotted [cut-off value, 324; HR=2.26 (1.65–3.1), 95% CI; log-rank P=2.2×10 −7 ]. The online Kaplan Meier Plotter software was used to construct the OS and FP graphs. *P<0.05, ***P<0.001. METTL1, methyltransferase-like 1; LUAD, lung adenocarcinoma; OS, overall survival; FP, first progression; TCGA, The Cancer Genome Atlas; GEO, Gene Expression Omnibus; HR, hazard ratio; CI, confidence interval.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Gene Expression, Software, Immunohistochemistry, Staining, Construct

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Association between METTL1 expression and the clinicopathological characteristics of patients with lung adenocarcinoma (n=517).

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: Cox regression multivariate analysis of METTL1 on OS and FP of patients with lung adenocarcinoma.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques:

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 promotes colony formation and A549 cell proliferation. (A) METTL1 expression was increased in A549 cells. (B) Western blot analysis demonstrated that METTL1 protein was upregulated and downregulated in the overexpression and knockdown experiments, respectively. Overexpression of METTL1 promoted A549 and H1993 cell (C) proliferation and (D) colony formation. METTL1 silencing attenuated A549 and H1993 cell (C) proliferation and (D) colony formation. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; si, small interfering; OD, optical density.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Western Blot, Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 inhibits autophagy in A549 cells. (A) METTL1 overexpression inhibited the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 overexpression may inhibit autophagy. (B) METTL1 knockdown enhanced the conversion of LC3-I to LC3-II and the degradation of p62/SQSTM1, suggesting that METTL1 knockdown may promote autophagy. (C) METTL1 knockdown increased both the size and number of GFP-LC3 puncta in HCC827/GFP-LC3 cells, suggesting that METTL1 knockdown may promote autophagy. *P<0.05, **P<0.01, ***P<0.001. METTL1, methyltransferase-like 1; SQSTM1, sequestosome 1; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Over Expression, Knockdown

Journal: Oncology Letters

Article Title: Methyltransferase-like 1 regulates lung adenocarcinoma A549 cell proliferation and autophagy via the AKT/mTORC1 signaling pathway

doi: 10.3892/ol.2021.12591

Figure Lengend Snippet: METTL1 activates the AKT/mTORC1 signaling pathway. GSEA analysis of the TCGA-LUAD dataset revealed that the gene sets (A) HALLMARK_MTORC1_SIGNALING and (B) HALLMARK_PI3K_AKT_MTOR_SIGNALING were enriched in the high-METTL1 expression group. (C) DAVID analysis of the GSE112180 dataset indicated that the gene cluster of the PI3K/AKT signaling pathway was enriched in the high-METTL1 expression group (D) METTL1 overexpression increased the protein levels of p-AKT and p-S6K, the effects of which were reversed following METTL1 silencing. Total protein levels of AKT and S6K remained unchanged compared with the control group. *P<0.05. METTL1, methyltransferase-like 1; p-AKT, phosphorylated protein kinase B; mTORC1, mechanistic target of rapamycin complex 1; GSEA, Gene Set Enrichment Analysis; TCGA, The Cancer Genome Atlas; LUAD, lung adenocarcinoma; DAVID, Database for Annotation, Visualization and Integrated Discovery; PI3K, phosphatidylinositol 3-kinase; si, small interfering.

Article Snippet: Proteins (30 μg) were separated by 12 or 15% SDS-PAGE gel, transferred onto polyvinylidene fluoride membranes and blocked with 5% non-fat milk at room temperature for 2 h. The membranes were incubated with the following primary antibodies: Rabbit anti-METTL1 antibody (cat. no. 14994-1; 1:2,000; ProteinTech Group, Inc.), rabbit anti-light chain (LC) 3B polyclonal antibody (cat. no. NB100-2220; 1:1,000; Novus Biologicals, LLC), rabbit anti-p62 polyclonal antibody (cat. no. 39749; 1:1,000), anti-AKT (cat. no. 9272; 1:1,000), anti-phosphorylated (p)-AKT (cat. no. 4060; 1:1,000), anti-S6K (cat. no. 9234; 1:1,000) and anti-p-S6K (cat. no. 9204; 1:1,000), overnight at 4°C (all purchased from Cell Signaling Technology, Inc).

Techniques: Expressing, Over Expression, Control

Journal: The Journal of Neuroscience

Article Title: HIV-1 Tat C Modulates Expression of miRNA-101 to Suppress VE-Cadherin in Human Brain Microvascular Endothelial Cells

doi: 10.1523/JNEUROSCI.4796-12.2013

Figure Lengend Snippet: Expression of VE-cadherin and other TJPs decreases upon Tat C treatment in a dose-dependent manner. A, Fold change in miR-101 expression level after treatment with increasing doses of HIV-1 Tat C protein and HI-Tat C in BMVECs. BMVECs were harvested for protein and RNA analysis after 12 h of Tat C treatment. Expression of miR-101 was determined by qPCR using the human miR-101-specific TaqMan assay. The expression level of a small RNA, RNU24, was used as a normalizer. Results are shown as the fold change compared with control. Changes in the expression levels of miR-101 are statistically significant. *p ≤ 0.05; **p ≤ 0.005; ***p ≤ 0.0005. B, Western blot analysis demonstrating no change in the VE-cadherin expression level in BMVECs after exposure to the HI-Tat C protein. C, Western blots analysis for VE-cadherin and other TJPs (claudin-5, ZO-1, occludin) of samples treated with increasing doses of Tat C showing a decrease at the protein expression level. D, Graph showing densitometry analysis of the results. Image density of Western blots has been normalized with β-tubulin using ImageJ software. Changes in TJPs and VE-cadherin in BMVECs exposed to the Tat C protein are statistically significant. **p ≤ 0.005; *p ≤ 0.05. For ZO-1, *p ≤ 0.05 at all doses; at 1.5 μg/ml Tat C treatment, **p ≤ 0.005. For occludin, downregulation is statistically significant at all doses of Tat C, *p ≤ 0.05; at 1.5 μg/ml Tat C treatment, **p ≤ 0.005. Downregulation of claudin-5 was statistically significant. *p ≤ 0.05. Results are representative of three biologically repeated experiments and are represented as mean ± SE. E, Fold change in miR-101 expression level in BMVECs after Tat C treatment and empty vector pET-21b purified in a similar manner, showing that upregulation of miR-101 is specific to Tat C. F, Western blot analysis of the pET-21b and Tat C-treated BMVECs showing that downregulation of VE-cadherin is specific to Tat C and is not induced by either buffer or empty vector. All experiments were repeated three times and are represented as mean ± SE.

Article Snippet: Antibodies against VE-cadherin (#ab76428; Abcam), anti-claudin-5 (#34-1600; Invitrogen), anti-ZO-1 (#61-7300; Invitrogen), anti-occludin (#33-1520; Invitrogen), anti-β-tubulin (#ab6046; Abcam), anti-Dicer (#ab14601; Abcam), anti-Drosha (#ab12286; Abcam), anti-TRBP (#ab42018; Abcam), and anti-Ago2 (#ab57113; Abcam) were used in this study.

Techniques: Expressing, TaqMan Assay, Control, Western Blot, Software, Plasmid Preparation, Purification

Journal: The Journal of Neuroscience

Article Title: HIV-1 Tat C Modulates Expression of miRNA-101 to Suppress VE-Cadherin in Human Brain Microvascular Endothelial Cells

doi: 10.1523/JNEUROSCI.4796-12.2013

Figure Lengend Snippet: Anti-miR-101 transfection rescues the VE-cadherin expression level in BMVECs. A, Anti-miR-101 transfection resulted in the reduced expression of miR-101. An ∼80% reduction in the expression of cellular miR-101 in anti-miR-101-transfected BMVECs was seen compared with scramble-transfected negative controls. *p ≤ 0.05. B, Western blot analysis to determine the regain in level of expression of VE-cadherin in anti-miR-101-transfected BMVECs. After 24 h of anti-miR-101 transfection in BMVECs, cells were again exposed to Tat C protein (500 ng/ml) to test the specificity of the miR-101-mediated control over the expression of VE-cadherin, but the expression of VE-cadherin remained unaffected due to the suppressive effect of anti-miR-101 on miR-101 (p ≤ 0.05). C, Western blot images of VE-cadherin were normalized with β-tubulin and are presented as graph bars. All experiments were repeated three times and are represented as mean ± SE. *p ≤ 0.05.

Article Snippet: Antibodies against VE-cadherin (#ab76428; Abcam), anti-claudin-5 (#34-1600; Invitrogen), anti-ZO-1 (#61-7300; Invitrogen), anti-occludin (#33-1520; Invitrogen), anti-β-tubulin (#ab6046; Abcam), anti-Dicer (#ab14601; Abcam), anti-Drosha (#ab12286; Abcam), anti-TRBP (#ab42018; Abcam), and anti-Ago2 (#ab57113; Abcam) were used in this study.

Techniques: Transfection, Expressing, Western Blot, Control

Journal: The Journal of Neuroscience

Article Title: HIV-1 Tat C Modulates Expression of miRNA-101 to Suppress VE-Cadherin in Human Brain Microvascular Endothelial Cells

doi: 10.1523/JNEUROSCI.4796-12.2013

Figure Lengend Snippet: Expression level of VE-cadherin influences the expression level of Claudin-5 directly. To distinguish the direct effect of expression level of VE-cadherin on the expression level of claudin-5, three sets of experiments were performed. A, The downregulation of VE-cadherin upon Tat C treatment decreases the expression level of claudin-5 in a dose-dependent manner. B, Transfection of anti-miR-101 in BMVECs showing the same trend of rescued expression of claudin-5 as in the regained expression of VE-cadherin, even in the presence of the Tat C protein. C, Overexpression of miR-101 decreases the expression level of claudin-5 in the same way as that of VE-cadherin. D, Densitometry analysis using ImageJ software of Western blot images for claudin-5 in anti-miR-101 transfection experiments. A significant recovery of claudin-5 expression level can be seen (p ≤ 0.005). All experiments were repeated three times and are represented as mean ± SE. **p ≤ 0.005.

Article Snippet: Antibodies against VE-cadherin (#ab76428; Abcam), anti-claudin-5 (#34-1600; Invitrogen), anti-ZO-1 (#61-7300; Invitrogen), anti-occludin (#33-1520; Invitrogen), anti-β-tubulin (#ab6046; Abcam), anti-Dicer (#ab14601; Abcam), anti-Drosha (#ab12286; Abcam), anti-TRBP (#ab42018; Abcam), and anti-Ago2 (#ab57113; Abcam) were used in this study.

Techniques: Expressing, Transfection, Over Expression, Software, Western Blot

Journal: The Journal of Neuroscience

Article Title: HIV-1 Tat C Modulates Expression of miRNA-101 to Suppress VE-Cadherin in Human Brain Microvascular Endothelial Cells

doi: 10.1523/JNEUROSCI.4796-12.2013

Figure Lengend Snippet: Proposed model for Tat C-mediated downregulation of VE-cadherin and claudin-5 in BMVECs through miR-101. We propose that expression of VE-cadherin is regulated through miR-101 in BMVECs exposed to the HIV-1 Tat C protein. After HIV-1 Tat C treatment, the expression level of miR-101 increases and targets the expression level of VE-cadherin directly, which in turn influences the expression level of claudin-5 and thereby the permeability in BMVECs.

Article Snippet: Antibodies against VE-cadherin (#ab76428; Abcam), anti-claudin-5 (#34-1600; Invitrogen), anti-ZO-1 (#61-7300; Invitrogen), anti-occludin (#33-1520; Invitrogen), anti-β-tubulin (#ab6046; Abcam), anti-Dicer (#ab14601; Abcam), anti-Drosha (#ab12286; Abcam), anti-TRBP (#ab42018; Abcam), and anti-Ago2 (#ab57113; Abcam) were used in this study.

Techniques: Expressing, Permeability

Journal: Cell stem cell

Article Title: Cell-Type-Specific Chromatin States Differentially Prime Squamous Cell Carcinoma Tumor-Initiating Cells for Epithelial to Mesenchymal Transition

doi: 10.1016/j.stem.2016.10.018

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Anti Rat Cy5 , Jackson ImmunoResearch , # 112-175-167, RRID:AB_2338264 , .

Techniques: Recombinant, DNA Library Preparation, Purification, Plasmid Preparation, Expressing, Sequencing, shRNA, Software

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: Antibodies used in the study

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques:

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: The induction of EndoMT in HUVEC in vitro by H 2 O 2 . A the OD450 absorbance in HUVEC exposed to different concentrations of H 2 O 2 (20, 50, 100, 200 μM). B the morphological changes of HUVEC were observed under a light microscope after stimulation with 100 μM H 2 O 2 or 200 μM H 2 O 2 for 24 h and 48 h, Scale bars = 650 μm. C immunofluorescence staining of F-actin was performed in HUVEC exposed to either PBS or 200 μM H 2 O 2, Scale bars = 275 μm. D immunofluorescence staining of Vimentin, α-SMA, CD31 and VE-Cadherin in HUVEC treated with PBS or 200 μM H 2 O 2, Scale bars = 275 μm. E the quantitative analysis of mean fluorescence intensity of aforementioned proteins. F qPCR analysis was performed to assess the mRNA levels of CD31, VE-Cadherin, Vimentin, α-SMA and SM22α in HUVEC stimulated with PBS or 200 μM H 2 O 2 . G immunoblot analysis was performed to assess the protein expression of CD31, VE-Cadherin, ZO-1, Vimentin, α-SMA and SM22α in HUVEC exposed to PBS, 50 μM, 100 μM or 200 μM H 2 O 2 . H The intensity of protein bands was quantified. Each experiment was conducted in triplicate. Data represented the mean ± SD of triplicates. * p < 0.05 , ** p < 0.01, *** p < 0.001

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques: In Vitro, Light Microscopy, Immunofluorescence, Staining, Fluorescence, Western Blot, Expressing

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: The impact of ADSC-Exo on H 2 O 2 -induced EndoMT in HUVEC. A immunofluorescence staining of F-actin was performed in HUVEC treated with 200 μM H 2 O 2 or combination with 20 μg/ml ADSC-Exo. Scale bars = 275 μm. B - F the mRNA levels of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α were quantified by qPCR analysis in HUVEC exposed to PBS, 200 μM H 2 O 2 or 200 μM H 2 O 2 + 20 μg/ml ADSC-Exo. G WB analysis was performed to assess the protein levels of CD31, VE-Cadherin, ZO-1, Vimentin, SM22α, and α-SMA in HUVEC exposed to PBS, 200 μM H 2 O 2 or 200 μM H 2 O 2 + 20 μg/ml ADSC-Exo. H the quantitative analysis of protein bands intensity. I immunofluorescence staining of Vimentin, α-SMA, CD31 and VE-Cadherin were performed in HUVEC stimulated with 200 μM H 2 O 2 or combination with 20 μg/ml ADSC-Exo. Scale bars = 275 μm. J the quantitative analysis of mean fluorescence intensity of aforementioned proteins. The experiment was conducted in triplicate. Data represented the mean ± SD of triplicates. * p < 0.05 , ** p < 0.01, *** p < 0.001

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: The impact of mir-486-3p on the expression of EndoMT-related markers and the functional characteristics of HUVEC. A , D qPCR analysis was performed to assess the mRNA expression of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in HUVEC transfected with mir-486-3p mimics or inhibitors. B - C , E – F the protein expression of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in HUVEC transfected with mir-486-3p mimics or inhibitors was analyzed by immunoblotting and the quantitative analysis of their protein bands intensity were performed by image J software. G the effect of mir-486-3p mimics/inhibitors on the proliferation, migration and tube formation of HUVEC. Scale bars = 650 μm, 500 μm and 275 μm. H the quantification of migrated cells in transwell assays. I the quantitative analysis of mean immunofluorescence intensity of Ki67 in HUVEC exposed to mimics and inhibitors. J - K the quantity analysis of branch points and tube length in tube formation assays. Each experiment was conducted in triplicate. Data represented the mean ± SD of triplicates. * p < 0.05 , ** p < 0.01

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques: Expressing, Functional Assay, Transfection, Western Blot, Software, Migration, Immunofluorescence

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: The effect of Sirt6 on EndoMT. A - C the mRNA and protein expression of Sirt6 in HUVEC exposed to different concentrations of H 2 O 2 (50 μM, 100 μM or 200 μM) with the quantitative analysis of protein bands intensity. D - F the mRNA and protein levels of Sirt6 in HUVEC treated with PBS, 200 μM H 2 O 2 or 200 μM H 2 O 2 + 20 μg/ml ADSC-Exo group and the quantitative analysis of protein bands intensity. G - H qPCR analysis of the mRNA levels of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in HUVEC transfected with either an overexpression plasmid for Sirt6 or a negative control. I - L immunoblot analysis of the protein levels of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in HUVEC transfected with either an overexpression plasmid for Sirt6 or a negative control. Data represented the mean ± SD of triplicates. * p < 0.05 , ** p < 0.01, *** p < 0.0001

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques: Expressing, Transfection, Over Expression, Plasmid Preparation, Negative Control, Western Blot

Journal: Cell Biology and Toxicology

Article Title: Exosomes derived from adipose tissue-derived stem cells alleviated H 2 O 2 -induced oxidative stress and endothelial-to-mesenchymal transition in human umbilical vein endothelial cells by inhibition of the mir-486-3p/Sirt6/Smad signaling pathway

doi: 10.1007/s10565-024-09881-6

Figure Lengend Snippet: The effect of ADSC-Exo and mir-486-3p were confirmed in a murine wound model. A the schematic representation of animal experiments. B - C the images depicted the temporal evolution of wound morphology at various time points (0D, 3D, 5D, 7D, 10D, 14D)( * PBS vs Exo, * p < 0.05 , ** p < 0.01; # Exo vs Exo + Lv-486-3p, ## p < 0.01 , ### p < 0.0001; † Exo + Lv-mir-486-3p NC vs Exo + Lv-mir-486-3p, † p < 0.05, †† p < 0.01 , ††† p < 0.0001). D - E the murine wound tissues were subjected to routine H&E staining and Masson trichrome staining in PBS group, ADSC-Exo (70 μg/100 μl) group, ADSC-Exo (70 μg/100 μl) plus lentivirus-mediated transfection with mir-486-3p mimics NC (1 × 10 9 TU/ml virus titer in PBS) group, ADSC-Exo (70 μg/100 μl) plus lentivirus-mediated transfection with mir-486-3p mimics groups (1 × 10 9 TU/ml virus titer in PBS). The Masson trichrome staining revealed the presence of a fibrotic region, characterized by a blue coloration. Scale bars = 2000 μm. F - J the mRNA levels of CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in wound tissues were quantified by qPCR in the aforementioned groups. K - N western blot analysis was performed to examine the protein expression of Sirt6, CD31, VE-Cadherin, α-SMA, Vimentin and SM22α in wound tissues from different experimental groups as mentioned above with the quantitative analysis of protein intensity measured by Image J software. O Immunofluorescence double-labeling staining was performed using antibodies against CD31 (an endothelial lineage marker, green fluorescence) and α-SMA (a fibroblast marker, red fluorescence). Scale bars = 650 μm, 275 μm. P the qualitative analysis of colocalization immunofluorescence staining (CD31/green and α-SMA/red) in above mentioned groups. Each group consisted of six mice (n = 6). Data represented the mean ± SD of triplicates. * p < 0.05 , ** p < 0.01, *** p < 0.0001

Article Snippet: Following transfer and blocking in defatted milk, the membranes were incubated with primary antibodies (dilution ratio/1:1000) against CD31(Proteintech, CST), VE-Cadherin (Boster, CST), α-SMA (Proteintech, CST), Nrf2 (Proteintech), Vimentin (Proteintech), SM22α (Boster), HO-1(Proteintech), ZO-1 (Proteintech), Sirt6 (CST), Nox4 (Proteintec), p-Smad2/3 (CST), Smad2/3 (CST), TGFβ1(Proteintech) and GAPDH (Zhuangzhi Bio) at 4°C overnight (listed in Table ).

Techniques: Staining, Transfection, Virus, Western Blot, Expressing, Software, Immunofluorescence, Labeling, Marker, Fluorescence