rabbit anti human col1  (Cell Signaling Technology Inc)

Journal: Stem Cell Research & Therapy

Article Title: Human pluripotent stem cell-derived cartilaginous organoids promote scaffold-free healing of critical size long bone defects

doi: 10.1186/s13287-021-02580-7

Figure Lengend Snippet: IL-1β priming allows successful fracture repair. Upon IL-1β treatment, a trend towards a decrease in chondromodulin was detected, while IL-1β upregulated VEGF and MMP13 ( A ). Safranin-O staining prior to implantation further revealed the increase in chondrocyte lacunae size, suggesting progressive maturation towards chondrocyte hypertrophy ( B ). Following orthotopic implantation, the cartilaginous organoids underwent progressive mineralization with bone bridging being detected in the µCT images after 4 weeks ( C ). µCT quantification revealed a significant increase ( p = 0.0095) in the volume of mineralized tissue in the defect area between BBT3 and BBT3 + IL-1β conditions after 8 weeks ( D ). Histological analysis using safranin-O further revealed accelerated bone formation with cartilage resorption being detected at week 4 ( E ). While bone union was detected after 8 weeks, some cartilaginous remnants could be detected, which indicates progressive cartilage resorption. Immunostaining of human collagen type I further revealed that the newly formed bone was host derived, while some hypertrophic chondrocytes expressed low levels of collagen type I ( F ). Mathematical modelling was used to confirm the cumulative effects of BMP, Wnt, TH and IL-1β in chondrocyte hypertrophy. An increase in hypertrophy chance, along with a decrease in variability, was detected following in vitro stimulation with aforementioned factors. DC: β-catenin Destruction Complex ( G ). The presented data have been collected from experiments ( n = 6) using CY2 cell line. Statistical significance is represented as follow: * p < 0.05; ** p < 0.01; *** p < 0.001, and **** p < 0.0001. The following abbreviations have been used to highlight the tissue composition in the sections: B: bone, C: cartilage, M: bone marrow, T: cartilage-bone turnover site (dotted rectangle) and CR: cartilage remnants

Article Snippet: Sections were then washed with PBS + 1% Tween 20 and blocked with 5% bovine serum albumin (BSA) for 30 min (Sigma, UK), before being incubated with a rabbit anti-human Col1 antibody (1:200, Thermo Scientific, USA) at 4 °C overnight.

Techniques: Staining, Immunostaining, Derivative Assay, In Vitro

Journal: International Journal of Molecular Medicine

Article Title: Quercetin reverses TNF-α induced osteogenic damage to human periodontal ligament stem cells by suppressing the NF-κB/NLRP3 inflammasome pathway

doi: 10.3892/ijmm.2021.4872

Figure Lengend Snippet: Reversing effect of Quer on TNF-α-induced suppression of osteogenesis. hPDLSCs were treated with Quer, TNF-α, or their combination for 7 days and/or 21 days in an OIM. (A) Protein levels of COL1, ALP and RUNX2 were measured by western blot analysis on day 7. (B) Band intensities were quantified using ImageJ software. ** P<0.01, *** P<0.001 vs. control; ## P<0.01, ### P<0.001 vs. TNF-α. (C) ALP activity detection following 7 days of osteogenic induction. ** P<0.01 vs. control; ## P<0.01 vs. TNF-α. (D) mRNA expression levels of COL1 and RUNX2 were measured by RT-qPCR day 7. ** P<0.01 vs. control; ## P<0.01 vs. TNF-α. (E) ALP staining detection following osteogenic induction for 7 days (scale bar, 100 μ m). (F) Alizarin Red staining detection following 21 days of osteogenic induction (scale bar, 100 μ m). hPDLSCs, human periodontal ligament stem cells; Quer, quercetin; OIM, osteogenic induction medium; TNF-α, tumor necrosis factor-α; ALP, alkaline phosphatase.

Article Snippet: Blocking was performed using 5% non-fat dry milk at 22°C for 1 h, then probed with rabbit anti-human GAPDH polyclonal (1:20,000, cat. no. 10494-1-AP; ProteinTech Group, Inc.), rabbit anti-human RUNX2 monoclonal (1:1,000, cat. no. ab23981; Abcam), rabbit anti human-COL1 mono- clonal (1:1,000, cat. no. #84336; Cell Signaling Technology, Inc.), rabbit anti human-ALP monoclonal (1:5,000, cat. no. ab108337; Abcam), rabbit anti human-NLRP3 polyclonal (1:1,000, cat. no. WL02635; Wanlei Biotech Co., Ltd.), rabbit anti human-procaspase-1 polyclonal (1:500, cat. no. WL02996; Wanlei Biotech Co., Ltd., China), rabbit anti human-caspase-1 polyclonal (1:500, cat. no. WL03450; Wanlei Biotech Co., Ltd.), rabbit anti human-phosphorylated (p-)p65 polyclonal (1:500, cat. no. WL02169; Wanlei Biotech Co., Ltd.), rabbit anti human-p-IκBα polyclonal (1:500, cat. no. WL02495; Wanlei Biotech Co., Ltd.), rabbit anti human-IκBα polyclonal (1:500, cat. no. WL01936; Wanlei Biotech Co., Ltd.), rabbit anti human-p65 monoclonal (1:1,000, cat. no. #59674; Cell Signaling Technology, Inc.) at 4°C for 24 h. This was followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:20,000, cat. no. 7074S; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein detection was performed using a chemiluminescent HRP (EMD Millipore) and the expression levels of target proteins were analyzed using ImageJ 1.47V software (National Institutes of Health) and normalized to GAPDH expression.

Techniques: Western Blot, Software, Activity Assay, Expressing, Quantitative RT-PCR, Staining

Journal: International Journal of Molecular Medicine

Article Title: Quercetin reverses TNF-α induced osteogenic damage to human periodontal ligament stem cells by suppressing the NF-κB/NLRP3 inflammasome pathway

doi: 10.3892/ijmm.2021.4872

Figure Lengend Snippet: Antagonistic effect of si-NLRP3 on the inhibition of the osteogenesis of hPDLSCs by TNF-α. (A) Protein level of NLRP3 in hPDLSCs transfected with si-NLRP3 or si-NC was examined by western blot analysis. ** P<0.01 vs. si-NC. (B) Protein levels of p-p65, p65, p-IκBα and IκBα were measured by western blot analysis. (C) Band intensities were quantified using ImageJ software. ** P<0.01, *** P<0.001 vs. control + si-NC; ## P<0.01 vs. TNF-α + si-NC, NS (no significant difference) vs. TNF-α + si-NC. (D) Protein levels of COL1, ALP and RUNX2 on day 7 of osteogenic induction. (E) Band intensities were quantified using ImageJ software. ** P<0.01 vs. control + si-NC; ## P<0.01 vs. TNF-α + si-NC. (F) mRNA expression levels of COL1, ALP and RUNX2 following osteogenic induction for 7 days. ** P<0.01 vs. control + si-NC; # P<0.05, ## P<0.01 vs. TNF-α + si-NC. (G) ALP staining following osteogenic induction for 7 days (scale bar, 100 μ m). (H) ALP activity analysis on day 7 of osteogenic induction. ** P<0.01 vs. control + si-NC; ## P<0.01 vs. TNF-α+si-NC.

Article Snippet: Blocking was performed using 5% non-fat dry milk at 22°C for 1 h, then probed with rabbit anti-human GAPDH polyclonal (1:20,000, cat. no. 10494-1-AP; ProteinTech Group, Inc.), rabbit anti-human RUNX2 monoclonal (1:1,000, cat. no. ab23981; Abcam), rabbit anti human-COL1 mono- clonal (1:1,000, cat. no. #84336; Cell Signaling Technology, Inc.), rabbit anti human-ALP monoclonal (1:5,000, cat. no. ab108337; Abcam), rabbit anti human-NLRP3 polyclonal (1:1,000, cat. no. WL02635; Wanlei Biotech Co., Ltd.), rabbit anti human-procaspase-1 polyclonal (1:500, cat. no. WL02996; Wanlei Biotech Co., Ltd., China), rabbit anti human-caspase-1 polyclonal (1:500, cat. no. WL03450; Wanlei Biotech Co., Ltd.), rabbit anti human-phosphorylated (p-)p65 polyclonal (1:500, cat. no. WL02169; Wanlei Biotech Co., Ltd.), rabbit anti human-p-IκBα polyclonal (1:500, cat. no. WL02495; Wanlei Biotech Co., Ltd.), rabbit anti human-IκBα polyclonal (1:500, cat. no. WL01936; Wanlei Biotech Co., Ltd.), rabbit anti human-p65 monoclonal (1:1,000, cat. no. #59674; Cell Signaling Technology, Inc.) at 4°C for 24 h. This was followed by incubation with a horseradish peroxidase-labeled goat anti-rabbit IgG secondary antibody (1:20,000, cat. no. 7074S; Santa Cruz Biotechnology, Inc.) at room temperature for 1 h. Protein detection was performed using a chemiluminescent HRP (EMD Millipore) and the expression levels of target proteins were analyzed using ImageJ 1.47V software (National Institutes of Health) and normalized to GAPDH expression.

Techniques: Inhibition, Transfection, Western Blot, Software, Expressing, Staining, Activity Assay

Journal: Molecular Medicine Reports

Article Title: Homeobox B9 facilitates hypertrophic scar formation via activating the mitogen-activated protein kinase signaling pathway

doi: 10.3892/mmr.2017.6836

Figure Lengend Snippet: Expression levels of laminin, FN, Col1 and HOXB9 are increased in HS tissue. Scar and healthy adjacent tissues were acquired from patients prior to surgical treatment. Expression of laminin, FN, Col1 and HOXB9 were significantly increased in HS tissue compared with autologous normal skin. Scale bar = 50 µm. HS, hypertrophic scar; FN, fibronectin; Col1, collagen type I; HOXB9, homeobox B9; H, healthy scar tissue.

Article Snippet: The sections were separately incubated for 2 h with rabbit anti-human fibronectin (FN; 1:1,000; catalog no. ab61214; Abcam, Cambridge, MA, USA), rabbit anti-human collagen type I (Col1; 1:200; catalog no. ABIN2473035; Antibodies-Online, Inc., Atlanta, GA, USA), rabbit anti-human laminin (1:300; catalog no. ab11575, Abcam) and rabbit anti-human HOXB9 (1:500; catalog no. ABIN952780; Antibodies-Online, Inc.) at room temperature overnight.

Techniques: Expressing

Journal: Molecular Medicine Reports

Article Title: Homeobox B9 facilitates hypertrophic scar formation via activating the mitogen-activated protein kinase signaling pathway

doi: 10.3892/mmr.2017.6836

Figure Lengend Snippet: HOXB9 regulates the expression of laminin, FN and Col1 in FBs. Reverse transcription-quantitative polymerase chain reaction and western blotting analyses were performed to assess differential mRNA and protein expression levels, respectively, in FB mock, FB-negative, FB-HOXB9si and FB-HOXB9over cells. (A) FBs were constructed to stably overexpress (FB-HOXB9over) or silence (FB-HOXB9si) HOXB9 using lentiviruses (P<0.001). (B) mRNA and protein expression levels of laminin were decreased in FB-HOXB9si cells and increased in FB-HOXB9over cells, compared with the FB-mock and FB-negative groups (P<0.01). (C) FN and protein expression levels were decreased in FB-HOXB9si cells and increased in FB-HOXB9over cells, compared with the FB-mock and FB-negative groups (P<0.01); however, no significant differences were observed in mRNA expression levels (P>0.05). (D) Col1 mRNA and protein expression levels were decreased in FB-HOXB9si cells and increased in FB-HOXB9over cells, compared with FB-mock and FB-negative cells (P<0.01). Data are presented as the mean ± standard deviation of three independent experiments. **P<0.01 and ***P<0.001 vs. FB-mock and FB-negative. FB, fibroblast; FN, fibronectin; HOXB9, homeobox B9; si, silenced; over, overexpressing; Col1; collagen type I; ns, non-significant.

Article Snippet: The sections were separately incubated for 2 h with rabbit anti-human fibronectin (FN; 1:1,000; catalog no. ab61214; Abcam, Cambridge, MA, USA), rabbit anti-human collagen type I (Col1; 1:200; catalog no. ABIN2473035; Antibodies-Online, Inc., Atlanta, GA, USA), rabbit anti-human laminin (1:300; catalog no. ab11575, Abcam) and rabbit anti-human HOXB9 (1:500; catalog no. ABIN952780; Antibodies-Online, Inc.) at room temperature overnight.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Construct, Stable Transfection, Standard Deviation

Journal: Molecular Medicine Reports

Article Title: Homeobox B9 facilitates hypertrophic scar formation via activating the mitogen-activated protein kinase signaling pathway

doi: 10.3892/mmr.2017.6836

Figure Lengend Snippet: Schematic diagram of HOXB9 facilitating the formation of a hypertrophic scar. HOXB9 activates the MAPK pathway by phosphorylating ERK, JNK and p38, potentially by interacting directly with p38. Activated MAPK leads to the transcription of laminin, FN and Col1 mRNA, increasing their protein expression levels, which reconstructs the ECM and contributes to hypertrophic scar formation. Increased laminin, FN and Col1 may in return increase p-ERK, p-JNK and p-p38 levels. FN, fibronectin; Col1, collagen type I; HOXB9, homeobox B9; ERK, extracellular signal-regulated kinase; JNK, c-Jun N-terminal kinase; p, phosphorylated; MAPK, mitogen-activated protein kinase.

Article Snippet: The sections were separately incubated for 2 h with rabbit anti-human fibronectin (FN; 1:1,000; catalog no. ab61214; Abcam, Cambridge, MA, USA), rabbit anti-human collagen type I (Col1; 1:200; catalog no. ABIN2473035; Antibodies-Online, Inc., Atlanta, GA, USA), rabbit anti-human laminin (1:300; catalog no. ab11575, Abcam) and rabbit anti-human HOXB9 (1:500; catalog no. ABIN952780; Antibodies-Online, Inc.) at room temperature overnight.

Techniques: Expressing