Journal: Regenerative Therapy

Article Title: Cartilage repair and inhibition of the progression of cartilage degeneration after transplantation of allogeneic chondrocyte sheets in a nontraumatic early arthritis model

doi: 10.1016/j.reth.2018.07.003

Figure Lengend Snippet: Macroscopic and histological findings, and gene expression in cell sheets. (A) Representative images of a cell sheet attached to a white polyvinylidene membrane. (B) Hematoxylin–eosin, Safranin O, toluidine blue, and collagen type II staining of the cartilage matrix. Collagen type I did not stain (×40, scale bar = 50 μm). (C) The expression of Col1A1 and ITGa10 increased, but there was no significant difference ( p > 0.05). Expression of Sox9 and Mmp13 increased significantly but not more than 2-fold (* p < 0.05, <2-fold). Expression of Col2A1 , Acan , Lect1 , and Comp increased significantly by >2-fold (* p < 0.05, >2-fold).

Article Snippet: The primers were as follows for TaqMan® Gene Expression Assays (Applied Biosystems, USA): Col1A1 (Rn01637087_m1), Col2A1 (Rn01463848_m1), Sox9 (Rn01751070_m1), Acan (Rn00573424_m1), ITGa10 (Rn01533928_m1), Mmp13 (Rn01448194_m1), Lect1 (Rn00578277_m1), Comp (Rn00563255_m1), and Gapdh (Rn01775763_g1).

Techniques: Gene Expression, Membrane, Staining, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Kaiso regulates osteoblast differentiation and mineralization via the Itga10/PI3K/AKT signaling pathway

doi: 10.3892/ijmm.2021.4874

Figure Lengend Snippet: Kaiso regulates the PI3K/AKT pathway via Itga10. Western blot analysis of Itga10 expression in (A) Kaiso-overexpressing or (B) Kaiso-knockdown MC3T3-E1 cells. (C) Chromatin immunoprecipitation analysis of the binding of Kaiso to the promoter of Itga10 in LV-ctr and LV-Kaiso MC3T3-E1 cells. Input DNA and DNA immunoprecipitated with IgG were included as positive and negative controls, respectively. The results are expressed as percentages of the input level. (D) WT and (E) Kaiso binding site-MUT Itga10 promoter-luciferase reporter vectors were co-transfected with Kaiso-overexpression vectors or control vector in MC3T3-E1 cells. Luciferase activity was normalized to Renilla luciferase activity and presented as the fold change to LV-ctr. (F) Expression levels of p85, AKT and p-AKT were determined via western blot analysis in Sh-Itga10-1, Sh-Itga10-2 and Sh-ctr transfectedMC3T3-E1 cells. (G) RT-qPCR analysis of ALP, OCN and BSP expression levels in Sh-ctr and Sh-Kaiso transfected MC3T3-E1 cells after 7 days culture with osteogenic medium. (H) ALP activity was measured on day 7. (I) ALP staining was performed on day 7, and Alizarin Red S staining was performed on day 21. All quantitative data are presented as the mean ± SD (n=3). ** P<0.01. WT, wild-type; MUT, mutant; LV, lentivirus; shRNA, short hairpin RNA; Ctr, control; p-, phosphorylated; ALP, alkaline phosphatase; OCN, osteocalcin; BSP, bone sialoprotein; Itga10, integrin subunit α10; RT-qPCR, reverse transcription-quantitative PCR.

Article Snippet: For gene silencing, Kaiso-targeting short hairpin RNA (shRNA) or Itga10-targeting shRNA were inserted into the PLKD-CMV-EGFP-Puro-U6 vector (Obio Technology Co., Ltd.).

Techniques: Western Blot, Expressing, Chromatin Immunoprecipitation, Binding Assay, Immunoprecipitation, Luciferase, Transfection, Over Expression, Plasmid Preparation, Activity Assay, Quantitative RT-PCR, Staining, Mutagenesis, shRNA, Real-time Polymerase Chain Reaction

Journal: International Journal of Molecular Medicine

Article Title: Kaiso regulates osteoblast differentiation and mineralization via the Itga10/PI3K/AKT signaling pathway

doi: 10.3892/ijmm.2021.4874

Figure Lengend Snippet: In vivo osteogenesis of LV-Kaiso, Sh-Kaiso and Sh-Itga10 infected MC3T3-E1 cells. Representative µ CT images (magnification, ×1) from each group: (A) Vehicle, (B) LV-ctr and LV-Kaiso, (C) Sh-ctr and Sh-Kaiso-1, (D) Sh-ctr and Sh-Itga10-1. (E) BMD of the implants was measured based on µ CT images. Representative hematoxylin and eosin staining images from each group: (F) Vehicle, (G) LV-ctr and LV-Kaiso, (H) Sh-ctr and Sh-Kaiso-1, (I) Sh-ctr and Sh-Itga10-1. Scale bar, 50 µ m. Thin arrows indicate osteoblast; arrowheads indicate newly formed osteoid; # indicates newly formed mature bone. Data are presented as the mean ± SD (n=3). * P<0.05 and ** P<0.01. BMD, Bone mineral density; CT, connective tissue; Itga10, integrin subunit α10; LV, lentivirus; shRNA, short hairpin RNA; Ctr, control.

Article Snippet: For gene silencing, Kaiso-targeting short hairpin RNA (shRNA) or Itga10-targeting shRNA were inserted into the PLKD-CMV-EGFP-Puro-U6 vector (Obio Technology Co., Ltd.).

Techniques: In Vivo, Infection, Staining, shRNA

Journal: Regenerative Therapy

Article Title: miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

doi: 10.1016/j.reth.2022.08.002

Figure Lengend Snippet: ITGA10 was dysregulated in OP (A) KEGG analysis of the pathway related to the differential genes. (B)The downregulation of ITGA10 in osteoporosis (n = 5) compared with the control (n = 5). (C) 30 common miRNAs were screened out by TargetScan and GEO dataset (GSE93883). GSE93883 was obtained from NCBI. (D) The upregulation of miR-4739 in osteoporosis (n = 12) compared with the control (n = 6).

Article Snippet: The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).

Techniques:

Journal: Regenerative Therapy

Article Title: miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

doi: 10.1016/j.reth.2022.08.002

Figure Lengend Snippet: ITGA10 was the target gene of miR-4739 (A) The activity of osteoblast with different transfections. (B) The binding sites between miR-4739 and ITGA10 were predicted by bioinformatics (C–D) The targeting relationship between miR-4739 and ITGA10 was detected by luciferase reporter assay. (E) The mRNA expression of ITGA10 in osteoblasts with different transfections was tested by qRT-PCR. (F–G) The ITGA10 protein expression in osteoblasts with different transfections was measured by Western blot. Data are expressed as the mean ± standard deviation. ˆP < 0.05 versus control group, ∗P < 0.05 versus NC group, ∗∗P < 0.01 versus NC group, +++P < 0.001 versus MC group, && P < 0.01 versus inhibitor group and ##P < 0.01 versus si-ITGA10 group.

Article Snippet: The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).

Techniques: Activity Assay, Transfection, Binding Assay, Luciferase, Reporter Assay, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Regenerative Therapy

Article Title: miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

doi: 10.1016/j.reth.2022.08.002

Figure Lengend Snippet: miR-4739 regulated osteoblast proliferation and apoptosis by targeting ITGA10 (A) Cell proliferation was measured with CCK8 assay. ∗∗P < 0.01 versus NC group, && P < 0.01 versus inhibitor group and ##P < 0.01 versus si-ITGA10 group. (B) The apoptosis of osteoblast was measured by flow cytometry.

Article Snippet: The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).

Techniques: CCK-8 Assay, Flow Cytometry

Journal: Regenerative Therapy

Article Title: miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

doi: 10.1016/j.reth.2022.08.002

Figure Lengend Snippet: miR-4739 regulated osteoblast differentiation by targeting ITGA10 . (A–B) The protein expression of ALP, OPN, Osterix and Runx 2 was measured by Western blot. Every experiment was performed in triplicate. Data were expressed as the mean ± standard deviation. ∗∗P < 0.01 versus NC group, && P < 0.01 versus inhibitor group and ##P < 0.01 versus si-ITGA10 group.

Article Snippet: The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).

Techniques: Expressing, Western Blot, Standard Deviation

Journal: Regenerative Therapy

Article Title: miR-4739/ITGA10/PI3K signaling regulates differentiation and apoptosis of osteoblast

doi: 10.1016/j.reth.2022.08.002

Figure Lengend Snippet: The effect of miR-4739/ITGA10 on the activation of PI3K/AKT signaling pathway (A–B) The relative expression levels of phosphorylated PI3K (p-PI3K), total PI3K (PI3K), phosphorylated AKT (p-AKT) and total AKT (AKT) proteins were determined by Western blot. Data were expressed as the mean ± standard deviation. ∗∗P < 0.01 versus NC group, && P < 0.01 versus inhibitor group and ##P < 0.01 versus si-ITGA10 group.

Article Snippet: The primary antibodies were as follows: anti-ITGA10 (AB6030, 1:1000, Merck), Alkaline phosphatase (ALP; ab229126, 1:1000, Abcam), Runx 2 (ab76956, 1:1000, Abcam), Osterix (ab209484, Abcam), Osteopontin (OPN; #88742, 1:1000, Cell Signaling Technology), p-PI3K (ab278545, 1:1000, Abcam), PI3K (#4249, 1:1000, Cell Signaling Technology), p-AKT (#4060, 1:2000, Cell Signaling Technology), AKT (#4691, 1:1000, Cell Signaling Technology), and GAPDH (ab8245, 1:5000, Abcam).

Techniques: Activation Assay, Expressing, Western Blot, Standard Deviation