Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: Primer sequences used for qRT-PCR.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques:

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: T -cell lymphoma invasion and metastasis 2 (TIAM2) is highly expressed in osteosarcoma (OS) tissue and is correlated with the prognosis of OS. A: TIAM2 expression levels of diverse human tumor types. B, C: Immunohistochemical (IHC) results showing that the expression of TIAM2 was significantly upregulated in OS tissue (200×); Assessment of staining intensity by positive cells ratio. D: The protein levels of TIAM2 in hFOB1.19, U2OS, and 143B cells detected using western blotting. Quantification of the band density ratio of TIAM2 to GAPDH. E: The mRNA expression of TIAM2 determined using qRT-PCR in hFOB1.19, U2OS, and 143B cells. G–I: Survival curve analysis of TIAM2 high- and low-expression groups in three databases. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: Expressing, Immunohistochemical staining, Staining, Western Blot, Quantitative RT-PCR

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: Expression of TIAM2 promotes proliferation, migration, and invasion of OS cells. A: Western blot showed TIAM2 protein levels extracted from TIAM2-OE and the control group in 143B and U2OS cells. B: Quantification of the band density ratio of TIAM2 to GAPDH. C, D: Detection of cell proliferation using the CCK-8 assay. E-F, H-I: The wound distance of TIAM2-OE and the control group (0 h, 24 h, and 48 h) determined using wound healing assays. G: The number of migrated cells in the TIAM2-OE and control groups determined using the transwell assay. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: Expressing, Migration, Western Blot, Control, CCK-8 Assay, Transwell Assay

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: Silencing of TIAM2 suppresses proliferation, migration, and invasion of OS cells. A: Western blot showed TIAM2 protein levels extracted from siTIAM2 and the control group in 143B and U2OS cells. B: Quantification of the band density ratio of TIAM2 to GAPDH. C, D: Detection of cell proliferation using the CCK-8 assay. E-F, H-I: The wound distance of siTIAM2 and the control group (0 h, 24 h, and 48 h) determined using wound healing assays. G: The number of migrated cells in the siTIAM2 and control groups determined using the transwell assay. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: Migration, Western Blot, Control, CCK-8 Assay, Transwell Assay

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: Expression of the TIAM2 gene affects the phosphorylation of key components in the JAK2/STAT3 pathway. A: Western blotting was used to detect the protein levels of JAK2, p-JAK2, STAT3, and p-STAT3 in siNC and siTIAM2 cells. B, C: Silencing of TIAM2 reduced the ratio of p-STAT3/STAT3 and p-JAK2/JAK2 in the 143B and U2OS cell lines. D: Western blotting was used to detect the protein levels of JAK2, p-JAK2, STAT3, and p-STAT3 in TIAM2-OE and control cells. E, F: Overexpression of TIAM2 increased the ratio of p-STAT3/STAT3 and p-JAK2/JAK2 in the 143B and U2OS cell lines. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: Expressing, Phospho-proteomics, Western Blot, Control, Over Expression

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: Silencing of TIAM2 can slow tumor growth. A: Western blot showed TIAM2 protein levels extracted from shTIAM2 and the control group in 143B cells. B: Quantification of the band density ratio of TIAM2 to GAPDH. C: Tumors obtained from nude mice after dissection. D: The real-time tumor volume. E: Comparison of tumor weight between the two groups. F-G: Hematoxylin-eosin (HE) and IHC staining of tumor tissues (400×); Assessment of staining intensity by positive cells ratio (F). * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: Western Blot, Control, Dissection, Comparison, Immunohistochemistry, Staining

Journal: Journal of Bone Oncology

Article Title: TIAM2 promotes proliferation and invasion of osteosarcoma cells by activating the JAK2/STAT3 signaling pathway

doi: 10.1016/j.jbo.2022.100461

Figure Lengend Snippet: JAK2 inhibitor can attenuate the tumor-promoting effects of TIAM2. A, B: Detection of cell proliferation using the CCK-8 assay. C-F: The wound distance of the four groups determined using wound healing assays. G: The number of migrated cells in the four groups determined using the transwell assay. * P < 0.05, ** P < 0.01.

Article Snippet: Briefly, 143B cells were seeded in 6-well plates (2 × 10 5 cells/well), and TIAM2 shRNA lentivirus (10 μL; Santa Cruz Biotechnology, Santa Cruz, CA, USA) was added to each well (virus titer: 6 × 10 8 TU/mL, shTIAM2 group), with nonspecific shRNA lentivirus added as a negative control (shNC group).

Techniques: CCK-8 Assay, Transwell Assay

Journal: Cellular signalling

Article Title: ROCK1 feedback regulation of the upstream small GTPase RhoA.

doi: 10.1016/j.cellsig.2012.03.005

Figure Lengend Snippet: Fig. 4. Tiam1 is required for the ROCK1 regulation of Rac1 and RhoA activity. PC-3 cells with no transfection (NT), mock transfection (M), a non-targeting, negative control siRNA (siCtrl), and Tiam1 or Tiam2 siRNA (siTiam1/2). Then, cells were treated with vehicle or fasudil (25 μM) at 37 °C for 5 min (designated as+F). (A) Examples of Western blots of GTP–RhoA, total RhoA, GTP–Rac1, total Rac1, Tiam1, and GAPDH from two independent experiments of Tiam1 siRNA. (B) Examples of Western blots of GTP–RhoA, total RhoA, GTP–Rac1, total Rac1, Tiam2, and GAPDH from two independent experiments of Tiam2 siRNA. (C) Average of RhoA activity (top) and Rac1 activity (bottom) from G-LISA™analysis from 3 separate experiments of 4 samples for each treatment as performed in panels A and B. The GTP–Rac1 and GTP–RhoA in siCtrl and siCtrl+F samples were not included in the Western blots; however, RhoA activity and Rac1 activity in these samples were measured by G-LISA. The RhoA activity and Rac1 activity were normalized to the untreated control cells. The results are mean±s.e.m.; ︷, not significantly different; *, significantly lower than control; #, significantly higher than control, pb0.05.

Article Snippet: Tiam1 and Tiam2 siRNA pools including primary antibodies against Tiam1 and Tiam2 raised in rabbit were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Activity Assay, Transfection, Negative Control, Western Blot, Control

Journal: Cellular signalling

Article Title: ROCK1 feedback regulation of the upstream small GTPase RhoA.

doi: 10.1016/j.cellsig.2012.03.005

Figure Lengend Snippet: Fig. 6. A proposed ROCK1 feedback loop for the regulation of Rac1 and RhoA activation. Under normal conditions, active (GTP-bound) RhoA activates ROCK1, which in turn inhibits Tiam1 function and subsequently suppresses the Rac1 activity. Direct ROCK1 inhibition by fasudil increases Tiam1 function to increase Rac1 activity and results in a diminished RhoA activity. The decreased levels of GTP–RhoA diminish the activity of ROCK1, resulting in a cyclical feedback mechanism. Fasudil and Y27632 were used to inhibit ROCK1 function and ROCK1 siRNA was used to knockdown ROCK1 protein and diminish its function. NSC23766 was used to inhibit the interaction of Tiam1/2 with Rac1 and Rac1 activation, and Rac1 siRNA was used to knockdown Rac1 protein and diminish its function. Tiam1/2 siRNA was used to knockdown Tiam1/2 proteins and diminish their ability to activate Rac1.

Article Snippet: Tiam1 and Tiam2 siRNA pools including primary antibodies against Tiam1 and Tiam2 raised in rabbit were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Activation Assay, Activity Assay, Inhibition, Knockdown