sik2 inhibitor  (Selleck Chemicals)

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 1. SIK2 promotes cell motility and metastasis in ovarian cancer. (A) SKOV3 cells were transfected with SIK2 siRNA for 48 h and knockdown efficiency was determined using western blot. (B) Wound healing as well as transwell migration and invasion assays were conducted in SKOV3 cells transfected with SIK2 siRNA; the representative images are shown on the left panel, while the quantitative values, which are presented as mean SD of three independent experiments, are shown on the right panel. Statistical values were calculated by one- way ANOVA. (C) The expression levels of SIK2 were determined in SKOV3 cells transfected with empty vector (EV) or SIK2 overexpression virus (SIK2 OE) for 48 h using western blot. (D) Transwell and wound healing assays were performed using SKOV3-SIK2 OE and SKOV3-EV cell subclones. The representative images are shown on the left panel, while the quantitative values, which were presented as means SD of three independent experiments, are shown on the right panel. Statistical values were calculated by student t-test (unpaired two-tailed) for two groups. (E) IMAGEJ software (NIH, Bethesda, MD, USA) was used to measure the cell areas of SKOV3 cell subclones with stable expression of shSIK2 or shNC. The representative images are shown on the left panel, while the right panel shows the areas of 100 cells in each group presented as means SD. Statistical values were calculated by Student t-test (unpaired two-tailed). (F) The cell area of SKOV3–EV and SKOV3– SIK2 were measured using IMAGEJ. The representative images are shown on the left panel, while the right panel shows the areas of 100 cells in each group presented as means SD. Statistical values were calculated by Student t-test (unpaired two-tailed) for two groups. (G) Immunoblotting of F-actin and MYL2-pS19 in SKOV3 after transfection with SIK2 siRNA for 72 h. (H) Immunoblotting of F-actin and MYL2-pS19 in SKOV3–EV and SKOV3–SIK2. (I) SKOV3-shSIK2 cells were stained for F-actin (red), MYL2-pS19 (green), and DAPI (blue), with the SKOV3- shNC cells being used as the controls. (J) SKOV3–EV and SKOV3–SIK2 cell subclones were stained using anti-F-actin (red), anti-MYL2-pS19 (green), and DAPI (blue); scale bar = 20 lm for 409. SIK2 OE represents SIK2 overexpression. All the experiments were repeated in three independent experiments. Bar plots represent the means SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Transfection, Knockdown, Western Blot, Migration, Expressing, Plasmid Preparation, Over Expression, Virus, Two Tailed Test, Software, Staining

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 2. SIK2 regulates cell motility and MYL2 phosphorylation through MYLK. (A) In vitro kinase assay with recombinant SIK2 protein and CRTC2-derived peptide (red) and MYL2 peptide (blue). (B) Immunoblotting assay of F-actin, MYL2-pS19, ROCK1 and ROCK2 after transfection with SIK2 siRNA for 72 h. (C) Cell lysates were first immunoprecipitated using anti-SIK2 or IgG followed by detection of MYLK expression using western blot. (D) The representative images of immunofluorescent staining for SIK2 (red) and MYLK (green) in SKOV3 and OVCAR8 cells; scale bar = 20lm for 40x. (E) The cell invasion and migration ability of SKOV3 and OVCAR8 cells was determined after MYLK siRNA transfection for 48 h using transwell assays with or without Matrigel. Representative images are shown on the left panel, and the results of quantitative analysis were presented as mean SD of three independent experiments. Statistical values were calculated by one-way ANOVA for groups more than two. (F) Immunoblot analysis of F-actin and MYL2-pS19 expression in SKOV3 and OVCAR8 at 72 h after MYLK siRNA transfection. (G) Immunoblot analysis of F-actin and MYL2-pS19 expression in the OVCAR8-shSIK2 subclone after transfection with control empty vector plasmid (EV) and MYLK overexpressing plasmid (MYLK OE) for 72 h. All the experiments were repeated in three independent experiments. Bar plots represent the means SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, In Vitro, Kinase Assay, Recombinant, Derivative Assay, Western Blot, Transfection, Immunoprecipitation, Expressing, Staining, Migration, Control, Plasmid Preparation

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 3. SIK2 phosphorylates MYLK on Ser343. (A) The putative SIK2 phosphorylation site containing the consensus sequence on the MYLK protein. (B) In vitro kinase assays were performed using wild-type or mutant (S343A) MYLK peptide as substrate to identify the phosphorylation site. The error bars indicate SD. (C) LS-MS/MS assay was performed using the wild-type or mutant (S343A) MYLK peptide as substrate to identify the phosphorylation site. (D) In vitro kinase assay of recombinant SIK2 protein (kinase) and recombinant MYLK protein (substrate) in the presence or absence of the SIK2 inhibitor, ARN-3236, (1 lM) or the MYLK inhibitor, ML-9, (2 lM). (E) The synthetic MYLK peptides bearing the S343 residue without phosphorylation (MYLK) or with phosphorylation (MYLK-pS343) were used in dot blot analysis to determine the validity and specificity of the anti-MYLK-p343 antibody. (F) Total and phosphorylated MYLK and MYL2 were detected in cells with SIK2 knockdown or overexpression. (G) Cell lysates were immunoprecipitated with anti-SIK2 or IgG followed by the detection of MYLK- pS343 using western blot. (H) Immunoblot analysis of the expression of motion-related proteins in the OVCAR8-shSIK2 subclone transfected with MYLK plasmid (WT, S343A, S343E) for 72 h. All the experiments were repeated thrice independently.

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Phospho-proteomics, Sequencing, In Vitro, Mutagenesis, Tandem Mass Spectroscopy, Kinase Assay, Recombinant, Residue, Dot Blot, Knockdown, Over Expression, Immunoprecipitation, Western Blot, Expressing, Transfection, Plasmid Preparation

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 4. ARN-3236 inhibits the MYLK/MYL2 axis and ovarian cancer cell motility. (A, B) SKOV3 and OVCAR8 cells were subjected to wound healing and transwell migration and invasion assays in the presence of SIK2 inhibitor, ARN-3236 (2 lM) or DMSO; the representative images are shown on the left panel, while the quantitative values, which are presented as mean SD of three independent experiments are shown on the right panel. Statistical values were calculated by student t-test (unpaired two-tailed). (C) Immunoblot analysis of F-actin and MYL2-pS19 expression in SKOV3 and OVCAR8 after treatment with ARN-3236 (2 lM) or DMSO for 48 h. All the experiments were repeated in three independent experiments. Bar plots represent the means SD (**P < 0.01, ***P < 0.001, ****P < 0.0001).

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Migration, Two Tailed Test, Western Blot, Expressing

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 5. Omentum-derived adipocytes promote cell motility and activate the SIK2/MYLK/MYL2 pathway. (A) A schematic diagram showing the steps involved in the isolation of adipocytes from the omentum of patients with benign ovarian tumours. (B) SKOV3 and OVCAR8 cells were cultured alone or with adipocytes in the upper chamber, and then transwell assays were performed with or without Matrigel to assess cell migration and invasion capability. The representative images are shown on the left panel, and the quantitative analysis are presented as mean SD of three independent experiments. Statistical values were calculated by Student t-test (unpaired two-tailed). (C) SKOV3 and OVCAR8 cells were cultured alone or with adipocytes for 12 h, and then the cell lysates were used for immunoblot analysis of the indicated proteins. All the experiments were repeated thrice independently. Bar plots represent the means SD (*P < 0.05, **P < 0.01).

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Derivative Assay, Isolation, Cell Culture, Migration, Two Tailed Test, Western Blot

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 6. There is a positive correlation between the expression of SIK2 and MYLK-pS343 in cancer cell lines and tissues. (A) Expression of SIK2, MYL2-pS19 and MYLK-pS343 in ovarian cancer cell lines and breast cancer cell lines were detected using western blot. (B) A plot showing the correlation between SIK2 and MYL2-pS19 in each cell line. Correlations between measured variables were analyzed using Spearman rank correlation test. (C) A plot showing the correlation between SIK2 and MYLK-pS343 in each cell line. Correlations between measured variables were analyzed using Spearman rank correlation test. (D) Representative images showing immunochemical analysis of paraffin-embedded, formalin-fixed ovarian cancer tissue array stained with anti-SIK2 and anti-MYLK-pS343 antibodies; scale bar = 500 lm for 49 and 100 lm for 409. (E) The correlation between SIK2 and MYLK-pS343 in ovarian cancer tissue microarray was analyzed using Pearson’s chi-squared test. (F) Quantification and correlation analysis of the expression of SIK2 and MYLK-pS343 protein in ovarian cancer tissue microarray. Correlations between measured variables were analyzed using Spearman rank correlation test. (G) Kaplan-Meier curves for overall survival time of FIGO stage III-IV patients with high co-expression of SIK2 and MYLK-pS343 vs low co-expression of SIK2 and MYLK-pS343. All the experiments were repeated thrice independently.

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: Expressing, Western Blot, Staining, Microarray

Journal: Molecular oncology

Article Title: SIK2 promotes ovarian cancer cell motility and metastasis by phosphorylating MYLK.

doi: 10.1002/1878-0261.13208

Figure Lengend Snippet: Fig. 7. SIK2 accelerates metastasis of ovarian cancer in vivo (A) Two pairs of ovarian cancer metastasis orthotopic models (SKOV3-EV, SKOV3-SIK2 OE and OVCAR8-shNC, OVCAR8-shSIK2 cancer cells) were orthotopically implanted into the right ovarian bursa of C.B-17 SCID mice. The images show the results of luciferase imaging of the primary and metastatic tumours 4 weeks after implantation. (B) Omental metastasis in mice that were orthotopically transplanted with SKOV3-EV, SKOV3-SIK2 OE and OVCAR8-shNC, OVCAR8-shSIK2 cancer cells (red arrow represents the primary lesion, whereas the white dotted circle represents metastatic tumours). (C–E) The primary and metastatic tumours of the OVCAR8-shNC and OVCAR8-shSIK2 groups. The weight of tumours in each group was also analyzed. Statistical values were calculated by student t-test (unpaired two-tailed). (F, G) Western blot and immunochemical analysis of the expression levels of SIK2, MYLK, MYLK-pS343, MYL2 and MYL2-pS19. (H) A schematic diagram depicting the regulation of cell motility and metastasis by SIK2 in ovarian cancer cells. All the experiments were repeated thrice independently. Bar plots represent the means SD. (**P < 0.01, ***P < 0.001).

Article Snippet: SIK2 inhibitor, ARN-3236, was purchased from Selleckchem (Houston, TX, USA), while MYLK inhibitor, ML-9, was purchased from Santa Cruz Biotechnology (Dallas, TX, USA).

Techniques: In Vivo, Luciferase, Imaging, Two Tailed Test, Western Blot, Expressing

Journal: BMC Pulmonary Medicine

Article Title: Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1186/s12890-022-01940-0

Figure Lengend Snippet: Inactivation of SIK2 led to the dephosphorylation and nuclear translocation of CRTC2 in human fetal lung fibroblasts (HFLs). HFLs or SIK2 overexpressed HFLs (HFLs-SIK2) were treated with DMSO (0.1%), ARN-3236 (0.5 μM) or transfected with SIK2 SiRNA (100 nM). Cells were then treated with TGF-β1 (5 ng/mL) for 12 h. A , B Representative western blot bands and quantitation of SIK2, pCRTC1, pCRTC2 and CRTC2 protein expressions in HFLs. C Representative confocal images and quantification of the nuclear translocated CRTC2 in HFLs and HFLs-SIK2 cells. Data are expressed as mean ± SEM of at least 4 independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: SIK2 inhibitor ARN-3236 was purchased from Medchemexpress (HY-120856) and Tsbiochem (T5993). pCREB inhibitor 666-15 was purchased from Medchemexpress (HY-101120) [ ], Tsbiochem (T5318) and Cayman Chem (30780).

Techniques: De-Phosphorylation Assay, Translocation Assay, Transfection, Western Blot, Quantitation Assay

Journal: BMC Pulmonary Medicine

Article Title: Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1186/s12890-022-01940-0

Figure Lengend Snippet: Inactivation of SIK2 inhibited the fibroblast differentiation and TGF-β1 induced pro-fibrotic mediator expression in HFLs. HFLs or SIK2 overexpressed HFLs (HFLs-SIK2) were treated with DMSO (0.1%), ARN-3236 (0.5 μM), 666-15 (0.5 μM) or transfected with SIK2 SiRNA (100 nM). Cells were then treated with TGF-β1 (5 ng/mL) for 12 h. Inactivation of SIK2 inhibited A mRNA expression of α-SMA, COL1A and fibronectin and B cell proliferation in HFLs. Data are expressed as mean ± SEM of at least 4 independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: SIK2 inhibitor ARN-3236 was purchased from Medchemexpress (HY-120856) and Tsbiochem (T5993). pCREB inhibitor 666-15 was purchased from Medchemexpress (HY-101120) [ ], Tsbiochem (T5318) and Cayman Chem (30780).

Techniques: Expressing, Transfection

Journal: BMC Pulmonary Medicine

Article Title: Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1186/s12890-022-01940-0

Figure Lengend Snippet: Inhibition of SIK2 reduced CRTC2 phosphorylation in mice. Male BALB/C mice were anesthetized and received intranasal instillation of BLM (5 mg/kg) or its saline vehicle, followed by receiving administration of ARN-3236 (10, 30 mg/kg) or its vehicle. A Representative western blot bands and quantitation of SIK2 and pCRTC2 protein expressions in lungs at day 14 and 28. B Representative western blot bands and quantitation of pCRTC2 protein expressions in lungs at day 28. Data are expressed as mean ± SEM of at least 6 independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: SIK2 inhibitor ARN-3236 was purchased from Medchemexpress (HY-120856) and Tsbiochem (T5993). pCREB inhibitor 666-15 was purchased from Medchemexpress (HY-101120) [ ], Tsbiochem (T5318) and Cayman Chem (30780).

Techniques: Inhibition, Saline, Western Blot, Quantitation Assay

Journal: BMC Pulmonary Medicine

Article Title: Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1186/s12890-022-01940-0

Figure Lengend Snippet: Inhibition of SIK2 attenuates bleomycin (BLM)-induced pulmonary fibrosis in mice. Male BALB/C mice were anesthetized and received intranasal instillation of BLM (5 mg/kg) or its saline vehicle, followed by receiving administration of ARN-3236 (10, 30 mg/kg), CREB inhibitor 666-15 (10 mg/kg) or their vehicle. Lung tissues were isolated at day 28 for analysis. A Lung coefficient measured at day 28. B Mice body weight and lung coefficient measured at day 0, 14 and 28. C The content of hydroxyproline in lung tissues. D Semiquantitative analysis of lung tissues by H&E staining. E Representative histopathological sections of lung tissues by H&E staining. Data are expressed as mean ± SEM of at least 6 independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: SIK2 inhibitor ARN-3236 was purchased from Medchemexpress (HY-120856) and Tsbiochem (T5993). pCREB inhibitor 666-15 was purchased from Medchemexpress (HY-101120) [ ], Tsbiochem (T5318) and Cayman Chem (30780).

Techniques: Inhibition, Saline, Isolation, Staining

Journal: BMC Pulmonary Medicine

Article Title: Salt-inducible kinase 2 (SIK2) inhibitor ARN-3236 attenuates bleomycin-induced pulmonary fibrosis in mice

doi: 10.1186/s12890-022-01940-0

Figure Lengend Snippet: Inhibition of SIK2 attenuates bleomycin (BLM)-induced pulmonary fibrosis in mice. Male BALB/C mice were anesthetized and received intranasal instillation of BLM (5 mg/kg) or its saline vehicle, followed by receiving administration of ARN-3236 (10, 30 mg/kg), CREB inhibitor 666-15 (10 mg/kg) or their vehicle. Lung tissues were isolated at day 28 for analysis. Semiquantitative analysis of A Masson’s trichrome staining in E , B α-SMA expression in F and C COL1A expression in G were scored. D mRNA expression of α-SMA, COL1A, TGF-β1 and fibronectin in lungs. E Representative histopathological sections and semiquantitative analysis of lung tissues by Masson’s trichrome staining. Representative images of the immunohistochemical F α-SMA and G COL1A expression in lung tissues. Data are expressed as mean ± SEM of at least 6 independent experiments. *** P < 0.001; ** P < 0.01; * P < 0.05

Article Snippet: SIK2 inhibitor ARN-3236 was purchased from Medchemexpress (HY-120856) and Tsbiochem (T5993). pCREB inhibitor 666-15 was purchased from Medchemexpress (HY-101120) [ ], Tsbiochem (T5318) and Cayman Chem (30780).

Techniques: Inhibition, Saline, Isolation, Staining, Expressing, Immunohistochemical staining