Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 promotes Akt/mTOR activation in ovarian cancer cells. (a) Western blot analysis of pAkt (S473), Akt, pmTOR (S2448), and mTOR levels in cells expressing control (ctrl) or RBM11 shRNA. (b) Flag-tagged RBM11 was transfected into OVCAR-3 and A2780 cells, and the expression of flag-RBM11 was validated by western blot assay. (c) Western blot analysis of indicated protein levels in cells transfected with empty vector (EV) or flag-RBM11.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Transfection, Plasmid Preparation

Journal: Oncotarget

Article Title: Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK -mutated neuroblastoma

doi:

Figure Lengend Snippet: A, Western blot analysis of pALK Y1604 in ALK F1174L -expressing Kelly NB cells treated with increasing concentrations of crizotinib for 6 hr. B, Western blot analyses of the indicated downstream signaling proteins in Kelly cells after treatment with increasing concentrations of crizotinib for 6 hr. C, Western blot analyses in Kelly ( ALKF1174L ) and NGP ( ALK-wild type ) cells of the indicated signaling proteins in cells in which ALK expression was depleted by shRNA knockdown.

Article Snippet: Antibodies for pALK Y1604 (3341), ALK (3333), pAKT S473 (9271), pAKT T308 (4056), AKT (9272), pRPS6 S240/244 (2215), RPS6 (2217), MYCN (9405), GAPDH (2118), β-Actin (4967), β-Tubulin (2128), 4E-BP1 (9644), p4E-BP1 T37/46 (9459), mTOR (2972), pmTOR S2448 (2971) and PARP (9542) were purchased from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, shRNA, Knockdown

Journal: Oncotarget

Article Title: Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK -mutated neuroblastoma

doi:

Figure Lengend Snippet: A, Western blot analyses of the indicated signaling proteins in Kelly cells in which MYCN expression was suppressed using shRNA knockdown. B, Composite of western blot analyses of the indicated proteins in SHEP NB cells expressing a doxycycline-repressible MYCN construct in which ALK F1174L was overexpressed using retroviral transduction. Controls are SHEP cells transduced with GFP. Doxycycline (1 μg/ml) was added for 24 hr. to repress MYCN expression. Repression of MYCN led to a 32% reduction in pRPS6 levels compared to cells expressing MYCN ( P = 0.017) as measured by densitometry. C, Western blot analyses of the indicated proteins in SHEP cells expressing ALK F1174L with (+) or without (−) MYCN repression treated with 1 μM crizotinib for the indicated duration.

Article Snippet: Antibodies for pALK Y1604 (3341), ALK (3333), pAKT S473 (9271), pAKT T308 (4056), AKT (9272), pRPS6 S240/244 (2215), RPS6 (2217), MYCN (9405), GAPDH (2118), β-Actin (4967), β-Tubulin (2128), 4E-BP1 (9644), p4E-BP1 T37/46 (9459), mTOR (2972), pmTOR S2448 (2971) and PARP (9542) were purchased from Cell Signaling Technologies.

Techniques: Western Blot, Expressing, shRNA, Knockdown, Construct, Retroviral, Transduction

Journal: Oncotarget

Article Title: Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK -mutated neuroblastoma

doi:

Figure Lengend Snippet: A, The growth of Kelly cell tumor xenografts was monitored in mice treated with vehicle, crizotinib, Torin2 or the combination, orally once daily for 3 cycles. Tumor volume measurements were truncated at the time point at which the first animal in a cohort had a tumor volume measuring >1000mm 3 . Tumor volumes are expressed as mean ± SEM. Volume comparisons were performed at day 18 (vehicle vs. crizotinib, P=not significant; vehicle vs. Torin2, *P<0.01; vehicle vs. combination, **P< 0.001; all comparisons by two-way ANOVA). B, Kaplan-Meier survival analysis of mice treated with control, single agents alone or the combination of crizotinib and Torin2 (crizotinib vs. combination, *P=0.03; Torin2 vs. combination, **P=0.005; all by log-rank test). C, Hematoxylin and eosin (H&E), pRPS6 and cleaved caspase-3 immunohistochemical staining on tumor sections following treatment with either single agents or the combination. The scale bar represents 50 μm. D, Immunoblot analysis of the indicated proteins in tumors of mice treated as indicated above. E, Bioluminescence measurements of human NB xenograft models established by intravenous injection of ALK F1174L / MYCN -positive NB cells. Animals were treated with the indicated agents. F, Kaplan-Meier survival analysis of mouse xenograft models of human NB treated with the indicated agents (crizotinib vs. combination, **P=0.001; by log rank test).

Article Snippet: Antibodies for pALK Y1604 (3341), ALK (3333), pAKT S473 (9271), pAKT T308 (4056), AKT (9272), pRPS6 S240/244 (2215), RPS6 (2217), MYCN (9405), GAPDH (2118), β-Actin (4967), β-Tubulin (2128), 4E-BP1 (9644), p4E-BP1 T37/46 (9459), mTOR (2972), pmTOR S2448 (2971) and PARP (9542) were purchased from Cell Signaling Technologies.

Techniques: Control, Immunohistochemical staining, Staining, Western Blot, Injection

Journal: Oncotarget

Article Title: Molecular rationale for the use of PI3K/AKT/mTOR pathway inhibitors in combination with crizotinib in ALK -mutated neuroblastoma

doi:

Figure Lengend Snippet: A, In ALK F1174L -mutant NB cells, AKT is phosphorylated at T308 and S473 by ALK-activated PI3K and mTORC2, respectively, promoting cell survival, transcriptional activation through release of 4E-BP1 from eIF4, and ribosome biogenesis through activation of RPS6. Activation of RPS6 in turn leads to inhibition of PI3K, creating a feedback loop that permits fine-tuning of proliferative and survival signaling. In MYCN -amplified, ALK F1174L -mutated NB cells, MYCN drives cellular proliferation, leading to increased dependence on RPS6 and 4E-BP1 activity. B, Treatment of these cells with crizotinib as a single agent inadequately inhibits mutant ALK, leading to marginally reduced PI3K (pAKT T308 ) and mTORC2 (pAKT S473 ) activities and a modest reduction in cell survival. Although the reduction in pAKT levels leads to a minimal reduction of pRPS6 in MYCN -amplified cells, this response is not sustained, as deregulated MYCN maintains upregulated mTORC1 activity. C, Single-agent treatment with specific ATP-competitive mTOR inhibitors, such as Torin2, blocks both mTORC1 and mTORC2 activity, downregulating pRPS6 and pAKT S473 , although amplified MYCN continues to promote persistent mTORC1 activation and some level of PI3K feedback inhibition. D, The combination of crizotinib and Torin2 in MYCN -amplified cells targets the activities of PI3K, mTORC1 and mTORC2 simultaneously; continued loss of feedback inhibition of PI3K activity via incomplete suppression of MYCN/mTORC1/RPS6 signaling synergizes with crizotinib-driven inhibition of ALK to downregulate PI3K activity and, in turn, AKT T308 phosphorylation.

Article Snippet: Antibodies for pALK Y1604 (3341), ALK (3333), pAKT S473 (9271), pAKT T308 (4056), AKT (9272), pRPS6 S240/244 (2215), RPS6 (2217), MYCN (9405), GAPDH (2118), β-Actin (4967), β-Tubulin (2128), 4E-BP1 (9644), p4E-BP1 T37/46 (9459), mTOR (2972), pmTOR S2448 (2971) and PARP (9542) were purchased from Cell Signaling Technologies.

Techniques: Mutagenesis, Activation Assay, Inhibition, Amplification, Activity Assay, Phospho-proteomics

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 is overexpressed in ovarian cancer tissues and is highly associated with poor survival in ovarian cancer patients. (a) RBM11 mRNA expression in ovarian cancer tissues was assessed with TCGA database. (b, c) RRM1 protein levels in normal ovary and ovarian cancer tissue were analyzed by Immunohistochemistry (IHC) staining. Representative image of staining (b) and quantitative analysis of immunoscore (c) were provided. (d) Overall survival assay in ovarian cancer patients with high RBM11 mRNA level and low RBM11 mRNA level was performed using the Kaplan-Meier plotter online server ( https://www.kmplot.com/analysis ).

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Immunohistochemistry, Staining, Clonogenic Cell Survival Assay

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: Knockdown of RBM11 inhibits ovarian cancer cell growth. (a) Western blot analysis of RBM11 protein level in OVCAR-3 and A2780 cells stably transfected with control (ctrl), RMB11 shRNA-1, or RBM11 shRNA-2. (b) Cell growth curve of OVCAR-3 and A2780 cells expressing ctrl shRNA, RBM11 shRNA-1, or RBM11 shRNA-2. (c) Colony formation assay of cells expressing ctrl shRNA or RBM11 shRNA. Left, the representative colony growth; right, the relative quantification of colony number. ∗∗ p < 0.01 and ∗∗∗ p < 0.001, by 2-tailed t -test, when compared with cells expressing ctrl shRNA.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Knockdown, Western Blot, Stable Transfection, Transfection, Control, shRNA, Expressing, Colony Assay, Quantitative Proteomics

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 promotes ovarian cancer invasion. (a) Transwell invasion analysis of OVCAR-3 and A2780 cells expressing ctrl or RBM11 shRNA. Left, representative staining of cells on the bottom membrane of the transwell insert was provided; right, cells on the bottom membrane per field were quantified. (b) Flag-tagged RBM11 was stably expressed in OVCAR-3 or A2780 cells; the expression of flag-RBM11 was examined by western blot. (c) Transwell analysis of OVCAR-3 or A2780 cells expressing flag-RBM11. Representative image (left) and quantitative analysis (right) were shown. ∗∗ p < 0.01 and ∗∗∗ p < 0.001, by 2-tailed t -test, when compared with cells expressing ctrl shRNA.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, shRNA, Staining, Membrane, Stable Transfection, Western Blot

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 promotes Akt/mTOR activation in ovarian cancer cells. (a) Western blot analysis of pAkt (S473), Akt, pmTOR (S2448), and mTOR levels in cells expressing control (ctrl) or RBM11 shRNA. (b) Flag-tagged RBM11 was transfected into OVCAR-3 and A2780 cells, and the expression of flag-RBM11 was validated by western blot assay. (c) Western blot analysis of indicated protein levels in cells transfected with empty vector (EV) or flag-RBM11.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Transfection, Plasmid Preparation

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 silence retarded tumor growth in the A2780 xenograft model. (a) A2780 cells expressing control (ctrl) or RBM11 shRNA were implanted into nude mice, the tumor volumes were measured, and the tumor growth curve was shown. (b) Immunohistochemistry (IHC) analysis of Ki67 levels in tumor tissue. ∗ p < 0.01 and ∗∗∗ p < 0.001, by 2-tailed t -test, when compared with cells expressing ctrl shRNA.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Expressing, Control, shRNA, Immunohistochemistry

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 promotes Akt/mTOR activation in ovarian cancer cells. (a) Western blot analysis of pAkt (S473), Akt, pmTOR (S2448), and mTOR levels in cells expressing control (ctrl) or RBM11 shRNA. (b) Flag-tagged RBM11 was transfected into OVCAR-3 and A2780 cells, and the expression of flag-RBM11 was validated by western blot assay. (c) Western blot analysis of indicated protein levels in cells transfected with empty vector (EV) or flag-RBM11.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Transfection, Plasmid Preparation

Journal: Disease Markers

Article Title: RNA-Binding Motif Protein 11 (RBM11) Serves as a Prognostic Biomarker and Promotes Ovarian Cancer Progression

doi: 10.1155/2021/3037337

Figure Lengend Snippet: RBM11 promotes Akt/mTOR activation in ovarian cancer cells. (a) Western blot analysis of pAkt (S473), Akt, pmTOR (S2448), and mTOR levels in cells expressing control (ctrl) or RBM11 shRNA. (b) Flag-tagged RBM11 was transfected into OVCAR-3 and A2780 cells, and the expression of flag-RBM11 was validated by western blot assay. (c) Western blot analysis of indicated protein levels in cells transfected with empty vector (EV) or flag-RBM11.

Article Snippet: Anti-RBM11 (17220-1-AP) antibody was purchased from Proteintech (Wuhan, China), and anti-pAkt S473 (#9271), anti-Akt (#4691), anti-pmTOR S2448 (#5536), and anti-mTOR (#2972) antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Expressing, Control, shRNA, Transfection, Plasmid Preparation