Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A promotes CRC tumor progression is dependent on RSK phosphorylation (A) Results from protein kinase microarray analysis of HCT116-MCS and HCT116-FAM65A cells. (B) Quantitative assessment of the protein kinase microarray data, n = 2. (C) Western blot analysis demonstrating the expression levels of p -RSK and total RSK in HCT116-MCS and HCT116-FAM65A cells. (D) Western blot analysis of p -RSK and RSK expression in HCT116-FAM65A cells treated with 4 and 8 μM BRD7389 or without treatment. (E) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of BRD7389, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Outcomes of the colony formation assay performed on HCT116-FAM65A cells treated with BRD7389 or without treatment. (G) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (H) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 100 μm. (I) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (J) Western blot analysis of Ki-67 expression in HCT116-FAM65A cells treated with BRD7389 or not. (K) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (L) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (M) Western blot analysis of the expression levels of cleaved Caspase 3, Bcl-2, and Bax in HCT116-FAM65A cells treated with BRD7389 or not. (N) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 50 μm. (O) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (P) Results from the wound healing assay performed on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (Q) Quantitative analysis of the wound healing assay results, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (R) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with BRD7389 or not. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: The phosphorylation of RSK was inhibited by the selective RSK family kinase inhibitor BRD7389 (Cat. # HY-12185, MedChemExpress, USA).

Techniques: Phospho-proteomics, Microarray, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A promotes CRC tumor progression is dependent on RSK phosphorylation (A) Results from protein kinase microarray analysis of HCT116-MCS and HCT116-FAM65A cells. (B) Quantitative assessment of the protein kinase microarray data, n = 2. (C) Western blot analysis demonstrating the expression levels of p -RSK and total RSK in HCT116-MCS and HCT116-FAM65A cells. (D) Western blot analysis of p -RSK and RSK expression in HCT116-FAM65A cells treated with 4 and 8 μM BRD7389 or without treatment. (E) Results from the CCK8 cell proliferation assay conducted on HCT116-FAM65A cells with and without the application of BRD7389, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (F) Outcomes of the colony formation assay performed on HCT116-FAM65A cells treated with BRD7389 or without treatment. (G) Quantitative analysis of the colony formation assay results, n = 3, ∗∗∗ p < 0.001. (H) Results from the EdU assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 100 μm. (I) Quantitative analysis of the EdU assay results, n = 3, ∗∗∗ p < 0.001. (J) Western blot analysis of Ki-67 expression in HCT116-FAM65A cells treated with BRD7389 or not. (K) Results from the apoptosis assay conducted on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (L) Quantitative analysis of the apoptosis experiments, n = 3, ∗∗∗ p < 0.001. (M) Western blot analysis of the expression levels of cleaved Caspase 3, Bcl-2, and Bax in HCT116-FAM65A cells treated with BRD7389 or not. (N) Results from the Transwell migration assay conducted on HCT116-FAM65A cells with and without the application of BRD7389. Scale bars, 50 μm. (O) Quantitative analysis of the Transwell migration assay results, n = 3, ∗∗∗ p < 0.001. (P) Results from the wound healing assay performed on HCT116-FAM65A cells treated with BRD7389 or not. Scale bars, 50 μm. (Q) Quantitative analysis of the wound healing assay results, n = 3, ∗∗ p < 0.01, ∗∗∗ p < 0.001. (R) Western blot analysis the expression of EMT markers in HCT116-FAM65A cells treated with BRD7389 or not. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -RSK , Cell Signal Technology , Cat# 11989; RRID: AB_2687613.

Techniques: Phospho-proteomics, Microarray, Western Blot, Expressing, Proliferation Assay, Colony Assay, EdU Assay, Apoptosis Assay, Transwell Migration Assay, Wound Healing Assay

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: FAM65A binds to Ras and activates the Ras/ERK signaling to mediate RSK activation (A) The volcano plot analysis results for the FAM65A high-expression and low-expression groups from the TCGA database were shown. (B) The KEGG and GO results were shown. (C) The GSEA results were shown. (D) GSEA on DEGs between the FAM65A high-expression group and low-expression group in the Reactome database were shown. (E) IP was performed to detect the binding of FAM65A and Ras/p-RSK. (F) IP was performed to detect the binding of Ras and FAM65A/p-RSK. (G) Immunofluorescence was performed to detect the co-localization of FAM65A and Ras. Scale bars, 20 μm. (H) Western blot analysis the Ras and p -ERK expression in FAM65A knockdown or overexpression cells. Data are presented as mean ± SEM of biologically independent experiments.

Article Snippet: p -RSK , Cell Signal Technology , Cat# 11989; RRID: AB_2687613.

Techniques: Activation Assay, Expressing, Binding Assay, Immunofluorescence, Western Blot, Knockdown, Over Expression

Journal: iScience

Article Title: FAM65A, as a potential predictor of prognosis, promotes colorectal cancer progression via activating Ras/ERK/RSK signaling

doi: 10.1016/j.isci.2026.114662

Figure Lengend Snippet: Knockdown of FAM65A inhibits tumor progression in vivo (A) LOVO-shCtrl and LOVO-shFAM65A cells were administered into the fourth fat pad of nude mice, and the resulting tumor growth curves were subsequently generated, n = 5, ∗ p < 0.05. (B) The tumors excised from mice across various experimental groups are presented. (C) Hematoxylin and Eosin (HE) staining results of lung tissue from the different groups is displayed. (D) A quantitative analysis of metastatic lung nodules is provided, n = 5, ∗∗ p < 0.01. (E) IHC results for FAM65A, Ki-67, p -RSK, p -ERK, Ras, N-cadherin, vimentin, cleaved Caspase 3, ZO-1, and E-cadherin in tumor tissues are illustrated. (F) A quantitative analysis of the IHC results is included. Data are presented as mean ± SEM of biologically independent experiments, n = 5, ∗∗∗ p < 0.001.

Article Snippet: p -RSK , Cell Signal Technology , Cat# 11989; RRID: AB_2687613.

Techniques: Knockdown, In Vivo, Generated, Staining

Journal: International Journal of Biological Sciences

Article Title: O-GlcNAcylation stabilizes RSK4 by antagonizing GSK3β-mediated phosphorylation to enhance radioresistance in esophageal squamous cell carcinoma

doi: 10.7150/ijbs.128078

Figure Lengend Snippet: FBXW7 is the physiological E3 ubiquitination ligase for RSK4. (A) Transfected 293T cells with Myc-tagged UBR5, FBXW7, β-TrCP, MG53, and ARIH1 plasmids, then assessed RSK4 ubiquitination levels. (B) Colocalization of RSK4 and FBXW7 was visualized by confocal microscopy in TE10 cells. Scale bars, 100 µm. (C) The interaction of RSK4 and FBXW7 was confirmed by an endogenous co-IP assay in TE10 cells. IgG served as a negative control. (D) Mapping analyses of full-length and truncated RSK4, supported by representative co-IP assays in 293T cells, revealed that the CTKD of RSK4 mediates its interaction with FBXW7. C, CTKD; K, kinase interaction motif (KIM); N, NTKD. (E) Following FBXW7 overexpression, cells were treated with MG132 to assess RSK4 protein levels. (F-G) Overexpression of FBXW7 in TE10 cells or its knockdown in ECA109 cells, combined with CHX treatment, enabled comparison of RSK4 protein half-life across experimental groups. (H) Perform ubiquitination experiments in TE10 and ECA109 cells pre-treated with MG132 to analyze the effect of FBXW7 expression on RSK4 ubiquitination levels. (I) 293T cells transfected with Myc-FBXW7, Flag-RSK4, V5-Ub, V5-Ub K48R, and V5-Ub K63R were immunoprecipitated with Protein A/G agarose incubated with anti-Flag antibody, followed by western blotting.

Article Snippet: The primary antibodies against RSK4 (Santa Cruz Biotechnology, sc-100424), FBXW7 (Santa Cruz Biotechnology, sc-293423), OGT (Proteintech, 11576-2-AP).

Techniques: Ubiquitin Proteomics, Transfection, Confocal Microscopy, Co-Immunoprecipitation Assay, Negative Control, Over Expression, Knockdown, Comparison, Expressing, Immunoprecipitation, Incubation, Western Blot

Journal: International Journal of Biological Sciences

Article Title: O-GlcNAcylation stabilizes RSK4 by antagonizing GSK3β-mediated phosphorylation to enhance radioresistance in esophageal squamous cell carcinoma

doi: 10.7150/ijbs.128078

Figure Lengend Snippet: GSK3β-mediated RSK4 phosphorylation instigates FBXW7-mediated RSK4 degradation. (A)The RSK4 protein sequence contains two potential FBXW7 recognition sites, Thr368-Ser372 and Thr402-Ser406. The red region indicates a conserved motif within the RSK4 sequence that may be subject to FBXW7-mediated downregulation. (B) GSK3β phosphorylated RSK4 at Thr402/Ser406 in vitro by an autoradiograph. The input was confirmed by silver staining. (C) 293T cells were transfected with HA-GSK3β, Flag-RSK4-WT, Flag-RSK4-T402A/S406A and V5-Ub. After MG132 treatment, co-immunoprecipitation was performed using Protein A/G agarose beads incubated with anti-Flag antibody, followed by western blotting analysis with anti-V5, anti-HA and anti-Flag antibodies, respectively. (D) 293T cells were transfected with V5-Ub, Myc-FBXW7, Flag-RSK4-WT and Flag-RSK4-K570R plasmids, and cell extracts were immunoprecipitated with an anti-Flag antibody. Ubiquitinated RSK4 was detected by immunoblotting. (E-F) In 293T cells, Flag-RSK4, Myc-FBXW7 and HA-GSK3β plasmids were transfected. The cells were treated with the GSK3β inhibitor AR-A014418 and MG132 as indicated. RSK4 protein levels were detected and immunoprecipitation assays were conducted to examine the effect of GSK3β inhibition on the interaction between FBXW7. (G) 293T cells pre-treated with MG132 were transfected with the indicated plasmids, and ubiquitinated RSK4 was detected by immunoblotting.

Article Snippet: The primary antibodies against RSK4 (Santa Cruz Biotechnology, sc-100424), FBXW7 (Santa Cruz Biotechnology, sc-293423), OGT (Proteintech, 11576-2-AP).

Techniques: Phospho-proteomics, Sequencing, In Vitro, Autoradiography, Silver Staining, Transfection, Immunoprecipitation, Incubation, Western Blot, Inhibition

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Endogenous LARG phosphorylation at S1288 and RhoA activation in GBM tissues . A , the phospho-LARG S1288 specific antibody was tested in U87MG cells transfected with Myc-LARG-WT, Myc-LARG-S1288 A, or Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min in the presence or absence of BI-D1870. B , the phospho-LARG S1288 antibody demonstrates specificity to the phosphorylated form of the protein. U87MG cells were transfected with Myc-LARG-WT of Myc-EV. After 24 h of starvation, cells were treated with EGF for 10 min. Protein was harvested and lysates were treated with λ phosphatase prior to immunoblotting. C , RSK2 phosphorylates LARG at S1288 in PDX-derived GBM39 cells. Cells maintained in complete medium with 10% FBS were treated with BI-D1870 for 4 h. Subsequently cells were starved in 0.1% FBS medium for 24 h prior to 10 min EGF treatment alone or after prior 4 h BID treatment. Immunoblotting was used to determine total expression and phosphorylation of target proteins. The results are representative of three independent experiments. D , levels of activated RhoA-GTP positively correlate with levels of phosphorylated LARG S1288 in human GBM tumor lysates. Endogenous total LARG, pLARG-S1288 and RhoA expression in seven human GBM tumor lysates were analyzed by immunoblotting. Activated RhoA-GTP was isolated by immunoprecipitation and detected with a RhoA antibody. Signals for each target protein expression were normalized to corresponding sample β-tubulin expression. A linear regression analysis was performed to assess the relationship between Activated RhoA expression and pLARG S1288 expression in all seven tumors.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Phospho-proteomics, Activation Assay, Transfection, Western Blot, Derivative Assay, Expressing, Isolation, Immunoprecipitation

Journal: The Journal of Biological Chemistry

Article Title: Phosphorylation at S1288 of leukemia associated RhoGEF (LARG/ARHGEF12) induces plasma membrane localization and promotes binding and activation of RhoA

doi: 10.1016/j.jbc.2025.110996

Figure Lengend Snippet: Model of LARG activation of RhoA in response to RSK2 phosphorylation . Schematic of the proposed signaling mechanism in which EGF stimulation activates RSK2, which leads to the phosphorylation of LARG at serine 1288. Phosphorylation of LARG at serine 1288 then promotes plasma membrane localization of LARG and binding to RhoA and subsequent RhoA activation.

Article Snippet: RhoA (sc-418) mouse, α tubulin (sc-8035) mouse, YB1 (sc-101198) mouse, RSK2 (sc-9986) mouse, LARG H-3 (sc-166318) mouse, GST (sc-374171) mouse monoclonal antibodies, and RSK2 inhibitor BI-D1870 were purchased from Santa Cruz Biotechnology.

Techniques: Activation Assay, Phospho-proteomics, Clinical Proteomics, Membrane, Binding Assay