Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: Fbxw7 deletion promotes the development of ESCC induced by 4NQO. (A) FBXW7 mutation hotspots in human esophageal squamous cell carcinoma (ESCC) tissues were analyzed by the cBioPortal database. (B and C) The genetically modified mouse models: mice with indicated genotypes were sacrificed at the age of 12 months, and the esophagus organs were isolated for photography (B and C). Macroscopic (B) and representative (C) images of the esophagus from the mice are shown. (D) Schematic diagram of the short‐term and long‐term 4NQO‐induced carcinogenesis mouse models of ESCC. (E–I) For the long‐term model, female mice at 6–7 weeks of age were administered 100 µg/mL 4NQO in their drinking water for 16 weeks, followed by tap water for 8 weeks. Esophagi were isolated for photography (E and F), H&E (G), and IHC (H and I) staining. Macroscopic (E) and representative (F) images of the esophagus from the long‐term model are shown. The number of tumors in the esophagus of each mouse is shown in (F). Representative images of Ki67 staining are shown (H), and the percentage of cells with Ki67‐positive staining from three random fields of the esophagus in each mouse is quantified (I). Scale bars: 1.25 mm (left, G), 100 µm (right, G), 100 µm (H), and 25 µm (inset, H); the insets show enlarged images of the red boxes. Data are shown as mean ± SEM and analyzed by Student's t test (F and I); WT: n = 18, Fbxw7 cKO: n = 22 (F), n = 5 (I); *** p < 0.001.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Mutagenesis, Genetically Modified, Isolation, Staining

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: Identification of SPT6 as a new substrate of FBXW7 in ESCC. (A and B) ESCC tumors from mice following long‐term treatment were harvested for mass spectrometry analysis (A). Volcano plot (B) shows differentially expressed genes (DEGs), with downregulated genes in blue and up‐regulated genes in red upon Fbxw7 deletion (Fold change > 1.5, p < 0.05). (C and D) KYSE30 and KYSE150 cells were treated with the indicated concentrations of MLN4924 for 24 h, followed by immunoblotting (IB) (C) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (D). (E and F) KYSE30 and KYSE150 cells were transfected with siRNAs targeting FBXW7 (siFBXW7s) or scrambled control siRNA (siNC) for 48 h, followed by IB analysis using the indicated antibodies (Abs) (E) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (F). (G and H) KYSE30 and KYSE150 cells were transfected with increasing amounts of FBXW7 plasmids for 48 h, followed by IB analysis with the indicated Abs (G) and the quantification of SPT6 levels from three independent experiments after normalization with β‐Actin as the loading control (H). (I–K) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IB analysis (I) and IHC staining (J and K). Representative images of Spt6 staining are shown (J), and the percentage of cells with Spt6 positive staining in three random fields of the esophagus in each mouse was quantified (K). Scale bars: 100 and 25 µm (inset). Data are shown as means ± SEM and analyzed by one‐way ANOVA (D, F, and H) and Student's t test (K); n = 3 (D, F, and H), n = 5 (K); * p < 0.05, ** p < 0.01, ns: no significance.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Mass Spectrometry, Western Blot, Control, Transfection, Immunohistochemistry, Staining

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: FBXW7 interacts with SPT6 via its consensus degron motif. (A and B) KYSE30 (A) and KYSE150 (B) cells were harvested and subjected to immunoprecipitation (IP) with FBXW7 or SPT6 Ab, along with normal control IgG, followed by IB analysis with the indicated Abs. (C and D) Schematic representation of FBXW7 (C) and SPT6 (D) and their truncate mutants. Numbers indicate the amino acid (AA) positions. FBXW7: DD, dimerization domain; F‐box, SKP1 binding region; WD40, substrate recognition and binding region. SPT6: SPT6‐C1, 1056–1726AA, SPT6‐C2, 1323–1726AA. The FBXW7 binding motif (SPNTE: 1143–1147 AA) on SPT6 is indicated (D). (E–H) Cells were transfected with the indicated constructs and then subjected to IP with SPT6 (E and H) or FBXW7 (F and G) Ab, followed by IB analysis with the indicated Abs. FL, full length. The quantification is performed with Image J and expressed as the relative level of FBXW7 binding with SPT6 after normalization to immunoprecipitated SPT6 (H). (I) KYSE30 and KYSE150 cells were co‐transfected with GFP‐FBXW7α and FLAG‐SPT6 for 48 h and stained with FLAG antibody. The images were captured using a fluorescence microscope. Scale bar: 10 µm. LE, longer exposure; SE, shorter exposure; WCE, whole cell extract.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Immunoprecipitation, Control, Binding Assay, Transfection, Construct, Staining, Fluorescence, Microscopy

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: FBXW7 negatively regulates SPT6 stability by promoting its ubiquitylation. (A and B) Cells were transfected with the indicated plasmids and siRNA oligos for 48 h (A). Cells were infected with lentivirus expressing the indicated shRNA and then transfected with the indicated plasmids for 48 h (B). After 6 h of MG132 treatment, cells were harvested for Ni‐NTA purification. Ni‐NTA affinity purified fractions and WCE were analyzed by IB with the indicated Abs. (C–E) KYSE150 cells were transfected with the indicated siRNA (C) or plasmids (D and E) for 48 h and then treated with cycloheximide (CHX) for the indicated time periods, followed by IB analysis. (F and G) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis (F) and the quantification of SPT6 levels from three independent experiments after normalization with β‐actin as the loading control (G). (H) KYSE150 cells were transfected with indicated plasmids for 48 h, and then treated with GSK3i‐IX or DMSO for 6 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (I) KYSE150 cells were transfected with indicated plasmids and siRNAs for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (J) KYSE150 cells were transfected with indicated plasmids for 48 h, followed by IP with FLAG beads and IB analysis with the indicated Ab. (K) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then treated with CHX for the indicated time periods, followed by IB analysis. (L) KYSE150 cells were transfected with the indicated plasmids and siRNA and then treated with MG132 and GSK3i‐IX for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (M) KYSE150 cells were infected with lentivirus expressing FBXW7 shRNA and then transfected with the indicated plasmids for 48 h, and then treated with MG132 for 6 h, followed by Ni‐NTA purification. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. PD, pull down. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (G). n = 3 (G), * p < 0.05, ** p < 0.01.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Transfection, Infection, Expressing, shRNA, Purification, Affinity Purification, Control

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: SPT6 knockdown inhibits the growth and colony formation of ESCC cells. (A–F) KYSE150 cells were transfected with siNC or siRNA targeting SPT6 (A, C, and D) or FBXW7 (B, E, and F), and then subjected to CCK8 (A and B) and colony formation assays (C–F). Images of representative plates are shown (C and E), and colony numbers were quantified (D and F). (G and H) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by flow cytometry analysis. The percentage of cells in the G2/M phase (G) and undergoing apoptosis (H) are plotted. (I) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (J–M) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to IB (J), CCK8 (K), and colony formation (L and M) assays. Images of representative plates are shown (L) and colony numbers are quantified (M). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, B, D, F, G, H, K, and M), n = 3 (A, B, D, F, G, H, K, and M). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Knockdown, Transfection, Flow Cytometry

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: ΔNp63 is regulated positively by SPT6 and negatively by FBXW7. (A and B) KYSE30 and KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB (A) and qRT‐PCR (B) analyses. (C) KYSE30 and KYSE150 cells were transfected with increasing amounts of SPT6 plasmid for 48 h, followed by IB analysis. (D) KYSE30 and KYSE150 cells were harvested for the ChIP assay with SPT6 antibody, followed by PCR (top) or qRT‐PCR analysis (bottom). (E) KYSE150 cells were transfected with the indicated siRNAs for 48 h, followed by IB analysis. (F and G) ESCC tumor tissues from WT or Fbxw7 cKO mice following long‐term treatment were subjected to IHC staining. Representative images of ΔNp63 staining are shown (F), and the percentage of cells with ΔNp63‐positive staining from three random fields of the esophagus in each mouse was quantified (G). Scale bars: 100 and 25 µm (inset). (H) KYSE150 cells were transfected with the indicated siRNAs and plasmids for 48 h and then harvested for Ni‐NTA purification after 6 h of MG132 treatment. Ni‐NTA affinity‐purified fractions and WCE were analyzed by IB with the indicated Abs. (I‐K) KYSE150 cells were transfected with the indicated siRNAs for 48 h and then subjected to CCK8 (I) and colony formation assays (J and K). Images of representative plates are shown (J), and colony numbers are quantified (K). (L–O) KYSE150 cells were transfected with the indicated siRNAs for 48 h, and then subjected to CCK8 assay (L), IB analysis (M), and colony formation assays (N, O). Images of representative plates are shown (N), and colony numbers are quantified (O). Data are shown as mean ± SEM and analyzed by one‐way ANOVA (B, I, L, K, and O) and Student's t test (D and G); n = 3 (B, D, I, L, K, and O); n = 5 (G). * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance.

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: Transfection, Quantitative RT-PCR, Plasmid Preparation, Immunohistochemistry, Staining, Purification, Affinity Purification, CCK-8 Assay

Journal: MedComm

Article Title: FBXW7 Targets the SPT6‐ΔNp63 Axis for Degradation to Inhibit Esophageal Tumorigenesis Induced by 4‐Nitroquinoline N‐Oxide

doi: 10.1002/mco2.70662

Figure Lengend Snippet: The FBXW7‐SPT6 axis regulates ESCC tumor growth in vivo and their inverse correlation in ESCC tumor tissues. (A–E) KYSE150 cells infected with lentivirus expressing the indicated shRNA were subcutaneously injected into nude mice. Tumor volume was monitored every 2–3 days (A), tumor masses were photographed (B), and tumor weights were measured (C) at the end of the experiment. Tumors were subjected to IHC staining (D and E). Representative images of SPT6 and ΔNp63 staining are shown (D), and the percentage of cells with positive staining from three random fields in each tumor was quantified (E). Scale bar: 100 µm. Data are shown as mean ± SEM and analyzed by one‐way ANOVA (A, C, and E). n = 3 (A), n = 6 (C), n = 5 (E); * p < 0.05, ** p < 0.01, *** p < 0.001, ns, no significance. (F and G) Human esophageal tumor tissue microarrays containing 86 tumor tissues stained for FBXW7 and SPT6. Representative staining images are shown (F). Quantification of positive staining was performed using ImageJ (G) ( r = −0.5036, p < 0.001, Pearson correlation coefficient). Scale bar: 100 µm. (H) Kaplan–Meier analysis of 51 ESCC tissue microarray samples showing an inverse correlation between FBXW7 and SPT6 staining. Lower FBXW7 positive staining, coupled with higher SPT6 positive staining, predicted worse overall patient survival. Log‐rank test, p < 0.01. (I) Working model for how the FBXW7‐SPT6‐ΔNp63 axis regulates the ESCC development (see the text for details).

Article Snippet: Antibodies were used as follows: FBXW7 (Bethyl, A301‐720A and A301‐721A), SPT6 (Santa Cruz, sc‐393920, and Novus, NB100‐2582), RBM7 (Proteintech, 21896‐1‐AP), LSM14A (Proteintech, 18336‐1‐AP), UAP1L1 (Proteintech, 25262‐1‐AP), WASHC4 (Proteintech, 51101‐1‐AP), PLEKHF2 (Proteintech, 25424‐1‐AP), PDPK1 (Proteintech, 17086‐1‐AP), EI24 (Proteintech, 20456‐1‐AP), NEDD8 (Abcam, ab81264), GSK3α/β (Cell Signaling Technology, 5676), c‐MYC (Cell Signaling Technology, 5605), p‐Ser/Thr‐Pro (05‐368, Upstate), FLAG (Sigma‐Aldrich, F1804), HA (Sigma‐Aldrich, A2095), β‐Actin (HUABIO, R1207‐1), GFP (ABclonal, AE012), cleaved‐NOTCH1 (Cell Signaling Technology, 4147), Cyclin B1 (Cell Signaling Technology, 12231P), PLK1 (Proteintech, 10305‐1‐AP), PARP (Cell Signaling Technology, 9542S), Cleaved PARP (Cell Signaling Technology, 9541S), caspase3 (Cell Signaling Technology, 9662S), cleaved caspase3 (Cell Signaling Technology, 9661S), ΔNp63 (Abcam, ab203826) and Ki67 (Abcam, ab16667).

Techniques: In Vivo, Infection, Expressing, shRNA, Injection, Immunohistochemistry, Staining, Microarray

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