Article Figures & Data
Figures
- Figure 1
PD166866 induces senescence of lung squamous cell carcinoma cells. (A) NCI-H226 and H1703 cells were treated with PD166866, and the tyrosine phosphorylation of FGFR was detected with immunoprecipitation and Western blot analyses. (B) NCI-H226 and H1703 cells were treated with PD166866, and anchorage-independent growth was detected with soft agar colony assays. (C, D) NCI-H226 and H1703 cells were treated with PD166866, and β-gal staining was used to detect cell senescence. Statistical analyses were performed. (E) FGFR 1 was knocked down in NCI-H226 cells, and β-gal staining was used to detect cell senescence. Statistical analyses were performed. **P < 0.01.
- Figure 2
Knockdown of RACK1 induces senescence of lung squamous cell carcinoma cells. (A) RACK1 expression in NCI-H226 and H1703 cells was detected. (B) RACK1 was knocked down in NCI-H226 and H1703 cells, and anchorage-independent growth was detected with colony formation assays. (C) β-gal staining was used to detect cell senescence. (D) RACK1 expression was knocked down in NCI-H226 and H1703 cells, subcutaneous tumorigenesis assays were performed, and the tumor weights were statistically analyzed. (E) Immunohistochemistry was used to detect the expression of Ki67 in tumors derived from RACK1 knockdown NCI-H226 cells (20x). Left: representative immunohistochemistry image; right: RACK1 and MDM2 protein levels in tumors. **P < 0.01.
- Figure 3
Interaction between RACK1 and MDM2. (A) The interaction between the GST-RACK1 fusion protein and MDM2 was detected with GST pull-down assays. Ten micrograms of the fusion protein GST-RACK1 was incubated with NCI-H226 cell lysate. (B) The interaction between myc-RACK1 and Flag-MDM2 expressed in NCI-H226 cells was detected by immunoprecipitation. Four micrograms of myc-RACK1 and Flag-MDM2 plasmids was transfected into the cells, and the proteins were collected for immunoprecipitation 48 h later. (C) The interaction between RACK1 and MDM2 expressed in NCI-H226 cells was detected by immunoprecipitation. (D) The interactions among FGFR, RACK1, and MDM2 expressed in NCI-H226 cells were detected by immunoprecipitation. (E) The interaction between the exogeneous proteins myc-RACK1 and Flag-MDM2 expressed in NCI-H226 cells was detected by immunoprecipitation. Four micrograms of myc-RACK1 and Flag-MDM2, HA-p53, and Flag MDM2 plasmids was transfected into the cells, which were collected 48 h after transfection.
- Figure 4
RACK1 promotes the ubiquitination of p53. (A) Overexpression of RACK1 in NCI-H226 cells promoted the ubiquitination of p53. (B) Overexpression of RACK1 in NCI-H226 and H1703 cells inhibited the p53 and p21 protein levels. (C) Treatment of NCI-H226 and H1703 cells with PD166866 increased the protein levels of p53 and p21. (D) The effects of PD166866 on the interaction between RACK1 and MDM2 were detected by immunoprecipitation.
- Figure 5
PD166866 and RG7112 cooperatively inhibit the malignancy of lung cancer. (A) PD166866 combined with RG7112 inhibited the colony formation of NCI-H226 and H1703 cells. (B) PD166866 combined with RG7112 promoted the expression of p53 and p21. (C) PD166866 combined with RG7112 inhibited the distal seeding of NCI-H226 cells. The gross morphology of lungs is shown. H&E staining was performed, and the numbers of foci were analyzed (10x). **P < 0.01; ##P < 0.01.
- Figure 6
Schematic of the proposed mechanism(s) for FGFR/RACK1 interaction with MDM2 to inhibit the senescence of lung squamous cell carcinoma. The interaction of FGFR/RACK1 with MDM2 promotes the degradation of P53 (left); the FGFR inhibitor PD166866 and the MDM2/p53 inhibitor RG7112 cooperatively decrease the interactions between RACK1 and MDM2; weaken the interaction between MDM2/p53; and induce the accumulation of p53 and the upregulation of p21 and consequently cell senescence.
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