Figure 4: WHSC1L1 interacts with nuclear EGFR and potentiates its interaction with PCNA through EGFR K721 mono-methylation in the nucleus of SCCHN cells. | Scientific Reports

Figure 4: WHSC1L1 interacts with nuclear EGFR and potentiates its interaction with PCNA through EGFR K721 mono-methylation in the nucleus of SCCHN cells.

From: WHSC1L1-mediated EGFR mono-methylation enhances the cytoplasmic and nuclear oncogenic activity of EGFR in head and neck cancer

Figure 4

(a) WHSC1L1 interacts with nuclear EGFR in SCCHN cells. Nuclear extracts from YD-10B and HN13 cells were obtained and immunoprecipitated using a WHSC1L1 specific antibody. Immunoprecipitates were blotted for EGFR. Expression of WHSC1L1 and EGFR was confirmed in YD-10B and HN13 nuclear extracts (input). H3 was used as a loading control. (b) WHSC1L1 expression correlates positively with EGFR K721 mono-methylation levels in the nucleus of YD-10B cells. Immunocytochemistry was performed in YD-10B cells treated with siNC or siWHSC1L1. Cells were stained with DAPI, anti-EGFRK721me1 and anti-WHSC1L1 antibodies. Results shown in 10x magnification. (c) WHSC1L1-mediated EGFR K721 mono-methylation enhances nuclear EGFR’s interaction with PCNA. 293T cells were cotransfected with FLAG-EGFR-WT and HA-Mock or HA-WHSC1L1, and FLAG-EGFR-K721A and HA-WHSC1L1 for 48 h. Protein extracts were sonicated and immunoprecipitated using a FLAG-antibody. FLAG-immunoprecipitates were immunoblotted for PCNA. (d) K721 mono-methylated EGFR interacts with PCNA in the nucleus of SCCHN cells. Nuclear extracts were obtained from YD-10B cells and PCNA immunoprecipitates were blotted for mono-methylated K721 EGFR. (e) siRNA-mediated WHSC1L1 knockdown, PCNA and Y211 PCNA phosphorylation levels in YD-10B and HN13 cells. Nuclear extracts were blotted for WHSC1L1, PCNA and Y211 PCNA. H3 was used as a loading control. (f) 293T cells were cotransfected with HA-WHSC1L1 and FLAG-EGFR-WT and immunocytochemistry was performed using anti-HA and anti-PCNA specific antibodies. HA-WHSC1L1 transfected (yellow arrows) and untransfected 293T cells are shown in correlation with PCNA levels. A representative histogram of corrected total cell fluorescence for PCNA in HA-WHSC1L1 transfected versus untransfected 293T cells is shown (Student’s t-test, p = 0.0019). Fluorescence intensity was analyzed using the Image J software and corrected total cell fluorescence was calculated as follows: Integrated density − (area of selected cell × mean fluorescence of background). (g) Correlation of PCNA and EGFRK721me1 levels. YD-10B cells were treated for 72 h with siNC versus siWHSC1L1 and plated in glass slide chambers. Immunocytochemistry was performed for PCNA and EGFR K721 mono-methylation. Results shown in 10x magnification. Cytofluorogram using the JacoP software is also presented (Pearson’s correlation co-efficient rho = 0.856, p < 0.0001).

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