FCHSD2 controls oncogenic ERK1/2 signaling outcome by regulating endocytic trafficking
Fig 3
FCHSD2 depletion-induced up-regulation of the RTKs is independent of their activities.
(A) FCHSD2 KD increases the transcription of EGFR and MET mRNA. All data were normalized to siCtrl and represent mean ± SEM (n = 5). Two-tailed Student t tests were used to assess statistical significance. *P < 0.05, ****P < 0.00005. (B) HCC4017 control or FCHSD2 siRNA-treated cells were incubated with EGFR inhibitor (afatinib) or MET inhibitor (crizotinib) at the indicated concentration for 24 h. Total ERK1/2 were served as the loading control as we have observed that the levels of total ERK1/2 remained consistent across different conditions (see Fig 4). (C) Quantification of EGFR/ERK or MET/ERK intensity ratios in the cells as described in (B). All data were normalized to siCtrl and represent mean ± SEM (n = 3). Two-tailed Student t tests were used to assess statistical significance. *P < 0.05, **P < 0.005, ****P < 0.00005. (D) KD of FCHSD2 did not enhance translocation of phospho-STAT3 into the nucleus. Cell lysates from control or FCHSD2 siRNA-treated HCC4017 cells were subjected to fractionation. (E) Loss of FCHSD2 did not increase the transcription of phospho-STAT3 target genes, HGF and c-Fos. All data were normalized to siCtrl and represent mean ± SEM (n = 5). Two-tailed Student t tests were used to assess statistical significance. The underlying data for this figure can be found in S1 Data. C, cytoplasmic fraction; c-Fos, proto-oncogene c-Fos; EGFR, epidermal growth factor receptor; ERK1/2, extracellular signal-regulated kinase 1 and 2; FCHSD2, FCH/F-BAR and Double SH3 Domain-Containing Protein; KD, knockdown; MET, proto-oncogene c-Met; N, nuclear fraction; n.s., not significant; RTK, receptor tyrosine kinase; siCtrl, control siRNA; siRNA, small interfering RNA; STAT3, signal transducer and activator of transcription 3.