Fig. 2: TRIB3 promotes EGFR recycling to enhance its stability and signaling activity.

a Control or TRIB3-silenced A549 cells were treated with cycloheximide (CHX) (10 µg ml⁻¹) at indicated intervals and protein stability of EGFR was analyzed by IB. Data are means ± SEM of four independent assays. b A549 cells stably expressed control-shRNA or TRIB3-shRNA plasmid were stimulated with or without EGF (100 ng ml⁻¹) for 1 h. Indicated proteins were analyzed by IB analysis. Data represent means ± SEM of three independent assays. c Confocal microscopy images show the distribution of EGFR and early endosome antigen 1 (EEA-1) in control or TRIB3-silenced A549 cells before and after 0.5 or 1 h of EGF (100 ng ml⁻¹) stimulation. Quantification of EGFR and EEA-1 colocalization was shown as Pearson’s coefficient. Data are means ± SEM of three independent assays. d Quantitative analyses of cell surface EGFR in A549 cells stably expressed control-shRNA or TRIB3-shRNA1. Cells were preincubated with DMSO or 10 μM monensin for 4 h, and then treated with or without EGF (100 ng ml⁻¹) for another 1 h. The Mean Fluorescence Intensity (MFI) of EGFR on cell surface was detected by flow cytometry analysis. Top: representative flow cytometry data. Bottom: the data were normalized to A549/control-shRNA cells without EGF stimulation, which was considered as 100% MFI signal. Data are means ± SEM of three independent assays. e EGFR recycling was detected in A549 cells stably expressed control-shRNA or TRIB3-shRNA1 plasmid. Data are means ± SEM of three independent assays. f Colocalization analysis of EGFR with Rab11 and LAMP-1 before or after 30 min EGF treatment in control and TRIB3-silenced A549 cells. Quantification of EGFR/Rab11 or EGFR/LAMP-1 colocalization was shown as Pearson’s coefficient. Data represent means ± SEM of three independent assays. Statistical significance between two groups was determined with two-tailed Student’s t test. Statistical significance among groups was determined by one-way ANOVA test. Source data are provided as a Source Data file.