Journal: The Journal of Cell Biology

Article Title: The endosomal transcriptional regulator RNF11 integrates degradation and transport of EGFR

doi: 10.1083/jcb.201601090

Figure Lengend Snippet: RNF11 is required for the EGF stimulation–induced up-regulations. Coexpression analysis was performed to predict candidate TRs of SEC23B, SEC24B, and SEC24D. To experimentally validate the prediction, Q-RT-PCR, luciferase assay, confocal microscopy, and the RUSH transport assay were performed in HeLa cells. (A) Top five–ranked candidate TRs coexpressed with SEC23B, SEC24B, and SEC24D. (B) Log2 mRNA fold changes of COPII paralogues and EGFR after siRNA-mediated RNF11 knockdown. Results were normalized to nonsilencing negative control siRNA and to GAPDH mRNA levels ( n = 8). (C) Luciferase assay was performed with luciferase reporter containing inner COPII or GAPDH gene promotor. The luciferase signal was analyzed as the ratio of relative luminescence units of RNF11-GFP–transfected cells to GFP-transfected cells in at least two independent biological experiments. (D and E) Representative cells (D) and quantification (E) of the RNF11 knockdown effect on the number of SEC24B- and SEC31A-labeled ER exit sites. Arrows in insets point to ER exit sites with SEC24B and SEC31A labeling, and arrowheads point to SEC31A sites with reduced SEC24B labeling. Images are max projections of z stacks covering the entire cell volume acquired on a confocal laser-scanning microscope. Bar, 10 µm. Data are means with a minimum of 100 analyzed cells ± standard deviation. (F) Quantification of the EGFP-EGFR transport efficiency in HeLa WT cells and in HeLa cells stably expressing mouse RNF11-mCherry (MUS-RNF11-mCherry) after knockdown of the human and mouse RNF11 gene (RNF11 siRNA HS/MUS) or of the human RNF11 gene (RNF11 siRNA HS; n = 3). Images for the quantification were acquired on an automated ScanˆR microscope. (G) Log2 mRNA fold changes of COPII paralogues and EGFR after RNF11 knockdown, followed by 12-h 200-ng/ml EGF stimulation ( n = 6). Results are represented as the percentage of fold change reductions normalized to the fold changes induced with nonsilencing negative control siRNA, followed by 12-h 200-ng/ml EGF stimulation (100%). (H) Quantification of the EGFR transport efficiency of cells after RNF11 knockdown, followed by 12-h 200-ng/ml EGF stimulation ( n = 3). All data, except when stated otherwise, are means ± SEM ( t test: *, P < 0.05; **, P < 0.01; ***, P < 0.001). a.u., arbitrary units.

Article Snippet: Luciferase assays were performed with the following promoter luminescent reporter gene constructs: SEC23A, SEC23B, SEC24B, SEC24C, SEC24D, and GAPDH (Active Motif).

Techniques: Reverse Transcription Polymerase Chain Reaction, Luciferase, Confocal Microscopy, Transport Assay, Knockdown, Negative Control, Transfection, Labeling, Laser-Scanning Microscopy, Standard Deviation, Stable Transfection, Expressing, Microscopy