Supplementary MaterialsS1 Data: Underlying numerical data and statistical analysis for figure sections Figs 1B, 1C, 1E, 1G, 2A, 2C, 3A, 3C, 3E, 4B, 4C, 4E, 4G, 5A, 5B, 5C and ?and6B;6B; S1, S2D and S2B Figs. recycling of TfnR was assessed in charge or FCHSD2 siRNA-treated HCC4017 and ARPE-19 cells in the lack or presence from the ERK1/2 inhibitor SCH772984 (10 M). Cells had been BAY 41-2272 pulsed for 30 min with 10 g/ml biotinylated Tfn, stripped, and reincubated at 37 C for the indicated situations Rabbit polyclonal to alpha 1 IL13 Receptor before measuring the rest of the intracellular Tfn. Percentage of recycled biotinylated Tfn was computed BAY 41-2272 relative to the original loading. Data signify indicate SEM (= 3). Two-tailed Pupil tests had been utilized to assess statistical significance versus siCtrl. * 0.05, ** 0.005. The root data because of this figure are available in S1 Data. ERK1/2, extracellular signal-regulated kinase 1 and 2; FCHSD2, Increase and FCH/F-BAR SH3 Domain-Containing Proteins; siCtrl, control siRNA; siRNA, little interfering RNA; Tfn, transferrin; TfnR, transferrin receptor.(TIF) pbio.3000778.s003.tif (343K) GUID:?D68A12B9-016C-4E45-9F09-0B97FEDC7D74 S2 Fig: FCHSD2 regulates EGFR endocytic trafficking in H1975 cells. (A) The KD of FCHSD2 in charge or FCHSD2 siRNA-treated H1975 cells. (B) Endocytic recycling of EGFR was assessed in charge or FCHSD2 siRNA-treated H1975 cells. Cells had been pulsed for 10 min or 30 min with 20 ng/ml biotinylated EGF, stripped, and re-incubated at 37 C for the indicated situations before measuring the rest of the intracellular EGF. Percentage of recycled EGF was computed relative to the original loading. Data signify indicate SEM (= 3). Two-tailed Pupil tests had been utilized to assess statistical significance. * 0.05, ** 0.005, *** 0.0005. (C) Representative confocal images of pEGFR and Light1 immunofluorescence staining in control or FCHSD2 siRNA-treated H1975 cells. Cells were incubated with 20 ng/ml EGF for 30 min at 4 C, washed, and re-incubated at 37 C for the indicated instances. Scale pub, 12.5 m. (D) Colocalization of pEGFR and Light1 immunofluorescence staining in the cells as explained in (C). Data were from at least 40 cells in total/condition and represent mean SEM. Two-tailed College student tests were used to assess statistical significance. * 0.05. The underlying data for this figure can be found in S1 Data. EGF, epidermal BAY 41-2272 growth element; EGFR, epidermal growth element receptor; FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; KD, knockdown; Light1, lysosome-associated membrane glycoprotein 1; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF) pbio.3000778.s004.tif (4.7M) GUID:?93518D16-12D7-4F06-8B56-EAF2281C7A34 S3 Fig: Representative confocal images of MET, EEA1, and Rab11 immunofluorescence staining in control or FCHSD2 siRNA-treated HCC4017 cells. Cells were incubated with 1 g/ml HGF for 30 min at 4 C, washed, and re-incubated at 37 C for the indicated instances. Scale pub, 25 m. Quantified results are demonstrated in Fig 2A. EEA1, early endosome antigen 1; FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; HGF, hepatocyte growth element; MET, proto-oncogene c-Met; Rab11, Ras-related protein Rab-11A; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF) pbio.3000778.s005.tif (4.3M) GUID:?C672A304-8CBA-4936-B79E-09B107181A07 S4 Fig: Representative confocal images of MET, EEA1, and LAMP1 immunofluorescence staining in control or FCHSD2 siRNA-treated HCC4017 cells. Cells were incubated with 1 g/ml HGF for 30 min at 4 C, washed, and re-incubated at 37 C for the indicated instances. Scale pub, 25 m. Quantified results are demonstrated in Fig 2A. EEA1, early endosome antigen 1; FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; Light1, lysosome-associated membrane glycoprotein 1; MET, proto-oncogene c-Met; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF) pbio.3000778.s006.tif (4.5M) GUID:?792C60C2-0FFE-46F3-A47B-ED1643B8AC66 S5 Fig: FCHSD2 depletion-induced up-regulation of the RTKs is independent of their activities. H1975 control or FCHSD2 siRNA-treated cells were incubated with EGFR BAY 41-2272 inhibitor (afatinib) or MET inhibitor (crizotinib) as indicated concentration for 24 h. EGFR, epidermal growth element receptor; FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; MET, proto-oncogene c-Met; RTK, receptor tyrosine kinase; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF) pbio.3000778.s007.tif (1.2M) GUID:?09FB6D06-CBE3-4776-84C3-3E913DB9E0C6 S6 Fig: FCHSD2 deficiency increases MET expression and ERK1/2 activity. The HCC4017 cells stably expressing shCtrl or shFCHSD2 at stable state. ERK1/2, extracellular signal-regulated kinase 1 and 2; FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; MET, proto-oncogene c-Met; shCtrl, control shRNA; shRNA, small hairpin RNA.(TIF) pbio.3000778.s008.tif (359K) GUID:?609C0238-4FA9-4197-A0A2-D0ED48FF6345 S7 Fig: Rab7 is essential for BAY 41-2272 FCHSD2 deficiency-induced up-regulation of MET. Rab7 KD by a different pool of siRNAs abolishes the MET up-regulation induced by FCHSD2 depletion. FCHSD2, FCH/F-BAR and Two times SH3 Domain-Containing Protein; KD, knockdown; MET, proto-oncogene c-Met; Rab7, Ras-related protein Rab-7A; siCtrl, control siRNA; siRNA, small interfering RNA.(TIF) pbio.3000778.s009.tif (498K) GUID:?C006A098-3E64-4340-9B32-69F9DC3B1596 Attachment: Submitted filename: ortholog, Nervous Wreck (homologue, also functions in endosomal trafficking, we first assessed recycling of transferrin receptor (TfnR), a canonical marker for the quantification of endosomal trafficking [17]. To further determine which step(s) are affected, we measured TfnR.