ZFIN Image: Sofou et al., 2021, Figure 4

Image

Figure Caption

Figure 4 Analysis of transferrin uptake and trafficking in fibroblasts

A, B

Analysis of the early endosome marker EEA1 in fibroblasts transduced with control or VPS16‐expressing lentivirus by confocal microscopy. (A) Representative confocal micrographs. Scale bar, 20 μm. (B) Quantification of EEA1‐stained puncta number and intensities (ca 80 cells analyzed for each of n = 3 biological replicates).

C

Analysis of transferrin uptake in fibroblasts transduced with the indicated lentiviruses. Representative confocal micrographs of fibroblasts fed with Alexa488‐conjugated Transferrin for 30 min. Scale bar, 10 μm.

D

Quantifications of the number and fluorescence intensities of intracellular Transferrin puncta (n = 28–30 fields from three independent experiments). Colored horizontal bars indicate median values and whiskers 5 and 95 percentiles.

E

Immunoblot for Transferrin receptor (TfR) levels in the indicated fibroblasts under basal and serum‐starved conditions.

F

Summary quantification of Transferrin receptor levels in non‐starved fibroblasts transduced with the indicated lentiviruses, normalized to actin (ACTB), and expressed as % of controls (n = 6 biological replicates).

G, H

Analysis of intracellular Transferrin trafficking. Fibroblasts fed with fluorescently labeled Transferrin were co‐stained with LysoTracker (G) or an antibody against LAMP2 (H). Representative images (left) of fibroblasts transduced with control virus, with magnified areas demonstrating co‐localized puncta (arrowheads). Scale bars 10 μm or 2 μm (inserts). Quantifications (right) of the co‐localization between Transferrin and LysoTracker or LAMP2, respectively, expressed as Pearson correlation coefficients. Colored horizontal bars indicate the median values and whiskers 5 and 95 percentiles (n = 10 optical sections from three independent experiments).

Data information: Bar graphs represent data as mean ±SEM. Statistical comparisons between the indicated groups by one‐way ANOVA with Holm–Sidak’s multiple comparisons tests. *P < 0.05; **P < 0.01; ***P < 0.001.

Acknowledgments

This image is the copyrighted work of the attributed author or publisher, and ZFIN has permission only to display this image to its users. Additional permissions should be obtained from the applicable author or publisher of the image. Full text @ EMBO Mol. Med.