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. 2019 May 29;27(1):310–328. doi: 10.1038/s41418-019-0357-y

Fig. 5.

Fig. 5

IFT20 is required for the transport of acid hydrolases to lysosomes. a, b Immunofluorescence analysis of control and IFT20KD Jurkat cells (a) or primary IFT20KO T cells (b) loaded 10 min at 37 °C with Lysotracker red (LyTr, 75 nM). Median optical sections are shown. Size bar: 5 µm. The graphs show the quantification of mean fluorescence intensity/cell (MFI/cell) (mean ± SD; ≥40 cells/sample, n = 3; Mann–Whitney test). c Flow-cytometric analysis of control and IFT20KD Jurkat cells loaded with the probe DQ-BSA (100 µg/ml) for 1.5, 3 or 5 h. The histogram shows the MFI (mean ± SD; n ≥ 4; Student’s t test). d–f Immunofluorescence analysis of control and IFT20KD Jurkat cells (≥55 cells/sample, n = 3; Mann–Whitney test) (d), or primary control and IFT20KO T cells (≥23 cells/sample, n = 3; Kruskal–Wallis test) (e), or IFT20KD Jurkat cells transiently transfected with a construct encoding GFP-tagged IFT20 or control and IFT20KD cells transiently transfected with the respective GFP vector (≥55 cells/sample, n = 3; Mann–Whitney test) (f), loaded 4 h at 37 °C with Magic red (1 µM). The graphs show the quantification of mean fluorescence intensity/cell (MFI/cell) (mean ± SD). g Immunoblot analysis of cathepsin D (Santa Cruz antibody) in lysates of ctr and IFT20KD Jurkat cells. Actin was used as loading control. The quantifications of pro-catD (precursor) and mature catD (normalized to actin), and the mature/total catD ratio are shown (mean fold ± SD; ctr value = 1) (n ≥ 3; one sample t test). h Quantification using Mander’s coefficient of the weighted colocalization of LAMP-1 and catD (Abcam antibody) in ctr and IFT20KD Jurkat cells (≥12 cells/sample, n = 3). Representative images (medial optical sections) are shown. Size bar: 5 μm. The data are expressed as mean ± SD (Student’s t test). i Immunoblot analysis of LAMP-1, catD (Abcam antibody), hexosaminidases A (hexA) and B (hexB), and lysosomal acid lipase (LAL) on lysosomes from control and IFT20KD Jurkat cells (equal amounts of proteins from the pool of the fractions enriched in LAMP-1; see Fig. S1). The quantification of the relative protein expression (mean fold ± SD; ctr value = 1) is reported below (n = 4; one sample t test). *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001