Fig. 6

Lysosomal and autophagic phenotypes of EIPR1 R279G fibroblasts. (A) Skin fibroblasts from a homozygous EARP-interacting protein 1 (EIPR1) R279G individual (Patient FI:1) and her heterozygous father (control) were cultured on glass coverslips. Cells were next immunostained for the endogenous lysosomal membrane protein l (LAMP1) (greyscale), along with the actin stain Alexa Fluor 488-phalloidin (green), and the nuclear DNA stain 4',6-diamidino-2-phenylindole (DAPI) (blue). Images were captured using confocal fluorescence microscopy. Scale bars = 10 μm. Images on the third column are enlarged views of LAMP1-labelled lysosomes. Scale bars = 1 μm. (B) Cells with increased LAMP1 expression were counted from three independent immunofluorescence experiments such as that shown in A. Values are the mean ± standard deviation (SD). Statistical significance was calculated using a Student’s t-test. **P < 0.01. (C) Control and patient fibroblasts were analysed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblotting for endogenous LAMP1 and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as a loading control. The positions of molecular mass markers (in kDa) are indicated on the left. (D) Quantification of LAMP1 normalized to GAPDH protein from three independent experiments such as that shown in C. Protein levels in control fibroblasts were set to 100%. Values are the mean ± SD. Statistical significance was calculated using the Student’s t-test. *P < 0.05. (E) Skin fibroblasts from a homozygous EIPR1 R279G individual (Patient FI:1) and her heterozygous father (control) were cultured on glass coverslips. Cells were immunostained for endogenous LC3B (grayscale) to label autophagic vesicles and with DAPI (blue) to label nuclear DNA. Images were obtained by confocal fluorescence microscopy. Scale bars = 10 μm. (F) The number of LC3B-positive puncta per cell was quantified from three independent experiments such as that shown in E. Values are the mean ± SD. Statistical significance was calculated using a Student’s t-test. ***P < 0.001. (G) Lysates from control and patient fibroblasts were analysed by SDS-PAGE and immunoblotting with antibodies to LC3B and GAPDH as a loading control. The positions of the LC3B-I and LC3B-II forms of LC3B are indicated. (H) Quantification of LC3B-II and LC3B-I normalized to GAPDH protein from three independent experiments. Protein levels in control fibroblasts were set to 100%. Values are the mean ± SD. Statistical significance was calculated using a Student’s t-test. ***P < 0.001, ns: not significant. (I) Control and patient fibroblasts were cultured on coverslips and immunostained for endogenous SQSTM1/p62 to mark autophagic vesicles (greyscale) and with DAPI for nuclear DNA (blue). Images were captured by confocal fluorescence microscopy. Scale bars = 10 μm. (J) The number of SQSTM1/p62 puncta per cell was quantified from three independent experiments such as that shown in I. Values are the mean ± SD. Statistical significance was calculated using a Student’s t-test. ***P < 0.001. (K) Lysates from control and patient fibroblasts were analysed by SDS-PAGE and immunoblotting with antibodies to SQSTM1/p62 and GAPDH as a loading control. (L) Quantification of SQSTM1/p62 normalized to GAPDH protein from three independent experiments such as that shown in K. Protein levels in control fibroblasts were set to 100%. Values are the mean ± SD. Statistical significance was calculated using the Student’s t-test. *P < 0.05. In C, G and K, the positions of molecular mass markers are indicated in kilodaltons.