Fig. 2: Transmission electron microscopy analyses of autophagosome biogenesis in cultured cells from individuals with bi-allelic variants in ATG4D.
a, b Representative TEM images of cultured primary fibroblasts from Control (GM09503) and Individual 1 (a) or lymphoblastoid cell lines from Control 1 (CCL-104), Control 2 (Family 2: I-2, mother of Individuals 2 and 3), Individual 2 (Family 2: II-3), and Individual 3 (Family 2: II-4) (B) treated with vehicle (DMSO) or 100 nM Torin 1 and 100 nM Bafilomycin A1 for 3 h to induce the formation of autophagosomes and to prevent their degradation, respectively (arrowheads). c–f Quantification of autophagosome area (c, d) and size (e, f) from the experiments represented in (a, b). The data are presented as dot plots with the median indicated by a horizontal line. Light gray data points represent measurements made on control cells; dark gray data points represent measurements made on affected cells. For the quantification of autophagosome area, 12 images (primary fibroblasts) or 19 images (lymphoblastoid cell lines) taken from each sample were assessed per condition. A Kruskal–Wallis test and Dunn’s multiple comparisons test were performed for relevant predefined dataset pairs. For the quantification of autophagosome size, data points represent individual autophagosomes measured from Torin 1- and Bafilomycin A1-treated conditions. For the primary fibroblasts, n = 98 and n = 152 autophagic structures were respectively measured for Control (GM09503) and Individual 1 (Family 1: II-2), and a two-tailed Mann–Whitney U test was performed. For the lymphoblastoid cell lines, n = 67, n = 78, n = 122, and n = 92 autophagomsomes were respectively measured for Control 1 (CCL-104), Control 2 (Family 2: I-2, mother of Individuals 2 and 3), Individual 2 (Family 2: II-3), and Individual 3 (Family 2: II-4), and a Kruskal–Wallis test and Dunn’s multiple comparisons test were performed for relevant predefined dataset pairs. Only statistically significant comparisons are shown. Magnification: ×4000. Scale bar: 500 nm. ****p < 0.0001; ***p < 0.001; Baf A1, Bafilomycin A1; DMSO, dimethyl sulfoxide; TEM, transmission electron microscopy.
