Fig. 6

TRIM32 mutations increase autophagy activity. a H&E staining of skeletal muscle from family A patients, family B patients and family C patient showed internalized nuclei, fibers splitting, fibrosis and atrophy. In some fibers small rimmed vacuoles were found (black arrowheads). Scale bars, 50 μm: lower magnification view; 20 μm: higher magnification view. b TEM analysis of skeletal muscle from family A patient, family B patients and family C patient revealed membrane-surrounded vacuoles (white arrowheads), some of them containing slightly electron-dense amorphous material (black arrowheads).c. TEM images of myoblasts at 8 days growing in proliferation medium from family A patients, family B patients and healthy controls. Lysosome load and autophagic vacuoles were increased in TRIM32^V591M and TRIM32^{N217S/F568del} myoblasts compared to control myoblasts. Lysosomes (black arrows), autolysosomes filled with partially degraded materials (black arrowheads), multi-vesicular/lamellated structures (red arrowheads), and fusion of electron-lucent vacuoles generating large vacuoles in the cytoplasm space (asterisk). Black scale bars, 2 μm; white scale bars, 1 μm. d Western blot analysis of muscle biopsy derived from family A patients, family C patients and healthy controls. Anti-LAMP1 (lysosome marker) revealed an increment of LAMP1 protein level in TRIM32^V591M and TRIM32^C39LfsX17 muscles compared with controls. An anti-GAPDH blot was used as a loading control. e Western blot of muscle biopsy from Family A and C patients. Anti-p62/ SQSTM1 and LC3-II showed reduced level in TRIM32-related myopathy patients compared to healthy controls. Although P62/SQSTM1 level in the only muscle sample available from Family C was not so reduced as in Family A, indeed it was slightly reduced since the GAPDH indicates more protein being present in the lane than the controls. An anti-GAPDH blot was used as a loading control. f Western blot experiments of primary myoblasts from AIII.2 patient and healthy controls incubated with vehicle or with 1 μM Baf-A1 for 6 h. Anti-TRIM32 antibody revealed that TRIM32^V591M protein level was rescued after treatment with Baf-A1. Anti-LC3 (autophagy marker) showed that degradation of LC3-II was partially inhibited, whereas LC3-I, as expected, was not affected in the presence of Baf-A1. Anti-GAPDH levels were used as loading control