Fig 2: miR-145 regulates glucocorticoid-induced apoptosis and differentiation of BMSC by down-regulating GABARAPL1 expression. (A)CCK8 assayed the activity of different groups of cells. (B) The proliferation level of BMSC cells was detected by EdU immunofluorescence double staining. (C) Flow cytometry was used to detect the apoptosis of cells in different groups. (D, E) The differentiation of BMSC cells was detected by ALP and alizarin red staining. (F) The expression of differentiation-related proteins (ALP, RUNX2, OPN, BMP2) in different groups were detected by western blot (Scale bar: 100 µm, DEX vs. control, ***P < 0.001, DEX + miR-145 inhibitor + sh-NC vs. control, ##P < 0.01, ###P < 0.001, DEX + miR-145 inhibitor + sh-GABARAPL1 vs. control, $P < 0.05, $$P < 0.01, $$$P < 0.001)

Fig 3: GABARAP, GABARAPL1 and GABARAPL2 colocalize with Nef in vesicular structures. (A) Live-cell fluorescence microscopy of HEK293 cells stably expressing Nef-CFP. Here, Nef was observed at cell-cell contact sites, bleb-like structures or at the plasma membrane (PM). Scale bar 20 µm. (B) Live-cell fluorescence microscopy of HEK293 cells transfected with peFP-C1/GABARAP (top), peYFP-C1/GABARAPL1 (middle) and peYFP-C1/GABARAPL2 (bottom) revealed a diffuse distribution pattern for all GABARAPs. Scale bar 20 µm. (C) Live-cell fluorescence microscopy of HEK293 cells stably expressing Nef-CFP (red) and transfected with peFP-C1/GABARAP (top), peYFP-C1/GABARAPL1 (middle) and peYFP-C1/GABARAPL2 (bottom) (all green). In this case, Nef and GABARAPs showed high colocalization levels at vesicular structures and at the plasma membrane (see merge and zoom). Scale bar 20 µm.

Fig 4: miR-145 targets downstream GABARAPL1 expression. (A)Double luciferase report of miR-145 targeting GABARAPL1. (B) Detection of luciferase activity in cells transfected with miR-145 mimic. (C) The expression of GABARAPL1 in serum of SANFH patients detected by qPCR. (D) The expression of GABARAPL1 in DEX induced cells was detected by western blot (***P < 0.001)

Fig 5: Nef selectively binds to GABARAPs in a direct manner and contacts the canonical ligand binding site of GABARAP. (A) Nef-conjugated or free control Sepharose beads were incubated with the purified recombinant ATG8 paralogs listed. The input, the unbound material of the flow through (U), the wash (W) fractions and the eluate (E) fractions have been analyzed by SDS-PAGE and CBB staining. Full-length gels are presented in Supplementary Fig. 4. (B) Mapping the Nef binding site on GABARAP by NMR titration experiments: overlay of 2D 1H-15N-HSQC spectra of [U-15N]-GABARAP (yellow) with increasing amounts of Nef (red and purple). GABARAP residues affected upon Nef binding are labelled. For better visibility the first titration step (ratio of 1:0.5 for [GABARAP]:[Nef]) has been omitted from this figure. (C) Combined chemical shift changes ?d(1H,15N) as a function of the GABARAP sequence. Secondary structure elements are shown on top of the diagram. Residues belonging to the hydrophobic pockets 1 and 2 are highlighted in light blue and blue, respectively. Residues for which no chemical shift changes could be determined and prolines are marked by a grey diamond. A cut-off of 0.05 ppm (continuous horizontal line) was used for significance. (D) Mapping of the Nef interaction site on a schematic representation of the secondary structure (top) and the molecular surface (bottom) of GABARAP (PDB ID: 1KOT). Residues strongly affected upon Nef binding are marked in purple, whereas non-interacting residues are shown in yellow.