Fig 2: Orpinolide-induced OSBP inhibition triggers alterations in the morphology and functionality of the Golgi apparatus.(a, b) Scanning confocal microscopy analysis of the effects of orpinolide treatment on OSBP localization and trans-Golgi morphology in HeLa cells. Cells were treated with orpinolide (100 nM or 1 μM) or DMSO for 4 h (a) or 8 h (b) and immunostained with antibodies against OSBP (488, green) and TGN46 (568, purple) while the nuclei were labelled with Hoechst (blue). The scale bar is 10 μm. Dotted line indicates cell outline. (c, d) Quantification of OSBP localization to the Golgi as identified by TGN46 (left) and trans-Golgi fragmentation measured by number of TGN46 spots per cell (right) after 4 h (c) and 8 h (d) orpinolide treatment. OSBP fluorescence intensity was measured in TGN46 region of interest (ROI) and normalized to total cellular OSBP. The graphs show one point per cell from at least 2 independent experiments for a total of 44 (DMSO; n = 3), 31 (100 nM orpinolide; n = 2) and 48 cells (1 μM orpinolide; n = 3) (c); 47 (DMSO; n = 3), 33 (100 nM orpinolide; n = 2) and 46 cells (1 μM orpinolide; n = 3) (d). Line indicates mean. One-way ANOVA was performed on log10 transformed data (see Supplementary Fig. 2a); P < 0.0001 with Dunnett’s multiple comparisons test; n.s. P = 0.8432 (c); n.s. P = 0.7773 (d). Source data

Fig 3: Functional inhibition of OSBP underlies the cellular efficacy of orpinolide.(a) CRISPR-based dropout screen in Jurkat cells constitutively expressing Cas9, transduced with a BFP-based dual knockout reporter. The reporter plasmid carries a combination of sgRNAs targeting OSBP, OSBP2 or AAVS1 locus to yield single or dual OSBP/ORP4 knockout. The bar plots depict relative BFP+ cells over 18 days, normalized to day 3 post transduction for each individual reporter system. (b) Top: Effect of orpinolide treatment (5 μM) on p-Akt and Akt protein levels in Jurkat and LOUCY cells. Phosphoinositide 3-kinase (PI3K) inhibitor taselisib (0.5 μM, 8 h treatment) was used as a positive control. Representative image of n = 3 independent experiments. Bottom: Quantification of the relative p-Akt/Akt protein levels normalized to the DMSO controls. Mean ± SD; n = 3 independent experiments. (c) Left: Effect of orpinolide treatment (5 μM) on p-Akt and Akt protein levels in Jurkat cells at different time points. Representative image of n = 3 independent experiments. Right: Quantification of the relative p-Akt/Akt protein levels normalized to the DMSO controls. Mean ± SD; n = 3 independent experiments. Source data

Fig 4: Oxysterol-binding protein OSBP is a putative target of orpinolide (W7).(a) Top: Dose-dependent stabilization of HiBiT-tagged OSBP at 52 °C in intact KBM7 cells (4 h treatment). n = 2 technical replicates; representative of n = 2 independent experiments. Bottom: Endogenous OSBP levels in KBM7 after dose-resolved orpinolide treatment and thermal destabilization at 52 °C. Representative image of n = 2 independent measurements. (b) Orpinolide (5 μM, 2 h) does not thermally stabilize HiBiT-tagged RAB33A (up) or TM9SF4 (down) in intact KBM7 cells. Mean ± s.e.m.; n = 3 technical replicates; representative of n = 3 independent experiments. (c) Orpinolide (5 μM, 2 h) does not thermally stabilize endogenous GALNT2 in intact KBM7 cells. Representative images of n = 3 independent measurements. (d) Fluorescence polarization competition assay measuring the displacement of 22-NDB-cholesterol from purified GST-OSBP(ORD), Aster-A/B/C and STARD1 upon orpinolide treatment (1 μM). n = 2 independent measurements. (e) Expression levels of HiBiT-tagged ORPs in KBM7 cells. Mean ± s.e.m.; n = 3 independent measurements. (f) Orpinolide (1 μM, 4 h) does not thermally stabilize HiBiT-tagged ORP1, ORP2, ORP9 or ORP11 in intact KBM7 cells. Mean ± s.e.m.; n = 3 technical replicates; representative of n = 2 independent experiments. (g) Effect of orpinolide treatment (1 μM) on OSBP protein levels in KBM7 cells. Representative image of n = 2 independent measurements. Source data

Fig 5: Orpinolide (W7) directly targets OSBP and inhibits its sterol transport function.a, TPP. Among 113 significantly altered melting profiles, 31 hits show a ΔTm exceeding 1 °C (highlighted). See also Supplementary Table 5. b, Overlay of TPP hits with TGN-localized (UniProtKB SL-0266) and sterol-binding (GO:0032934) proteins reveals OSBP as the putative target of orpinolide. c, Orpinolide (5 μM, 2 h) stabilizes endogenous OSBP in intact KBM7 cells. Representative images of n = 3 independent measurements are shown. d, Orpinolide (1 μM, 4 h) stabilizes HiBiT-tagged OSBP in intact KBM7 cells. Data are mean ± s.e.m.; n = 3 independent experiments; n = 3 technical replicates. e, Fluorescence polarization competition assay. Orpinolide displaces 22-NDB-cholesterol from purified GST-ORD. Data are mean ± s.e.m.; n = 3 independent experiments; n = 3 technical replicates; IC50, half-maximal inhibitory concentration. f, FRET-based in vitro cholesterol transfer assay. g, Orpinolide (500 nM) disrupts the transfer of TF-Chol from the donor (LD; 16 μM) to the acceptor (LA; 16 µM) vesicles mediated by ORD (500 nM). The OSBP inhibitor OSW-1 (500 nM) was used as a positive control. Data are representative of n = 2 independent experiments. h, Orpinolide-induced (1 μM, 4 h) thermal stabilization of HiBiT-tagged ORP4L in intact KBM7 cells. Data are mean ± s.e.m.; n = 2 independent experiments; n = 3 technical replicates.Source data