Extended Data Fig. 10: The C-terminal domain of NSP6 is involved in the recruitment of DFCP1 in a PtdIns3P-independent manner.
From: The role of NSP6 in the biogenesis of the SARS-CoV-2 replication organelle
a, Fluoromicrographs of cells co-expressing GFP-DFCP1 and Flag-NSP6(1–157). b, Fluorescent images of HeLa cells transfected with the indicated DFCP1 mutants alone or in combination with NSP6 (as indicated). A schematic representation of DFCP1 mutants is reported on top. Arrowheads, DFCP1 signal in the NSP6 compartment. Insets, enlarged merge of boxed areas. Numbers indicate the percentage of co-localization between DFCP1 mutants and NSP6. Mean ± SD (see Methods). c, Fluorescent micrographs of cells expressing mCherry-DFCP1 and Flag-NSP6 treated with SAR405 or with wortmannin. Anti-Flag immunostaining. d, Fluoromicrograph showing LC3 staining in one transfected and one non-transfected cell from Flag-NSP6-expressing HeLa cells. Inset, anti-Flag immunostaining. Graph, quantification of LC3 puncta in non-transfected (CTRL) and NSP6-transfected cells (number of LC3 spots/cell). Means ± SEM. n = 74 cells examined over three independent experiments. Two-tailed unpaired t-test with Welch’s correction. ns, not significant. e, HeLa cells expressing HA-NSP3 and GFP-NSP4, or HA-NSP3, GFP-NSP4 and Flag-NSP6, were loaded with BODIPY-DA-C12 and washed out for 6 h (see Methods). Dotted yellow lines, LDs; dotted white lines, NSP4 puncta. Graph, quantification of the percentage of NSP4 puncta positive for BODIPY-DA-C12. Single values are plotted, Means ± SEM are indicated, N = 3, n = 90, two-tailed Mann–Whitney test. f, Western blot of protein extracts from mock-treated (CTRL) and DFCP1-KD cells. Actin was used as loading control. The graph shows the level of DFCP1 protein expressed as percentage of control (set at 100). Mean ± SEM. N = 3, Two-tailed unpaired t-test with Welch’s correction. g, Western blot of protein lysates from untreated (CTRL), scramble- and DFCP1 siRNA-treated (DFCP1-KD) SARS-CoV-2-infected Calu-3 cells detected with an anti-DFCP1 antibody. GAPDH was used as loading control. The graph shows the level of DFCP1-KD expressed as percentage of control (set at 100). Mean ± SEM. N = 3, two-tailed unpaired t-test with Welch’s correction. h, Quantification of viral titres in SARS-CoV-2-infected Calu-3 cells untreated (-), transfected with scramble siRNA (CTRL) or DFCP1 siRNA (DFCP1-KD). Mean ± SEM, N = 7, two-tailed unpaired t-test with Welch’s correction. Scale bar a–e, 10 μm. Western blot images are representative of three independent experiments.
