Fig 2: APE1 mediates ABS-induced YAP1 activation. A Representative immunofluorescence images of APE1 (red) and YAP1 (green) in FLO-1 cells exposed to ABS (200 µM, pH 4, 20 min) followed by 3 h recovery with or without APE1 knockdown. DAPI (blue) was used for nuclear staining. B Quantification of immunofluorescence stain intensity using ImageJ. C Cytosolic and nuclear fractions from FLO-1 (left) and OE33 (right) cells transfected with si-Ctrl or si-APE1 and treated with ABS (200 µM, pH 4, 20 min) followed by recovery in complete media for indicated timepoints were evaluated by western blot for the levels of APE1 and YAP1. ß-tubulin and p84 were used as a loading control for cytosolic and nuclear fractions, respectively. D 8xCTIIC luciferase reporter assay was used to measure YAP1 transcriptional activity in FLO-1 and OE33 cells transfected with si-Ctrl or si-APE1 and exposed or not to ABS treatment (200 µM, pH 4, 20 min) followed by 3 h recovery. The luciferase reporter activity values were normalized to Renilla expression levels. Values are represented as mean ± SEM of three independent experiments. * P < 0.05; ** P < 0.01; *** P < 0.001

Fig 3: EMC physically interacts with DENV NS4B post-cleavage and knockout of EMC leads to an aberrant topology of NS4A and NS4B.(A) Immunoblot for processed products of transfected NS2B-4B-GFP construct in WT and EMC4 KO cells in presence or absence of BZ. Anti-GFP antibody was used to detect processed viral proteins and p84 was used as loading control. (B) Co-immunoprecipitation of EMC4-FLAG from lysates of uninfected (UI) or DENV infected HEK293FT EMC4-FLAG cells. Cells were harvested after 96 hr and lysates were incubated with anti-FLAG magnetic beads. Eluates were immunoblotted for different viral proteins (prM, NS4B, NS5). p84 was used as loading and non-specific binding control. (C) Co-immunoprecipitation of EMC4-FLAG from lysates of uninfected cells (UI), cells infected with WT DENV (WT) or infected with EMC adapted DENV (AD). (D) Protease protection assay for different NS4A-4B-eGFP fusion constructs. Experimentally determined topology according to Miller et al. (2006); Miller et al. (2007) and generated constructs are displayed. Different shades of blue indicate NS4A, 2K and NS4B in the model and red triangle points to signal peptidase cleavage site. Constructs were transfected into WT or EMC4-KO cells. After 24–48 h cell pellets were resuspended in buffer with digitonin, and subsequently treated with proteinase K (PK) or left untreated. Finally, samples were lysed and immunoblotted using anti-GFP antibody. (E) Deglycosylation assay using PNGase F to probe luminal localization of NS4A TMD2 using constructs containing the NS4A WT sequence or A6665G mutation. Opsin tags containing a N-K-T sequon were inserted downstream of NS4A TMD2. WT or EMC4-KO cells were transfected with WT or mutant construct and harvested after 24 hr. Lysates were treated with PNGase F to remove N-linked oligosaccharides or left untreated following analysis by immunoblot using anti-FLAG antibody. Upper bands represent NS4A with glycosylated and thus luminally localized opsin tag while lower bands are non-glycosylated forms. (F) Possible model for the role of EMC in flavivirus infection. In infected WT cells, the EMC co-translationally engages with NS4A and NS4B TMDs to facilitate or maintain correct insertion and topology of TMDs , thus ensuring stable expression. In the absence of the EMC, an aberrant topology is obtained during biogenesis, leading to misfolding and degradation of the NS4A-4B viral proteins.

Fig 4: The isoprenylcysteine carboxylmethyltransferase inhibitor C75 disrupts progerin-AKT interactions, increases AKT activation, and mislocalizes progerin from the nuclear membrane to the nucleoplasm.(a) Western blot showing amounts of phosphorylated and total AKT (a.k.a protein kinase B) in whole-cell lysates of human wild-type (WT) and Hutchinson-Gilford progeria syndrome (HGPS) cells and mouse Zmpste24-deficient fibroblasts. (b, c) Left panels, western blots (WB) showing amounts of phosphorylated and total AKT in WT and HGPS cells (b) and Zmpste24-deficient (c) fibroblasts incubated with C75 for 20–30 days; ß tubulin was the loading control. Right panels, quantification of the ratio of phosphorylated and total AKT from densitometry analyses of protein bands. Data are mean of two independent cell lines, each analyzed in duplicate. (d) Immunoprecipitation (IP) and WB analyses showing that C75 disrupts the association between AKT and progerin. The lysates were also used directly for WB with total AKT antibodies (input). (e) WB showing amounts of progerin in HGPS cells incubated with C75 for 20 days. (f) WB showing amounts of progerin remaining in HGPS cells incubated with vehicle and 5 µM C75 for 20 days and then with cycloheximide to stop protein synthesis. (g) Quantification of progerin amounts from the experiment in panel f and two others like it. (h, i) Left panels, WB showing amounts of progerin and prelamin A in nuclear membrane and nucleoplasm fractions of HGPS (h) and Zmpste24-deficient (i) fibroblasts, respectively, incubated with vehicle and 5 µM C75 for 20 days. Lamin B2 and nuclear P84 were loading controls for nuclear membrane and nucleoplasm fractions, respectively. Right panels, quantification of nucleoplasmic progerin/prelamin A from densitometry analyses. *p<0.05; **p<0.01; ***p<0.001.

Fig 5: ATP7A expression is diminished in Commd1 but not Commd9 intestinal knockout mice. (A) Immunoblot analysis for ATP7A expression in Commd1 and Commd9 intestinal knockout mice (top). ATP7A quantification after normalization by the loading control (P84 or actin) (bottom). (B) Representative images of immunofluorescence staining for ATP7A (red) and nuclei (Hoechst, blue) in intestinal tissues of Commd1?IEC (n=5) or Commd9?IEC (n=3) mice (bottom row) or corresponding control floxed animals, Commd1fl/fl (n=4) and Commd9fl/fl (n=6) (top row). Scale bar: 25 µm. Bar graphs represent quantification of ATP7A signal intensity in immunofluorescence staining of intestinal tissues, expressed as the fold change over the nuclear staining. Results for individual mice are plotted along with the mean and s.e.m. for each group; *P<0.05 (unpaired two-tailed Student's t-test).