EMBO/SourceData huggingface dataset:
Figure 4A) Enrichment of the putative LRP6 binding sites bound to AGO2 in miR-cluster-Dox-MCF-10A cells after the miR-424/503 cluster is induced with 100ng/ml of dox for three days. A CDC25A-binding site is included as a positive control and a region of B2M and GAPDH are included as negative controls. The error bars show the standard deviation.B) The WT-blot shows the expression of LRP6 upon upregulation of miR-424/503 in miR-cluster-Dox-MCF-10A cells (Dox+) when one or both of the conserved microRNA binding sites (#1 and #3) are blocked by target protection.C) The WT-blot shows the expression of LRP6 and β-catenin in cells in miR-cluster-Dox-MCF-10A cells cultured in the presence of Dox and with and without WNT3a ligand (100ng/ml for 6 hours) when the microRNA binding sites #1 and #3 are blocked by target protection.
Figure 1Representative composite immune-fluorescent images of whole hair follicle units from non-lesional and lesional scalp psoriasis patients. K15 (green), c-JUN (red A, C) and JUNB (red B, D) and DAPI (blue).Confocal images of the bulge region of human psoriatic hair follicles from non-lesional and lesional regions of the scalp. K15 (green), c-JUN (red E, G) and JUNB (red F, H) and DAPI (blue).Percentage of HF-SCs (K15+) that express c-JUN (I) and JUNB (J) in the bulge region of human psoriatic hair follicles from non-lesional and lesional scalp in comparison with healthy scalp. n=2-6 hair follicles in anagen per group from five psoriatic patients and two healthy patients.
Figure 2Composite immunofluorescent images showing GFP expression of whole-ear sections from DKO*-mT/mG mice at day 5, 7, 15 and 30. Increased GFP expression is shown at day 7 followed by progressive decrease of GFP+ keratinocytes (white dotted line represents basal layer, and red dotted line represents outermost skin layer). n=3 per time point. Quantification analysis of GFP expression (top) and epidermal thickness (bottom) of DKO* mice at different time points during psoriasis-like disease progression. n=6 per time point. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).Quantification analysis of cleaved Caspase-3 (cCas3 top) and Ki67 (bottom) in ear skin of DKO* mice at different time points during psoriasis-like disease progression. n=3 per time point.Intravital confocal time-lapse imaging of non-mutant Tomato keratinocytes and mutant GFP keratinocytes of DKO*-mT/mG at day 7 after first tamoxifen injection. Dotted circles emphasize areas in which mutant GFP+ keratinocytes are replaced by non-mutant Tomato+ keratinocytes.Percentage of GFP+ area eliminated during intravital confocal time-lapse imaging at day 5, 7 and 15 in DKO*-mT/mG mice (Time-lapse 1:8h). Positive value: Area of GFP increased. Negative value: Area of GFP reduced.Fluorescence imaging of DKO*-mT/mG ear skin at day 30 after first tamoxifen injection shows that remaining mutant GFP+ keratinocytes reside along the hair follicles. White dotted line separates epidermis and dermis. White arrows represent GFP+ hair follicles.Intravital confocal time-lapse imaging of DKO*-mT/mG at day 15 after first tamoxifen injection. Mutant GFP+ Keratinocytes (white arrows) are maintained around hair follicles.
Figure 3Ear pictures of control, DKO*, and DKO*K15 mice after 15 days of induction show clear signs of inflammation and psoriatic-like scales in both models.Representative images of hematoxylin and eosin (H&E) staining of ear sections from control, DKO* and DKO*K15 mice.Ear thickness measurement at different time points during psoriasis-like development in control, DKO* and DKO*K15 mice. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to control group).FACS quantification of CD45+ cells from ear skin of control, DKO* and DKO*K15 mice at different time points during psoriasis-like development. n > 5 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of IL-17A, IL-6 and LCN2 production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n > 4 per group. Statistical significance *p<0.05, **p<0.01, ***p<0.001 (t-student two tailed-test relative to controls).Quantification of total number of GFP+ epidermal cells by FACS analysis from DKO*-mT/mG and DKO*K15-mT/mG mice at different time points during psoriasis-like progression compared to Co-mT/mG mice. n = 3-6 per group and time point.Representative immunofluorescence images of ear sections from CoK15-mT/mG and DKO*K15-mT/mG mouse models after 15 days of induction. Red (Tomato), green (GFP) and blue (DAPI). Hyperplasia of Tomato+ keratinocytes in psoriatic-like DKO*K15-mT/mG mouse with clusters of GFP+ keratinocytes are derived from hair follicle K15+ GFP+ stem cells. n=3 per group. Dotted lines separate epidermis and dermis. Quantification analysis of cleaved Caspase-3 (cCas3) and Ki67 in ear skin of DKO*K15-mT/mG mice at different time points during psoriasis-like disease progression.
Figure 4Average number of live and dead (TOPRO-3+ Dapi+) primary keratinocytes derived from HF-SCGFP at different time points of time lapse capture during 48 hours. There is a slight increase over time of live HF-SCs from DKO* mice when compared to control cells, and no difference in average of dead cells. n = 4 fields from two independent experiments. Average number of live and dead (TOPRO-3+ DAPI+) primary keratinocytes derived from basal keratinocytesGFP show a significant increase in DKO* basal live keratinocytes at 12 hours, followed by a significant increase in cell death at 48 hours.Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ bulge HF-SCs at different time points of time lapse capture during 48 hours. Co-culture of HF-SCGFP with HF-SCTom from DKO*-mT/mG mice shows a significant increase of HF-SCTom, while the HF-SCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with HF-SCGFP from control mice. Average number of live primary keratinocytes from co-cultures of GFP+ with Tomato+ basal keratinocytes at different time points of time lapse capture during 48 hours. Co-culture of b-KCGFP with b-KCTom from DKO*-mT/mG mice show significant increase of b-KCTom, while these b-KCTom from DKO*-mT/mG mice show a normal growth when they are co-culture with b-KCGFP from control mice.Average total number of live Tomato+ basal keratinocytes (from DKO* or control mice) at different time points cultured in the bottom part of trans-well system cultures with GFP+ basal keratinocytes (from DKO* or Control mice) cultured in the upper trans-well (Tw). n = 5 fields from three independent experiments. Tom+ basal keratinocytes significantly increased when these were cultured with mutant GFP+ basal keratinocytes in the trans-well in comparison with control GFP+ basal keratinocytes.
Figure 5Heat map for specific genes relative to biological process enriched based on Gene Ontology IDs for DEGs that are: i) exclusively up-regulated in HF-SCGFP; ii) exclusively up-regulated in HF-SCTom; iii) commonly up-regulated in HF-SCGFP and HF-SCTom.Gene expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice.protein expression of TSLP in different epidermal cell subpopulations sorted at mid-term (D7) or late-term (D30) of psoriasis-like progression in DKO* mice. n=2-3.Quantification of TSLP production in sera of control, DKO* and DKO*K15 mice at day 30 of psoriasis-like disease by ELISA. n = 4 per group.
Figure 6TSLP production in conditioned media quantified by ELISA three days after infection with Ad-empty or Ad-cre. n=3 independent experiments.Percentage of EdU+ Tomato+ keratinocytes after blocking with anti-TSLP. Anti-TSLP neutralizes the hyper-proliferation of non-mutantTom KCs in vitro after infection with Ad-cre.Gene expression of Tslpr after TSLP neutralization is reduced. Gene expression of IL-7ra after TSLP neutralization is increased. Gene expression of VEGFα after TSLP neutralization is reduced.Representative images of treated ears after 9 days of anti-TSLP or IgG treatment.Longitudinal analysis of ear thickness in DKO* mice at different time points during psoriasis-like disease progressionPercentage of EdU+ epidermal cells (CD45-CD49f+) after blocking with anti-TSLP in DKO* and DKO*15 miceRepresentative immune-fluorescence images of ear sections from control or DKO* mice treated with IgG or anti-TSLP) stained for Ki67. Ki67 expression was reduced in mice treated with anti-TSLP (n =3).
Figure 7Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients. c-JUN (purple), K-15 (green), TSLP (red) and DAPI (blue).TSLP intensity measurement in hair follicle units and epidermis by ImageJ from healthy scalp, non-lesional and lesional scalp psoriasis patients. Representation of fold change relative to control healthy scalp. n= 3 samples.Representative composite immune-fluorescent images of whole hair follicle units by confocal from non-lesional and lesional scalp psoriasis patients (D) and magnification images of specific regions from the HFs (E). CD200 (green), c-JUN (white), TSLP (red), DAPI (blue).
figf1b) The affinity of the UVRAG-C2 domain for phospholipids was assessed using a protein-lipid overlay assay. The left panel indicates the identity of lipid species on PtdIns strips. Bacterial-purified GST fusion of UVRAG-C2 (wild type, WT), but not GST, binds PtdIns(3)P, PtdIns(4)P and PtdIns(5)P. The K78A/R82A mutant of C2 is defective for phospholipid-binding, whereas the K87A/N88A mutant is partially impaired. Binding of the FYVE and PH domains of Hrs and FAPP1, respectively, to PtdIns(3)P and PtdIns(4)P served as quality controls (right). PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPA, lysophosphatidic acid; LPC, lysophosphatidylcholine; S1P, sphingosine 1-phosphate; PA, phosphatidic acid; PS, phosphatidylserine.(c) The relative affinity of the GST-fusion proteins for phosphoinositides was quantified by densitometric analysis of the GST immunoblots in b from five independent experiments (n = 5). Error bars represent standard deviation (s.d.). A.U., arbitrary units.(d) Binding of full-length UVRAG (untagged) to lipid blots. The same PtdIns strips (in b) were used and immunoblotted with antibodies against IgG (right) and UVRAG (left).(g) Pulldown of UVRAG-C2 domain GST-fusion proteins by liposomes bearing the indicated phosphoinositides. GST-Hrs-FYVE and GST-FAPP1-PH are also shown.
figf2(a-k) Confocal analyses of the subcellular co-localization of endogenous UVRAG with ER markers, including endogenous calnexin (red, a), endogenous PDI (red, b), and overexpressed DSred-ER (red, c); with Golgi markers, including endogenous GM130 and p115 (red, d,e, cis-Golgi), overexpressed GFP-Man II (green, f, medial-Golgi), endogenous TGN46 (red, g, trans-Golgi), and overexpressed PtdIns(4)P probe, GFP-FAPP1-PH (green, h); with endogenous coatomer proteins, including Sec31 (red, i, COPII-related) and β′-COP (red, j, COPI-related); and with the endosome marker GFP-p40phox(PX) (green, k) in HeLa cells. The insets show a high magnification of the selected areas. Scale bars, 10 μm. (l) Confocal microscopic quantification of co-localization of UVRAG with the indicated markers (data are mean±s.d., n = 150 cells obtained by gathering data from three independent experiments); **P0.01; ***P0.001.
figf3a) UVRAG is released from the ER membrane in cells overexpressing MTMR3. HeLa cells transfected with MTMR3-mStrawberry or the catalytically inactive mutant MTMR3C413S were stained with anti-UVRAG antibody and processed for confocal microscopy. Asterisks indicate MTMR3-transfected cells (left). The percentage of transfected cells with diffused staining of UVRAG was quantified (middle). Data are mean±s.d.; n = 240 cells collected from four independent experiments; **P0.01. Western blot analysis shows the levels of endogenous UVRAG in the presence or absence of MTMR3 (right).(b) Wortmannin treatment releases UVRAG from the ER. HeLa cells were treated with 100 nM wortmannin for 1h, and processed for confocal microscopy using anti-UVRAG and anti-PDI antibodies (top panel). UT, untreated. Insets highlight the relative localization of UVRAG at the ER. Confocal co-localization is indicated (bottom left panel). Data are mean±s.d.; n = 75 cells for each group from three independent experiments; ***P0.001. Endogenous UVRAG and PDI expression before and after wortmannin treatment were confirmed by immunoblotting (bottom right panel).(c) Mutation of the C2 domain leaves UVRAG unable to associate with the ER. HeLa cells expressing Flag-UVRAG (WT) or its K78A/R82A mutant were fixed and processed for confocal microscopy for co-localization with the ER marker PDI (top panel). Insets highlight the relative distribution of UVRAG at the ER. UVRAG and endogenous PDI expression were confirmed by immunoblotting (bottom panel). See Supplementary Fig. S9 for uncropped data. Scale bars, 10 μm.
figf4(a) The UVRAG C-terminal 270-442 region interacts with RINT-1. Cells (HEK293T) were co-transfected with HA-RINT-1, together with Flag-UVRAG or its mutant derivatives, and whole-cell lysates (WCLs) were immunoprecipitated (IP) with anti-Flag, followed by immunoblotting (IB) with an anti-HA antibody.c) Interaction between endogenous UVRAG and RINT-1. WCLs of HEK293T cells were used for immunoprecipitation with control serum (control) or an anti-UVRAG (left) or anti-RINT-1 antibody (right), followed by immunoblotting with the indicated antibodies. The bottom panel shows endogenous protein expression.d) ER localization of RINT-1, ZW10 and UVRAG. HeLa cells were stained with anti-UVRAG (green), anti-PDI (ER marker, red), and anti-ZW10 or anti-RINT-1 (blue), followed by confocal microscopy. Insets highlight UVRAG co-localization with RINT-1 and ZW10 at the ER. Scale bars, 10 μm.(e) Interaction between endogenous UVRAG and the RINT-1-ZW10-NAG tethering complex proteins. WCLs of HEK293T cells were used for immunoprecipitation with control serum (IgG) or an anti-UVRAG (first panel), anti-RINT-1 (second panel), anti-ZW10 (third panel) and anti-NAG (fourth panel), followed by immunoblotting with the indicated antibodies. As input, 10% of the WCLs was used. Syn 18, syntaxin 18; Syn 6, syntaxin 6. See Supplementary Fig. S9 for uncropped data.
figf5(a) UVRAG knockdown impairs the interaction of the RINT-1-ZW10 complex with COPI in an autophagy-independent manner. Cells (HEK293T) were transfected with control shRNA, UVRAG shRNA or Atg16L1 shRNA for 72 h. Whole-cell lysates (WCLs) were used for immunoprecipitation with anti-RINT-1 (first and third panels), anti-ZW10 (second panel), or anti-UVRAG (fourth panel), followed by immunoblotting with the indicated antibodies. Input represents 10% whole-cell lysates. Note that β′-COP interaction was suppressed by UVRAG knockdown but unaffected by Atg16L1knockdown. See Supplementary Fig. S9 for uncropped data. ((b) Retrograde transport of tsO45-VSVG-KDELR-YFP from the Golgi to the ER is inhibited by UVRAG knockdown. HeLa cells serially transfected with UVRAG shRNA or control shRNA, then with VSVG-KDELR-YFP expression vector, were incubated at 32°C (permissive condition) overnight. On shifting to 40°C (non-permissive condition), the ER-like distribution pattern of VSVG-KDELR-YFP was registered. Representative confocal images of the distribution pattern of VSVG-KDELR at 40°C and its co-localization with PDI are shown (upper panel). Data are mean±s.d., n = 100 cells obtained from three independent experiments; **P0.01 (bottom panel). Scale bars, 10 μm.(c) UVRAG interaction with bothPtdIns(3)P and RINT-1 is required for COPI-dependent retrograde transport, as shown by the redistribution of VSVG-KDELR from the Golgi to the ER. HeLa cells were transfected with control shRNA or UVRAG shRNA. The UVRAG-depleted cells were then complemented with empty vector, Flag-UVRAG, UVRAGΔ270−442, UVRAGK78A/R82A or UVRAGΔC2, along with the transfection of VSVG-KDELR-YFP. The ER pattern for the chimaeric KDELR was quantified (left panel). Data are the mean±s.d.; n = 200 cells obtained from three independent experiments; *P0.05; **P0.01. Endogenous and reconstituted UVRAG expression was confirmed by immunoblotting (right panel).(d) PtdIns(3)Pdepletion inhibits Golgi-to-ER retrograde transport of VSVG-KDELR. HeLa cells were transfected with empty vector, wild-type MTMR3, the C413S mutant of MTMR3, or treated with wortmannin or rapamycin, and then the ER pattern of VSVG-KDELR was quantified. UT, untreated. Data are the mean±s.d.; n = 100 cells for each group obtained from three independent experiments; **P0.01.e) Interaction between endogenous UVRAG and the ER tethering complex under wortmannin treatment. WCLs of HEK293T cells treated with wortmannin were used for immunoprecipitation with anti-UVRAG or anti-RINT-1 antibody, followed by immunoblotting with the indicated antibodies. The input panel shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.
figf6(a) Golgi morphology on UVRAG depletion. HeLa cells were transfected with control or UVRAG-specific shRNA for 72 h and subjected to electron microscopy analysis (left panel). When compared with control cells (left), the Golgi structure was swollen and fragmented in cells lacking UVRAG. The level of Golgi fragmentation was quantified (right panel). Data represent mean±s.d.; n = 50 cells obtained by gathering data from two independent experiments; *P0.05. Scale bars, 500 nm.(b) Depletion of UVRAG leads to cis-Golgi dispersion. HeLa cells were transfected with control shRNA or UVRAG-specific shRNA expressing GFP as an expression marker and then stained for GM130 (left panel; red; highlighted by arrows). Nuclei were stained with DAPI (blue). The dispersed distribution of GM130 in shRNA-transfected cells was quantified (middle panel). Data are the mean±s.d.; n = 500 cells obtained from five independent experiments. The expression of UVRAG and GM130 in treated cells is also shown (right panel). Scale bars, 10 μm.(c) The effect of UVRAG on membrane trafficking from the ER to the Golgi. HeLa cells serially transfected with UVRAG shRNA RINT-1 shRNA or control shRNA, then with VSVG-GFP expression vector, were incubated at 40°C (non-permissive condition) overnight. The cells were then shifted to 32°C (permissive condition) for 30 min to allow cargo transit from the ER to the Golgi. Before and after the temperature shift, the cells were fixed and labelled with anti-PDI and anti-GM130 antibodies to define the ER and Golgi region, respectively. Representative confocal microscopy images of the distribution pattern of the retrograde cargo at 40°C and 32°C are shown (left panel). The Golgi-like distribution pattern of VSVG-GFP was registered (right panel). Data are mean±s.d.; n = 100 cells from three independent experiments; **P0.01. Scale bars, 10 μm.(d,e) UVRAG interaction with PtdIns(3)P and RINT-1 is required or Golgi integrity. (d) HeLa cells were transfected with control shRNA (first row) or UVRAG shRNA (second to sixth rows). The UVRAG-depleted cells, as indicated by Flag expression, were then transfected with empty vector (second row), UVRAGK78A/R82A-UVRAG (third row), UVRAGΔ270−442mutant (fourth row), UVRAGK78A/R82A mutant (fifth row), or UVRAGΔCCD mutant (sixth row), followed by confocal microscopy using anti-GM130 (red) for cis-Golgi staining, anti-Flag (purple) for the ectopically expressed UVRAG protein expression, and UVRAG for nuclei (blue). Asterisks indicate shRNA-transfected cells and arrows denote the cells reconstituted with GM130 (wild-type or mutant) expression. (e) The dispersed distribution of GM130 was quantified, and data represent the mean±s.d.; n = 200 cells from four independent experiments; *P0.05; **P0.01.
figf7(a,b) Differential interaction of UVRAG with the RINT-1-ZW10-NAG tethering complex and the beclin-1-Bif-1-PI(3)KC3 autophagy complex after treatment with rapamycin (a) and HBSS (b). Cells (HEK293T) treated with rapamycin (50 nM) in a or with HBSS in b were subjected to immunoprecipitation with anti-UVRAG (first panel), anti-RINT-1 (second panel) or anti-PI(3)KC3 (third panel), followed by immunoblotting with the indicated antibodies. Input (10%) shows endogenous protein expression. See Supplementary Fig. S9 for uncropped data.(c) Gel filtration analysis of UVRAG complex formation under normal conditions (untreated, UT) and after rapamycin-induced autophagy (50 nM). Affinity-purified UVRAG complexes from HCT116 cells stably expressing Flag-UVRAG were fractionated by Superose-6 gel filtration column and the eluates were analysed by western blotting for UVRAG, RINT-1, ZW10, beclin 1, PI(3)KC3 and Bif-1 proteins. Whole-cell lysates (2.5%) were used as the input (lane-1). The elution profile of each protein was quantified by densitometry analysis and normalized. Relative densitometry units were plotted against fraction number. Black arrows indicate the positions of the molecular weight size markers. Red arrows indicate the peak shift of the UVRAG eluates.
figf8(a-c) UVRAG interaction with beclin 1 and PtdIns(3)P, but not with RINT-1, is required for autophagy-induced Atg9 dispersal. (a) HeLa cells were stably transfected with control shRNA (first row) or UVRAG shRNA (second-sixth rows). The UVRAG-depleted cells were then transfected with empty vector (second row), Flag-UVRAG (third row), UVRAGΔ270−442 mutant (fourth row), UVRAGK78A/R82A mutant (fifth row) or UVRAGΔCCD mutant (sixth row), and incubated in complete medium (untreated, UT, first panel), starvation medium (HBSS 2 h, third panel), or treated with rapamycin (50 nM 2 h, second panel) or SMER28 (50 μM, 2 h, fourth panel). The distribution pattern of endogenous Atg9 (green) in the UVRAG (wild-type or mutant)-transduced cells (red) was analysed by confocal microscopy (a). Asterisks denote condensed (non-dispersed) Atg9 in UVRAG-depleted HeLa cells and arrows denote the cells complemented with wild-type or mutant UVRAG.(b,c) The percentage of cells with Atg9 translocation was quantied (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the means:d:; n D300 cells from three independent experiments; NS, not signicant; P <0:01; P <0:001. Scale bars, 10 m.(b,c) The percentage of cells with Atg9 translocation was quantied (c) and the expression of UVRAG and actin was determined by immunoblotting (b). Data represent the means:d:; n D300 cells from three independent experiments; NS, not signicant; P <0:01; P <0:001. Scale bars, 10 m.(d) Effect of knockdown of UVRAG, beclin 1 andRINT-1 on the translocation of Atg9 to the LC3-labelled autophagsomes during autophagy. Control-, UVRAG-, BECN1- or RINT-1-knockdown HeLa cells were incubated in complete medium (normal condition), starvation medium (HBSS, 2 h), or treated with rapamycin (100 nM, 2 h) or SMER28 (50 μM, 2 h). The cells were then stained for endogenous Atg9 (green) and LC3 (red) followed by confocal microscopy analysis (top and bottom left panels). The percentage of Atg9 vesicles co-localized with LC3+-autophagic puncta was quantified (bottom middle panel). Endogenous UVRAG, beclin 1 and RINT-1 protein expression are shown (bottom right). Data represent mean±s.d.; n = 200 cells obtained from three independent experiments. NS, not significant; *P0.05; **P0.01; ***P0.001. Scale bars, 10 μm.
Figure 1(B) H&amp;amp;E staining of representative atherosclerotic lesions in aortic root (upper panel-4x magnification) and brachiocephalic arteries (lower panel-10x magnification) at 14 week harvest. Lesion width of roots highlighted with black bars. (C) Quantification of root lesion size (left panel, *p=0.002), and of brachiocephalic lesion size (right panel, *p=0.001), both quantified as percent of total lumen area. Data in (C) are averaged lesion percentages from 4-6μm serial sections from each animal, n=9, 13, 15. All of the data represent the mean + SEM, *p<0.05 versus LNA-control tissue. Level of significance was determined using one-way ANOVA with Bonferroni's post test.