EMBO/SourceData huggingface dataset:
Figure 5A. Representative blots of ATG13 (left) and VPS34 (right) phosphorylation levels in wt and KO-OFD1 cells transiently expressing GFP-ATG13 and FLAG-VPS34 in FM.B. Wild-type and KO-OFD1 cells were starved in HBSS with/without Baf-A1 (100nM, 2h) and chloroquine (CQ) (50μM, 2h) and total lysates were analyzed by immunoblotting using anti-OFD1, -LC3B (LC3B-I 18 kDa and LC3B-II 16 kDa) and -ACTIN antibodies. (Right) ACTIN-normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of wt cells. n=5 independent experiments.C. Representative images of LC3B and WIPI2 puncta in wt and KO-OFD1 cells (HBSS, 90min). Green, LC3B; red, WIPI2; blue, Hoechst, nuclei. Scale bar=10μm. (Right) Quantification of LC3B and WIPI2 puncta is shown. ≥100 cells analyzed/sample, n=5 independent experiments.D. Transmission electron microscopy (left) and quantification (right) of autophagic vacuoles (arrows) in wt and KO-OFD1 cells (HBSS, 90min).E. Representative images of siOFD1 depleted and control (Ctrl) HeLa cells transiently expressing mRFP-eGFP-LC3B (HBSS, 90min). Insets show higher magnification of colocalization in selected areas. Scale bar=10μm. Bottom, tandem structure of the mRFP-GFP-LC3B construct and quantification of RFP+GFP+ and RFP+GFP- puncta in siOFD1-depleted cells and controls, cultured in full medium (FM) or kept in HBSS for 2h (STV) are depicted. n≥90 cells/sample analyzed, n=5 independent experiments.F. Representative images of WIPI2 staining in wt HK2 cells transiently expressing eGFP-OFD1 (HBSS,90min). Green, eGFP-OFD1, red, WIPI2; blue, Hoechst labels nuclei. On the right, quantification of WIPI2 puncta in eGFP-OFD1 transfected cells compared with non-transfected cells (Ctrl) is shown. Scale bar=10μm. n≥90 cells analyzed/sample, n=3 independent experiments.G. Representative images of WIPI2 and OFD1 co-staining in KO-OFD1 cells transfected with empty vector (Empty), 3xFLAG-OFD1 (OFD1) or 3xFLAG-OFD1∆LIR (OFD1∆LIR) (HBSS,90min). Red, WIPI2; green, OFD1; blue, Hoechst labels nuclei. Scale bars: 10μm. On the right, quantification of WIPI2 puncta. ≥100 cells analyzed/sample, n=4 independent experiments.
Figure 6A. Western blot of LC3B in lymphoblasts of OFD type I patients and controls, either untreated (-) or exposed to Baf-A1 (10nM, 2h)(+). Cells were cultured in complete medium (FM, top) or under serum starvation for 4h (STV, bottom). ACTIN and GAPDH were used as loading controls.B. (Left) Graphs show LC3B-II levels normalized on ACTIN in patients lymphoblasts and controls cultured in complete medium (FM). (Right) Graphs show LC3B-II levels normalized on GAPDH in patients lymphoblasts and controls in serum starvation for 4h (STV). Normalized LC3B-II levels are expressed as fold change versus untreated conditions (-) of control cells, n=4 patients and 3 controls.C. Western blot of ATG13 and ATG101 proteins in serum starved lymphoblasts from controls and OFD type I patients. (Right) Protein levels relative to ACTIN (loading control) are expressed as fold change versus controls, represented by the dashed line, n=4 patients and 3 controls.
Figure 7A. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1fl/y;creKsp and control mice. (Right) LC3B-II protein levels normalized to Actin (loading control) are expressed as fold increase versus control mice, n=4 mice/group.B. Representative images of GFP-LC3 puncta (autophagosomes, green) in aquaporin2-positive (AQP2, red) renal distal tubules from P8 Ofd1fl/y;creKsp;GFP-LC3 and Ofd1fl/y;GFP-LC3 mice. Scale bar=10μm. Hoechst labels nuclei. Bar graphs on the left show quantitative analysis of GFP-LC3 puncta; n=4 mice/group for a total of 300 AQP2 positive nuclei/group analyzed.C. Western blot of LC3B in kidney homogenates from leupeptin-treated (40mg/kg, 6h) fasted Ofd1-IND and control mice. (Right) LC3B-II protein levels relative to βTubulin (loading control) are expressed as fold increase compared with control mice, n=5 mutant/8 control mice .D. Western blot of Atg13 in Ofd1fl/y;creKsp and control kidneys at pre-cystic (P8, left) and cystic (P22, right) stages. Gapdh was used as loading control. Atg13 protein levels are expressed as fold change compared with control mice; n=7 mutant/5 control mice at P8, n= 4mice/group at P22.E. Haematoxylin & eosin staining of kidney sections from Ofd1fl/y;Atg7+/+;creKsp and Ofd1fl/y;Atg7fl/fl;creKsp mice. (Right) Graph shows fold change of the cysts number observed in Ofd1fl/y;Atg7fl/fl;creKsp animals compared with Ofd1fl/y;Atg7+/+;creKsp; n=12 sections/animal were analyzed; n=6 mice/group. Scale bars=1mm.F. Blood Urea Nitrogen was quantified on blood withdrawn from Ofd1fl/y;Atg7fl/fl;creKsp, Ofd1fl/y;Atg7+/+;creKsp and control mice at P45. n=8 mice per group.
Figure 1Agar plugs containing actively growing cultures of wild type S. sclerotiorum (strain 1980) and the OA deficient A2 mutant were inoculated onto Col-0 and ced-9 expressing Arabidopsis leaves. (A) Wild type inoculations onto Col-0 plants resulted in typical lesions for this pathogen including a rapid, spreading cell death; however, infection was completely suppressed in ced-9 expressing plants. The expression of this gene had no effect on the A2 phenotype.(B) Trypan blue staining indicates the extent of cell death for each genotype/strain combination, including an agar plug control. All images were recorded 48 hours post inoculation.
Figure 2Microscopic examination of cross sections of tomato leaves at the leading edge of the lesion following fungal inoculation.S. sclerotiorum A2 (A) and wild type (B) strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were post-fixed in osmium tetroxide, and embedded in Spurr's epoxy resin. A microtome was used to cut 400 nm sections. Toluidine blue stain was used to reveal fungal hyphae. H = hyphae. The dotted line represents the leading edge of the visible lesion. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227.
Figure 3S. sclerotiorum A2 strain induces autophagic structures in plants.S. sclerotiorum wild type and A2 strains were inoculated onto tomato leaves using colonized agar plugs. 24 hours post inoculation; leaves were stained with 100 µM final concentration of MDC (Sigma) in PBS for 30 min. Fluorescence was visualized using an Olympus IX81 inverted fluorescence confocal microscope (Olympus systems, Germany), with an excitation wavelength of 335 nm and an emission wavelength of 508 nm. Images were collected using an Olympus DP 70 camera and processed with Olympus DP Controller software, version 2.2.1.227. Scale bar = 10 µm.
Figure 4Transmission Electron Microscopy (TEM) fungal inoculated tomato leaves.Representative TEM images from four independent experiments. (A, G); Healthy non-inoculated leaf tissue. (B-F) Tomato leaves inoculated with the OA deficient A2 strain. (H,I) Tomato leaves inoculated with wild type S. sclerotiorum. Arrows, autolysosomal/autophagosomal-like structures; C, chloroplast; V, vacuole; N, nucleus; Circle, active dismantlement of chloroplast; Rectangle, chromatin condensation within the nucleus. Black scale bars = 2 µm, white scale bars = 1 µm. Sections were examined with a Phillips Morgagni 268 transmission electron microscope at an accelerating voltage of 80 kV. Digital images were recorded with a MegaViewIII digital camera operated with iTEM software.
Figure 5(A,B) Agar plugs containing actively growing cultures of the OA deficient A2 mutant were inoculated onto leaves of Arabidopsis Col-0 and select Arabidopsis autophagy mutant plants. These mutants showed enhanced susceptibility to the normally non-pathogenic A2 strain. Lesion diameter was monitored over time and all images were recorded 48 hours post inoculation.(C) Tomato leaves were either pre-infiltrated with water (control) or autophagy inhibitors Wortmannin, LY294002, Chloroquine (CQ), and 3-methyladenine (3-MA). Agar plugs containing actively growing A2 were placed on the infiltrated leaves to initiate infection. (B) 48 hours post inoculation; Trypan blue was used to determine the extent of fungal colonization and cleared with acetic acid and ethanol (1: 3, v/v). Images were taken 48 hours post inoculation.
Figure 6(A) NBT treated Arabidopsis (Col-0 and two independent atg8a mutant lines) following agar plug inoculation with the A2 mutant. Images were collected 48 hours post inoculation. Dotted lines represent the edge of the observable legion.(B) RT-PCR was used to evaluate the transcript levels of three catalases (CAT1, 2, and 3) and three superoxide dismutases in Col-0 plants following inoculation with wild type S. sclerotiorum (black bars) and the A2 mutant strain (grey bars). *>2 fold change, **>5 fold change.
Figure 1(A, B) Representative images of the expression of NEDD4L in the skins from IMQ-treated mice (A) or psoriasis patients (B) as detected by IHC on the left. The NEDD4L expression level was calculated by multiplying the staining intensity score and the extent score and is shown on the right. (A, n=5, B, normal, n=15, psoriasis, n=36). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.(C, D) Representative images of the expression of EZH2 in the skin epidermis from the IMQ-treated mice (C) and the psoriasis patients (D). The number of EZH2-positive cells per HPF (high power field) in each mouse and patient is shown on the right. (C, control, n=3, IMQ, n=6. D, normal, n=13, psoriasis, n=27). Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test ** P＜0.01, *** P＜0.001. NS, no significance.(E) WB analysis of NEDD4L, EZH2 and EZH1 expression in the EZH2-specific siRNA (siEZH2)- or negative control siRNA (siNC)-transfected NHEKs with IMQ stimulation. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments Scale bar, 200 μm (100×) or 50 μm (400×).
Figure 2(D) Representative images of HE staining of the back tissues are shown on the left. The epidermal thickness measured using the scale in the microscope is shown on the right. (E) Representative images of Ki67 staining in back tissues are shown on the left. The number of Ki67-positive cells per HPF in each mouse is shown on the right. (F) Representative images of HE staining of the ear tissues are shown on the left. The epidermis thickness measured using the scale in the microscope is shown on the right. (G) Representative images of Ki67 staining in ear tissues (same specimen as in F) are shown on the left. The number of Ki67 positive cells per HPF in each mouse is shown on the right. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments. For back tissues, scale bar, 200 μm (100×) or 50 μm (400×). For ear tissues, scale bar, 200 μm (100×) or 200 μm (200×). Significant differences were tested using a two-tailed Student's t test Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance.
Figure 3NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs. WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. cell signaling pathway activation induced by IL-6 were detected by WB analyses Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments(G, H) Primary keratinocytes were separated from the WT and Nedd4l KO mice. (G) WB analysis of the protein expression in IL-6-induced cell signaling pathway activation. (H) WB analysis of the proteins in the IL-6 signaling pathway. Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments
Figure 4Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Representative images of Ki67 staining in back tissues are on the left. The number of Ki67-positive cells per HPF is shown on the right. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test C, control, n=5, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.Six- to eight-weeks old WT and Nedd4l cKO mice were treated with IMQ for 6 consecutive days and at the beginning of the treatment, the mice were injected intra-epidermally with 10 μg anti-GP130 antibody or with same dosage of isotype on every other day. Keratinocyte proliferation associated genes in the skin tissues are detected using real-time PCR analysis. Data information: Data are shown as mean ± s.e.m., and are representative of two independent experiments. Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance.
Figure 5(A) Representative pictures of the expression of NEDD4L, GP130 and p-STAT3 in the epidermis of a normal skin section (same specimen) and sections from psoriasis patients. Isotype are IgG antibodies from same species served as negative control. (B) Semiquantitative scoring of NEDD4L, GP130 and p-STAT3 in the epidermis detected by IHC. Data information: Each symbol represents one patient Scale bar, 200 μm (100×) or 50 μm (400×). All data are shown as the mean ± s.e.m., and significant differences were analyzed using two-tailed Student's t test (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.
Figure 6(F) Full length Flag-GP130, GP130-aa 616-918, GP130-aa 616-889, GP130-aa 616-764, GP130-aa 616-734 were transfected with Myc-NEDD4L into HEK293T cells, and whole cell lysates were co-IP with ANTI-FLAG M2 magnetic beads, followed by WB with anti-Flag or anti-Myc antibodies.
Figure 7(C) WT or NEDD4L KO NHEK cells was infected with lentivirus that overexpress NEDD4L-WT, NEDD4L WW domain truncated mutant (NEDD4L-ΔWW) or its enzymatic activity mutant (NEDD4L-C943S), the cells were followed by denaturation-IP with anti-GP130 antibody and WB analysis with anti-Ub antibodies.(E) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with full length Myc-NEDD4L, HA-Ub and Flag-GP130 or its lysine mutants (K-R) in the intracellular domain, followed by denaturation-IP with ANTI-FLAG M2 magnetic beads and WB analysis with anti-HA antibodies.
Figure 2(A-D) The serial dilutions of post-second immunization serum samples were analyzed by ELISA using plates coated with purified RSV rA2-Line19F-FFL virions. The serum samples were serially diluted, and the detection of antibodies were measured by optical density (OD) at 490 nm. End-point titers of the serum samples were determined as the reciprocal of the highest dilution providing an absorbance twice that of the negative control (PBS immunized animal).
Figure 3(A) Lung RSV titers (PFU/gram of lung tissue) were determined in individual lungs (n = 5 mice per group) collected at 5 days post-RSV infection. Results were determined and presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.
Figure 4(A) Serum samples collected from individual mouse (M 1-5) immunized with F 110-136 peptide were tested for antibody binding against F-p27 (110-136) peptide or F 1-34 peptide (control) in ELISA.(B) A549 cells were infected with RSV (MOI = 0.1) for 16 h, and fixed. Cells were treated with mock control rabbit sera (left panels), or rabbit anti-sera against F (center panels), or against F-p27 (110-136) peptide (right panels), followed by Alexa 594 conjugated anti-Rabbit IgG (red). Nuclei are stained with DAPI (blue). Scale bar = 10 µm. The number of cells positive for RSV-F (middle panel) and RSV-p27 (right panel) upon counting of 200 cells were used to calculate percentage of cells stained for each antibody staining are shown in the 'merge' panel. The experiments were performed twice and variation between the two independent expeiremnts was <6%.
Figure 6(A) Lung tissues from individual mice (n=5) at day 5 post-RSV challenge (or uninfected control) were used to evaluate CD4/CD8 T cells separately for airway and distal lungs. Lung slides were fixed and stained with DAPI to visualize nuclei (blue) and either anti-CD4 (green) or anti-CD8 (red) T cells. The number of CD4/CD8 T cells were determined in each lung airway and distal region. Individual CD4+/CD8+ T cells data are presented as number of positive cells per 174 x 174 µm of lung tissue for each mice. Results are presented as box and whisker plots, where boxes extends from 25th to 75th percentile, whiskers show minimum to maximum value and central band represents the median value for the group.
Figure 1B Phospholipid transport/synthesis, as measured by 3H-serine incorporation, in human fibroblasts (mean±SE; n=9, with 5 replicates/experiment) and in primary mouse hippocampal neurons (mean±SE; n=3, with 4 or 5 replicates/experiment). *, p<0.05 vs E3.C Comparison of 3H-serine incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 3 replicates/experiment).D Duramycin sensitivity in ApoE ACM-treated fibroblasts. Note increased sensitivity after 20 min to 5 μM duramycin in ApoE4-treated cells, which was blocked upon the addition of 15 μM exogenous PtdEtn (mean±SD; n=3, with 3 replicates/experiment).
Figure 2B Fractions from the discontinuous gradient were blotted with anti-ApoE (WUE-4) antibody; the doublet is a characteristic feature on ApoE western blots due to ApoE sialylation [16].C An ApoE-containing fraction (fraction 5 [+]), an ApoE-negative fraction (fraction 3 [-]), and recombinant lipid-free ApoE protein were applied to human fibroblasts. Note increase in phospholipid synthesis using an ApoE4-containing lipoprotein fraction, but not with either an ApoE4-negative fraction or with lipoprotein-free recombinant ApoE3 or ApoE4 protein (mean ± SE; n=3, with 3 replicates/experiment).
Figure 3A Cholesteryl ester synthesis, as measured by 3H-oleate incorporation. Note increased CE in ApoE4-treated cells, whereas there was essentially no difference in triglyceride (TAG) production (mean±SE; n=3. with 3 replicates/experiment). †, p=0.06 vs E3.B Comparison of 3H-oleate incorporation in ApoE ACM-treated WT- and Mfn2-KO MEFs (mean±SE; n=3, with 5 replicates/experiment).C ApoE ACM-treated fibroblasts were stained with LipidTox green neutral lipid stain, and lipid droplets were visualized (example at left) and counted (right). Note significantly more lipid droplets in ApoE4-treated cells (mean ± SD, n=3).
Figure 1A. 293T cells were co-transfected with CMV-renilla, 8X-TBS (eight repeats of the TEAD-binding-sequence) luciferase reporter, Flag-YAP, HA-TEAD2 and wild-type (WT) or catalytically inactive (CS) USP9X-V5. One day after transfection, luciferase activity was measured and normalized with respect to renilla activity. The value for Flag-YAP/HA-TEAD-transfected cells (2nd column) was adjusted to 1. Shown below is a representative Western blot showing that the expression levels were comparable between samples (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).B. 293T cells were transfected with control or USP9X-targeting siRNAs. One day after siRNA transfection, the cells were co-transfected with CMV-renilla, 8X-TBS luciferase, Flag-YAP and HA-TEAD2. Reporter activity was quantified as described in (A) (n=4). Error bars indicate the S.E.M. (***p < 0.001; paired Student's t-test).C. RPE or MCF10A cells were transfected with the indicated siRNAs for 24 hours and then re-seeded to either sparse or dense culture conditions. At 48 hours after siRNA transfection, the cells were harvested and cell extracts were analyzed by Western blotting for the indicated proteins. L.E., long exposure, S.E., short exposure.D. Cells were treated as described in (C), the indicated mRNAs were analyzed with RT-qPCR, and the results were normalized with respect to the β-actin mRNA (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).E. RPE cells were transfected with the indicated siRNAs for 24 hours, and then co-transfected with CMV-renilla and 8X-TBS luciferase. One day after the latter transfection, the cells were re-seeded to either sparse or dense conditions, and reporter activity was measured at 24 hours after re-seeding (n=3). Error bars indicate the S.E.M. (**p < 0.01; paired Student's t-test).
Figure 2A. Representative anti-YAP immunofluorescence images of sparsely cultured RPE cells transfected with the indicated siRNAs. Green, YAP. Blue, DAPI. Scale bar, 10 μm.B. The nuclear YAP intensities of the cells shown in (A) were quantified using the Image J software. At least 70 cells from five or six random fields were analyzed. Quantified values were normalized by adjusting the average of "siControl Dense cells" to 1. We performed two independent experiments and obtained similar results. Data from one representative experiment are presented. The red bar indicates the average value. S, Sparse. D, Dense (***p < 0.001; paired Student's t-test).C. Sparsely cultured RPE or MCF10A cells were transfected with the indicated siRNAs, fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.D. Representative anti-YAP immunofluorescence images of densely cultured RPE cells transfected with the indicated plasmids. Green, GFP or V5. Red, YAP. Blue, DAPI. Scale bar, 10 μm.E. The nuclear YAP intensities of transfected cells in (D) were quantified using the Image J software. At least 70 cells from five to six random fields were analyzed. Quantified values were normalized by adjusting the average of "EGFP transfected cells" to 1. Similar results were obtained from two independent experiments. Data from one representative experiment are presented. The red bar indicates the average value. (***p < 0.001; paired Student's t-test).F. Densely cultured stable RPE clones integrated with empty vector (Vec), USP9X-V5 WT or CS mutant were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.G. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were fractionated into cytosolic and nuclear extracts, and analyzed by Western blotting for the indicated proteins.
Figure 3A. RPE cells were analyzed by Western blotting for the indicated proteins.B. Indicated stable RPE clones in densely cultured conditions were analyzed by Western blotting for the indicated proteins.C. Densely cultured RPE cells transduced with CRISPR-SAM targeting USP9X were analyzed by Western blotting for the indicated proteins.D. RPE or MCF10A cells expressing the control or USP9X shRNA were transfected with the LATS1/2 siRNA as indicated. Cells were re-seeded to low density and harvested after one day.E. RPE cells transduced with Flag-YAP WT or Flag-YAP 5SA were transfected with the indicated siRNAs. Cells were re-seeded to low density and harvested after one day.F. RT-qPCR of CTGF mRNAs in the cells described in (E) (n=3). Error bars indicate the S.E.M. (*p< 0.05, paired Student's t-test).
Figure 4A. RPE or MCF10A cells were transfected with the indicated combinations of siRNAs and re-seeded to low density, and cell extracts were analyzed by Western blotting for the indicated proteins.B. The cells described in (A) were analyzed by RT-qPCR for the indicated target genes (n=4). Error bars indicate the S.E.M. (*p< 0.05, **p < 0.01; paired Student's t-test).C. Sparsely cultured RPE cells transfected with the indicated siRNAs were immunoprecipitated with an anti-AMOTL2 antibody and analyzed by Western blotting for the indicated proteins. WCL, whole cell lysates.