EMBO/soda-nel huggingface dataset:
Clipped histone H3 is integrated into nucleosomes of DNA replication genes in the human malaria parasite Plasmodium falciparum (2019)
Figure 4

Figure 4: In P. falciparum, clipped Histone H3 is targeted to the 5' UTR of genes regulating DNA replication. A. Genome-wide distribution of ectopically expressed PfH3p-HA in P. falciparum schizont stages is represented as fold-enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input in blue; y-axis scale 1-10) or over PfH3n (PfH3p-HA / Input in teal; y-axis scale 1-10) calculated using MACS2. The coverage of PfH3p-HA (red; y-axis scale 0-50), Input (black; y-axis scale 0-30) and PfH3n (green; y-axis scale 0-30) is also shown and represents average reads per million over 1000 nt bins of the genome. The x-axis represents chromosome size in Mb. Major MACS2-derived peaks identified in all replicates are indicated using an orange arrowhead. B. Principal Component Analysis of the different ChIP-seq replicates (bigwig files derived from deduplicated bam files) was performed using the plotPCA function of deepTools on a multibigwig summary file, over 150 nt bins. The Eigen values of the top two principal components PC1 and PC2 are shown and meaningful clustering of replicates highlighted. Rep = Biological Replicate. C. Summary of the PfH3p-enriched peaks - and corresponding genes - identified in the PfH3p-HA versus Input or PfH3p-HA versus PfH3n comparisons using MACS2 peak-calling analysis. The overlap between the two comparisons is indicated using a Venn diagram. FE = Fold Enrichment; q-value represents the Benjamini and Hochberg false discovery rate. D. The genomic context of the six peaks identified in all three PfH3p-HA ChIP-seq replicates is shown. The fold enrichment of PfH3p-HA ChIP-seq signal over Input (PfH3p-HA / Input; blue) or PfH3n (PfH3p-HA / PfH3n; teal) is shown, as are the coverage plots of PfH3p-HA (red), PfH3n (green) and Input (black), which are represented as average reads per million over 1000 nt bins of the genome. DNA replication genes: Replication Protein A 1 (RPA1), Proliferating Cell Nuclear Antigen 1 (PCNA1), Single Stranded DNA binding protein (SSB), Topoisomerase I (TOPO-I), DNA polymerase alpha subunit (DNA pol a), and Topoisomerase II (TOPO-II) are highlighted and their directionality indicated. Note that the data in part D correspond to Biological Replicate 1.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 1

Fig 1. Downregulated NEDD4L expression in the epidermis from IMQ-treated mice and psoriasis patients.

(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).

(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).

(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.

(F, G) NHEK were treated with or without IMQ (1ug/ml) for 6h. A ChIP assay was conducted to analyze the local enrichment of histone H3K27me3 in specific regions of the NEDD4L promotor in mouse primary keratinocytes (F) and NHEKs (G). n=3 technical replicates.

Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments (A, C, E) or two independent experiment (F-G). Scale bar, 200 μm (100×) or 50 μm (400×). Significant differences were tested using a two-tailed Student's t test (A-D, F, G). ** P＜0.01, *** P＜0.001. NS, no significance. See also Fig EV1.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 2

Fig 2. NEDD4L is critical for inhibiting IMQ-induced keratinocyte hyperplasia in mice.

Six- to eight-weeks old WT and Nedd4l cKO mice were treated with or without IMQ for 6 consecutive days on the back and the ears.

(A) Representative pictures of WT and Nedd4l cKO mice treated with or without IMQ.

(B) Erythema, scaling and thickness of the back skin were scored daily on a scale from 0 to 4, and the cumulative score (erythema plus scaling plus thickness) is depicted.

(C) Ear thickness of the right ear was measured on the days indicated.

(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 Mann-Whitney U test (B, C) or two-tailed Student's t test (D-G). Control, n=3, IMQ, n=6. ** P＜0.01, *** P＜0.001. NS, no significance. See also Fig EV2.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 3

Fig 3. NEDD4L inhibits the activation of the IL-6 signaling pathway in keratinocytes.

(A-F) NEDD4L was knocked out by the CRISPR Cas9 method in NHEKs.

(A) Cell proliferation induced by IL-17A and IL-6 was detected by CCK-8 assays.

(B, C) Western blot (WB) analysis of protein expression in NHEKs induced by IL-17A (B) and IL-6 (C).

(D) WT and NEDD4L KO NHEKs were precoated with anti-IL-6 neutralization antibody or an isotype, and the cell proliferation induced by IL-17A was detected by CCK-8 assays.

(E, F) WT and NEDD4L KO NHEKs were infected with lentivirus overexpressing NEDD4L-WT or NEDD4L-C943S mutants. Cell proliferation (E) and cell signaling pathway activation (F) induced by IL-6 were detected by CCK-8 and WB analyses, respectively.

(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.

(I) Cell proliferation assay of GP130 KO NHEKs transfected with siNEDD4L induced by IL-17A.

Data information: Data are shown as the mean ± s.e.m., and are representative of three independent experiments (A-I). Significant differences were tested using a two-tailed Mann-Whitney U test (A, D, E, I). P＜0.01, ** P＜0.01, *** P＜0.001. A, D, E I, n=4 biological replicates. See also Fig EV3.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 4

Fig 4. Nedd4l deficiency promotes IMQ-induced keratinocyte hyperplasia via IL-6/GP130 signaling.

(A-D) 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.

(A) Representative pictures of the mice treated with IMQ.

(B) Erythema, scaling and thickness of the back skin was scored daily on a scale from 0 to 4, and the cumulative score (Erythema plus scaling plus thickness) is depicted.

(C) 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.

(D) 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 Mann-Whitney U test (B) or two-tailed Student's t test (C,D). C, control, n=5, IMQ, n=6. D, control, n=3, IMQ, n=6. * P＜0.05, ** P＜0.01, *** P＜0.001. NS, no significance. See also Fig EV4.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 5

Fig 5. The expression level of NEDD4L negatively correlates with the levels of GP130 and p-STAT3 in the epidermis from clinical samples.

The skin tissues from psoriasis patients were harvested and subjected to IHC staining. Normal skin sections (Normal) consisted of healthy tissue from the resection edges of cutaneous squamous cell carcinoma (SCC) that appeared to be healthy at the histological level.

(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.

(C) Correlation analysis for NEDD4L and GP130 in the normal and psoriasis groups.

(D) Correlation analysis for NEDD4L and p-STAT3 in the normal and psoriasis groups.

(E) Correlation analysis for GP130 and p-STAT3 in the normal and psoriasis groups.

Data information: Each symbol represents one patient (B-E). 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 (B) and two-tailed Pearson's χ2 test (C-E) (normal, n=15, psoriasis, n=36). * P＜0.05, ** P＜0.01, *** P＜0.001.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 6

Fig 6. NEDD4L interacts with GP130 and promotes its degradation.

(A) NEDD4L promotes GP130 degradation in a dose and enzymatic activity dependent manner. HA-GP130 was transfected with 0.5 μg, 1 μg or 2 μg of Myc-NEDD4L or 2 μg of its enzymatic activity mutant into HEK293T cells and 36 h later, MG-132 (25 μM) was added for further 12 h, and the cell lysates were harvested for WB analysis.

(B) Flag-GP130 was transfected with Myc-NEDD4L into HEK293T cells, 36 h later, cyclohexane (CHX, 50 ng/ml) was added into the cells for indicated time. The cell lysates were harvested and analyzed by WB and the gray values were measured to calculate half-life of GP130.

(C) Whole cell lysates of NHEKs were coimmunoprecipitated (co-IP) with anti-IgG or anti-GP130 antibodies, which was followed by WB detection with anti-NEDD4L and anti-GP130 antibodies.

(D) Recombinant Flag-GP130-his (Flag-GP130) proteins were mixed with HA-GFP-his (HA-GFP) or HA-NEDD4L-his (HA-NEDD4L) proteins, and then, the proteins were pulled down with ANTI-HA agarose beads and detected using anti-Flag and anti-HA antibodies.

(E) Full length Flag-NEDD4L, NEDD4L-ΔC2, NEDD4L-ΔWW or NEDD4L-ΔHECT truncation mutants were co-expressed with Myc-GP130 in 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.

(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.

Data information: A representative result from three independent experiments is shown. See also Fig EV5.
E3 ubiquitin ligase NEDD4L negatively regulates keratinocyte hyperplasia by promoting GP130 degradation (2021)
Figure 7

Fig 7. NEDD4L mediates Lys-27-linked polyubiquitination of GP130.

(A) Recombinant Flag-GP130, Myc-NEDD4L, Myc-NEDD4L-ΔC2, Myc-NEDD4L-ΔWW, Myc-NEDD4L-ΔHECT or Myc-NEDD4L-C943S, and ubiquitination-associated proteins were mixed in adenosine 5'-triphoshpate buffer and incubated at 30℃ for 2 h. Then the reaction aliquots were quenched and measured by WB with anti-Flag antibodies.

(B) Whole cell lysates of WT and NEDD4L KO NHEKs were immunoprecipitated with anti-GP130 antibody, and the immune-precipitates were analyzed by WB with anti-Ub antibodies.

(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.

(D) WB analysis of the polyubiquitination of GP130 in HEK293T cells co-transfected with Flag-GP130, Myc-NEDD4L, HA-Ub or its mutants expressing only one lysine (K-only), respectively, which followed by denaturation-IP with ANTI-FLAG M2 beads and WB analysis with anti-HA 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.

Data information: A representative result from three independent experiments is shown. See also Fig EV5.

Expanded View Figure Legends
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation (2021)
Figure 2

Figure 2. VPS41 patient and HeLa VPS41 knockout cells contain more but smaller lysosomes.

A. Immunofluorescence microscopy of control, parental and patient fibroblasts labeled for LAMP-1. Patient fibroblasts (VPS41S285P/R662* and VPS41c.1423-2A>G/R662*) show more LAMP-1 puncta, which are distributed throughout the cell. (A') Quantification shows a significant increase in the number of LAMP-1 positive compartments for both patients. >15 Cells per condition were quantified (n=3). Scale bars, 10µm.

B. HeLa VPS41 knockout (HeLaVPS41KO) cells made using CRISPR/Cas9 methodology. Western blot analysis confirms a full knockout. The same HeLaWT and HeLaVPS41KO samples were analyzed in Appendix Figure S8C, showing the same actin control.

C. LAMP-1 immunofluorescence of HeLaWT and HeLaVPS41KO cells. Similar to patient derived fibroblasts more LAMP-1 positive compartments are seen in HeLaVPS41KO cells (quantified in C'). >10 cells per cell line per experiment were quantified (n=3). Scale bars, 10µm.

D. Immuno-electron microscopy of VPS41WT/WT and VPS41S285P/R662* fibroblasts incubated for 2 hours with BSA conjugated to 5 nm gold (BSA-Au5) and labeled for LAMP-1 (10 nm gold particles). Lysosomes are recognized by the presence of degraded, electron-dense material. LE = late endosome, LY= lysosome, PM = plasma membrane. Scale bar, 200nm.

E. Morphometrical analysis showing that lysosomes in patient fibroblasts are significantly smaller than in control and parental cells. >100 Randomly selected lysosomes per condition were quantified.

F. Relative labeling density of LAMP-1. Number of LAMP-1 gold particles per lysosome was divided by the number of grid intersections, representing lysosomal size. No significant difference was found between VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* or VPS41S285P/R662* fibroblasts. >53 Lysosomes per condition were quantified.

G. Quantitation of LAMP-1 gold particles per lysosome. VPS41S285P/R662* lysosomes have significantly less LAMP-1 then control and parental cells. >53 Lysosomes per condition were quantified. Similar results were obtained for LAMP-2 (Appendix Fig S2).

Data information: Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ****P < 10-5. One-way ANOVA with Tukey's correction (A', E, F and G) or Unpaired t-test (C'). Exact p-values are reported in Appendix Table S3.
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation (2021)
Figure 4

Figure 4. Patient fibroblasts and VPS41KO cells show decreased autophagic flux and response to autophagic stimuli.

A. Western blot of LC3 expression levels in control and patient fibroblasts. In steady state (0 hours) conditions, patient fibroblasts have a higher ratio of lipidated LC3 (LC3II):LC3I than control cells. Induction of autophagy by nutrient starvation (2 hours incubation with minimal EBSS medium) results in a raise in LC3II:LC3I ratio in both control and patient cells, but in VPS41S285P/R662* fibroblasts this increase is only modest (quantified in A') (n=2).

B. Immunofluorescence of LC3 inVPS41WT/WT and VPS41S285P/R662* fibroblasts under steady state and starved conditions. At steady state conditions (0 hours), patient fibroblasts contain more LC3-positive compartments. Nutrient starvation (2 hours) increases the number of LC3-positive autophagosomes in control fibroblasts 13.4-fold, and in VPS41S285P/R662* fibroblasts only 3.8-fold, indicating a reduced responsiveness to starvation (quantified in B'). >54 Cells per cell line were quantified. Scale bars, 10µm.

C. Western blot analysis of HeLaVPS41KO cells shows a 4-fold increase in LC3II protein levels in steady state conditions (0 hours) compared to HeLaWT cells. In contrast to control cells, nutrient starvation (2 hours) did not increase LC3II protein levels in HeLaVPS41KO cells, indicating irresponsiveness to nutrient availability (quantified in C') (n=3).

D. Rescue experiments. HeLaVPS41KO cells transfected with EV-APEX2-V5, VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5 and labeled for V5 and LC3 by immunofluorescence microscopy. Rescue with VPS41WT-APEX2-V5 decreases the number of LC3-positive compartments in steady state conditions (0 hours) and restores responsiveness to nutrient starvation (2 hours) and replenishment (2 hours starvation followed by 15 minutes restimulation). Neither of the mutant VPS41 variants rescues this autophagy phenotype (quantified in D'). >15 Cells per condition were quantified (n=3). Scale bars, 10µm.

E. Immunofluorescence of HeLaWT and HeLaVPS41KO cells transfected with LC3GFP/RFP tandem construct. The increased percentage of GFP/RFP-positive compartments in HeLaVPS41KO cells indicate a block in autophagic flux (quantified in E'). >12 Cells per cell line were quantified. Scale bars 10µm; zoom, 1µm.

Data information: Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 10-4, ****P < 10-5. Unpaired t-test (A' and E') or one-way ANOVA with Tukey's (B') or Bonferroni correction (C' and D'). Exact p-values are reported in Appendix Table S3.
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation (2021)
Figure 5

Figure 5. Patient fibroblasts and VPS41KO cells exhibit mTORC1 inhibition towards TFE3.

A. Immunofluorescence of control, parental and patient fibroblasts labeled for LAMP-1 and mTOR. In steady state conditions (0 hours) VPS41S285P/R662* fibroblasts show less colocalization between mTOR and LAMP-1. VPS41WT/WT, VPS41WT/S285P and VPS41WT/R662* show an appropriate mTOR response upon nutrient deprivation (2 hours) or restimulation (2 hours starvation followed by 15 minutes restimulation) (Appendix Fig S5A). >10 Cells per cell line were quantified (A'). Quantifications are performed relative to colocalization under steady state conditions per cell line (n=3). Scale bars, 10µm; zoom, 1µm.

B. Immunofluorescence of VPS41WT/WT, VPS41WT/S285P, VPS41WT/R662* and VPS41S285P/R662* fibroblasts labeled for TFE3. In VPS41S285P/R662* fibroblasts, TFE3 is constitutively localized in the nucleus regardless of nutrient state (quantified in B'). >25 Cells per condition were quantified (n=3). Scale bars, 10µm.

C. TFE3 immunofluorescence in HeLaWT and HeLaVPS41KO cells. In HeLaVPS41KO cells TFE3 constitutively localizes in the nucleus (quantified in C') >83 Cells per condition were quantified (n=3). Scale bars, 10µm.

D. Rescue experiments of HeLaVPS41KO cells. Expression of VPS41WT-APEX2-V5, VPS41S285P-APEX2-V5 or VPS41R662*-APEX2-V5. Reintroduction of VPS41WT rescues TFE3 localization, whereas expression of mutant VPS41 has no effect (quantified in D'). >83 Cells per condition were quantified (n=3). Scale bars, 10µm.

E. Western blot of phosphorylated mTORC1 substrates S6K and 4EBP1 after starvation (2 hours) and restimulation (10, 30 or 60 minutes). HeLaVPS41KO cells show comparable levels of phospho-S6K and phospho-4EBP1, with no difference in recovery after restimulation (quantified in E') (n=3).

Data information: Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 10-4, ****P < 10-5. One-way ANOVA with Bonferroni correction (A' - D') or unpaired t-test (E'). Exact p-values are reported in Appendix Table S3.
Neurodegenerative VPS41 variants inhibit HOPS function and mTORC1-dependent TFEB/TFE3 regulation (2021)
Figure 6

Figure 6. VPS41S285P rescues regulated secretion in PC12 VPS41KO cells.

A. Western blot of pulldown of AP3 incubated with HIS-6 VPS41WT or VPS41S285P. There is no difference in affinity between VPS41WT and VPS41S285P (See Appendix Fig S10 for MBP-AP3 pulldown input).

B. Western blot on cellular or secreted secretogranin II (SgII) of PC12 cells VPS41KO, or VPS41KO transduced with HA-VPS41WT or HA-VPS41S285P lentivirus. Cells were washed and incubated for 30 minutes in Tyrode's solution containing 2.5 mM K+ (basal) or 90 mM K+ (stimulated). No difference in secreted sgII levels was observed between VPS41WT and VPS41S285P.

C, D. Cellular (C) and secreted (D) secretogranin II (SgII) were measured by quantitative fluorescence immunoblotting (n=3). VPS41S285P rescues intracellular SgII levels and regulated secretion of SgII to the same extent as VPS41WT.

Data information: Data are represented as mean ± SEM. *P < 0.05, **P < 0.01, ***P < 10-4. One-way ANOVA (C and D). Exact p-values are reported in Appendix Table S3.