EMBO/soda-nel huggingface dataset:
Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis (2020)
Figure 5

Figure 5. Paladin is induced by VEGF-A and regulates Erk phosphorylation in pathological angiogenesis.

(a) Eyes from Pald1+/LacZ mice collected at P17 from untreated animals or animals with oxygen-induced retinopathy (OIR). Pald1-promoter driven LacZ expression and X-gal staining generated signals in capillaries, veins and arteries in the OIR retina at P17 compared to the normoxia control with predominantly arterial LacZ expression. Scale bar: 1 mm.

(b) Paladin levels in primary human umbilical vein endothelial cells (HUVEC) untreated or treated for 24 h with VEGF-A (50 ng/ml), FGF2 (50 ng/ml), or SDF1α (30 ng/ml), quantified by immunoblotting for Paladin and β-actin (loading control). Mean±SEM, one way-ANOVA, n=5 biological replicates.

(c) Eyes from adult Pald1LacZ/+ mice collected at 72 h after single-bolus intravitreal injection of 1 μg VEGF-A, and PBS in the contralateral eye, followed by X-gal staining. Arrow indicates Pald1 promoter activity in veins (outlined by dashed line) specifically after VEGF-A treatment. A, artery; V, vein. Scale bar: 100 μm.

(d-f) Representative images of isolectinB4-stained P17 retinas from OIR-challenged Pald1+/+ and Pald1-/- mice (detailed view in the insets) (d). Scale bar: 1 mm (inset 250 µm). Quantification of neovascular tuft area (e) and avascular area (f). Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 4 Pald1+/+ and 3 Pald1-/- pups.

(g) Representative images of retina vasculature immunostained for isolectinB4 and pT202/pY204 Erk1/2 (pErk1/2). pErk1/2 staining within the vessels is also visualized using a 16-color heatmap to display staining intensity. Scale bar: 30 µm (inset 10µm).

(h) Quantification of pErk1/2 immunostaining as shown in (g) within isolectinB4-positive vessels, as fold-change of pErk1/2 stained area. Each dot represents the mean of both retinas per mouse. Mean±SEM, unpaired t-test, n=3 litters, 5 Pald1+/+ and 5 Pald1-/- pups.

Data information: *p < 0.05, **p < 0.01.

FIGURES LEGENDS Expanded View 1-5
Functional screen reveals essential roles of miR-27a/24 in differentiation of embryonic stem cells (2014)
Figure 1

Figure 1 Screening of target miRNAs silencing ESC self-renewal.

Screening strategy. miRNAs potentially targeting important ESC pluripotency factors meanwhile enriched in MEFs or up-regulated during EB differentiation were selected as candidate miRNAs. Candidate miRNAs silencing ESC self-renewal were identified by the function analyses in Dgcr8-/- ESCs.

Distribution of candidate miRNAs. There were 44 miRNAs enriched in MEFs (red region), 53 miRNAs up-regulated during EB differentiation (blue region) and 454 miRNAs with binding sites to the 3′ UTR of pluripotency factors (yellow region). The gray region represents 52 selected candidate miRNAs in silencing self-renewal.

List of candidate miRNAs. Left panel showed candidate miRNAs enriched in MEFs comparing with ESCs. Right panel showed candidate miRNAs up-regulated during EB differentiation. Gray highlighted the known miRNAs suppressing ESC self-renewal. Red marked the same miRNAs existed in both of the two panels. XY and XX indicated ESCs derived from male or female mouse respectively. NA meant D5/D0 was extremely high as the expression value in D0 was zero.

Colony formation assay of miRNA mimic transfected-Dgcr8-/- ESCs. There were 17 miRNAs that decreased the colony forming ability, 15 miRNAs that did not affect colony formation and 8 miRNAs enhanced colony formation of ESCs. Representative pictures are shown. Full data are shown in Supplementary Fig S1.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 1

Figure 1. FLCN is required for starvation dependent peri-nuclear clustering of lysosomes.

(a) Widefield immunofluorescence images of PFA fixed HeLa cells showing endogenous FLCN and LAMP1 staining in (top) normal growth or (bottom) starvation conditions. Orange arrows highlight association of FLCN with LAMP1 positive membranes in the peri-nuclear region. White dotted line shows cell periphery in starved condition. Scale bar = 10μm.

(b) (left) Quantification of FLCN/LAMP1 association in growth and starvation conditions. A cell is scored as positive if 5 or more discrete FLCN/LAMP1 puncta were observed at 100x magnification using a widefield microscope. Data represents 60 cells from 3 independent experiments, error bars show S.E.M, *** = p<0.001. (right) Western blot shows levels of phosphorylated of S6K and 4EBP in whole cell extracts under the same conditions.

(c) Western blot showing typical efficiency of FLCN knockdown in HeLa cells using a pool consisting of 4 oligonucleotides and 2 independent oligonucleotides from the pool. Graph shows relative FLCN expression (n=3, error bars show S.E.M.)

(d) Representative confocal fluorescence images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of FLCN or control cells transfected with a non-targeting siRNA. The location of the Golgi (green) is highlighted by giantin staining.

(e) Schematic illustrating application of the cumulative intensity distribution method for quantification of lysosome distribution.

(f) Graph showing cumulative distribution of LAMP1 intensity in normal growth and starvation conditions in HeLa cells. P-value is determined by the extra sum of F-squares test following non-linear regression and curve fitting. Error bars show S.E.M. from 30 cells in 3 replicates.

(g) Graph showing cumulative distribution of LAMP1 intensity in starvation conditions for control cells transfected with non-targeting siRNA or cells depleted of FLCN. Error bars show S.E.M. from 30 cells in 3 replicates.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 2

Figure 2. Distribution and dynamics of FLCN-GFP in HeLa cells.

(a) Representative spinning disk confocal image showing FLCN-GFP and lysosomes (Lysotracker-Red) in a live FLCN-GFP/HA-FNIP2 transfected HeLa cell (from Movie EV1). Top panels show the first frame of a 180 frame (1fps) image series. Bottom panels show a maximum intensity projection of the image series to highlight mobile and immobile components. Boxed and magnified regions show typical peri-nuclear (i) and peripheral (ii) regions in the cell as well as a FLCN-GFP tubule (iii) Scale bar = 5μm.

(b) Spinning disk confocal image series of a region live FLCN-GFP/HA-FNIP2 transfected HeLa cell highlighting dynamic FLCN-GFP tubules (Movie EV2). First merge panel shows FLCN-GFP and lysotracker-Red at T=0 Subsequent panels show only FLCN-GFP at 30 second intervals.

c) Confocal image of a region of a methanol fixed FLCN-GFP/HA-FNIP2 transfected HeLa cell showing Rab7 (red) and LAMP1 (blue) association with FLCN-GFP tubules. Scale bar = 1μm.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 3

Figure 3. FLCN supports Rab34 dependent peri-nuclear lysosome clustering.

(a) Graphs showing cumulative distribution of LAMP1 intensity in control GFP (black), GFP-Rab34 (green) or GFP-Rab7 (blue) transfected cells. Note black line and symbols partially obscure blue.

(b) GST-RILP pull-down analysis from HeLa cells in normal and starved conditions showing an enhanced capacity of endogenous Rab34 to bind RILP in starvation conditions. Quantification is mean of 3 independent experiments. Error is S.E.M.

(c) Representative confocal fluorescence images of FLCN depleted or control cells, transfected with GFP-Rab34. Scale bar = 10μm.

(d) (left) Graphs showing mean GFP-Rab34 expression (grey values) and cumulative distribution of LAMP1 intensity (right) in FLCN depleted or control siRNA cells transfected HeLa cells expressing GFP-Rab34. Error bars show S.E.M.. from 30 cells in 3 replicates. Scale bar = 10μm.

(e) Western blot and graph showing typical efficiency of Rab34 siRNA knockdown after 48 hours in HeLa cells. Error bars show S.E.M from 3 experiments, *** = p<0.001.

(f) Representative confocal fluorescence images showing LAMP1 (magenta) distribution in HeLa cells under normal growth or starvation conditions in cells depleted of Rab34 or control cells transfected with a non-targeting siRNA. Giantin is shown in green. Scale bar = 10μm.

(g) Graphs showing cumulative distribution of LAMP1 intensity in normal growth or starvation conditions for control cells or cells depleted of Rab34 using siRNA.

(h) Graph showing percentage of LAMP1 in cis-medial Golgi-region (giantin overlap) in the indicated conditions. Error bars show S.E.M from 30 cells in 3 replicates, *** = p<0.001.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 4

Figure 4. Peri-nuclear Rab34 positive membranes contact lysosomes

(a) (left) Representative confocal fluorescence image showing methanol fixed HeLa cell transfected with GFP-Rab34 and stained for LAMP1. Linescan analysis shows relative intensity of GFP and LAMP1 signals. Scale bar = 10μm.

(b) Maximum intensity projection images from a confocal Z-stack showing LAMP1 and endogenous Rab34 in P-M fixed HeLa cells from normal and starved conditions. Whole cells and fields from which these panels are derived are shown in Figure EV4A. Boxes highlight regions shown in Figure 4C. Scale bar = 2μm.

(c) Single confocal sections from the Z-stack in figure 4B. Orange arrows show close LAMP1/Rab34 association.

(d) Analysis of Lysotracker-Red dynamics using PCC decay in HeLa cells transfected with either GFP or GFP-Rab34. Data is from 10 cells per condition. Error bars show S.E.M.

(e) Live–cell spinning disk confocal image series at 10 sec intervals (from Movie EV3) showing GFP-Rab34 positive compartment making dynamic contacts with lysosomes. Scale bar = 1μm

(f) Live–cell spinning disk confocal image series at 10 sec intervals (from Movie EV4) of FLCN-GFP and mCherry-Rab34 in the peri-nuclear region of a FLCN-GFP/HA-FNIP2/mCherry-Rab34 transfected HeLa cell. Blue arrow highlights FLCN-Rab34 association moving on a linear trajectory. Yellow arrow highlights shorter saltatory movements. White arrow highlights dynamic associations between distinct FLCN-GFP and Rab34 positive structures. Scale bar = 1μm

(g) Confocal image showing localisation of endogenous FLCN and Rab34 in starved HeLa cells.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 5

Figure 5. High-resolution image analysis of Rab34-lysosome contacts

Images from fixed GFP-Rab34 transfected cells showing contacts between GFP-Rab34 positive peri-nuclear membranes and lysosomes.

(a) Spinning disk confocal microscopy images acquired at 160x magnification. Left panels show a maximum intensity projection imaged of a Z-stack, sampled according to the Nyquist criterion and deconvolved. Right panels shows a 3D volume rendering of the same data.

(b) A projection of 4 planes from a structured illumination microscopy super-resolution Z-stack of the same cells.

(c) A thin section electron microscopy image of the peri-nuclear region of a GFP-Rab34 transfected HeLa cell. * indicates lysosomes.

(d) A cryo-immuno-EM image of a GFP-Rab34 transfected HeLa cell. GFP-Rab34 (dark black dots) is identified by immunostaining for GFP and detection with gold conjugated protein A. The lysosome is intimately associated with Rab34 positive membranes. Sites of contact are highlighted by arrows.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 6

Figure 6. FLCN directly promotes the association of Rab34 and RILP

(a) Confocal Immunofluorescence images of HeLa cells transfected with Rab34-dsRED-Mito, FLCN-GFP and HA-FNIP2. White arrows highlight FLCN-GFP/HA-FNIP2 co-localisation distinct from Rab34-mitochondria/FLCN-GFP localisation. Scale bar = 10μm.

(b) Confocal immunofluorescence images of Rab34-dsRED-Mito (WT) and FLCN-GFP DENN only or ΔDENN transfected HeLa cells showing DENN domain dependent recruitment to mitochondria. Scale bar = 10μm.

(c) Western blot analysis of GST-RILP pull down showing association of endogenous FLCN, Rab34 and Rab7 with RILP. Input lane shows 1% input cell extract.

(d) Western blot analysis of GST-RILP pull down from UOK257 cells showing DENN domain dependent association of FLCN with RILP N-terminal, Rab binding domain (RBD) and C-terminal fragments.

(e) Western blot showing binding of His-tagged FLCN-DENN domain in E.coli extract to GST-RILP.

(f) Western blot analysis of GST-RILP pull down showing direct interaction between RILP and GTP hydrolysis deficient (Q111L) Rab34 (at 200nM, in the presence of 1mM GTPγS) and FLCN-DENN domain (1μM), but not low nucleotide affinity (T66N) Rab34. FLCN-DENN enhances association of RILP with Q111L Rab34. Graph shows quantification of relative RILP / Rab34 Q111L binding. Error bars show SEM. ** = p<0.01.

(g) Western blot analysis of GST-RILP / Rab34 Q111L pull down (Rab34 at 200nM) showing that enhanced association of with RILP is achieved with low concentrations of FLCN.

(h) Western blot analysis of GST-RILP pull down experiments with GTPγS loaded (in the presence of 1mM GTPγS) and GDP loaded (in the presence of 1mM GDP) wildtype Rab34, showing FLCN-DENN (500nM) induced interaction with RILP.

(i) Western blot analysis of a GFP-Trap immunoprecipitation experiment from HeLa cells showing that expression of HA-RILP promotes the association of GFP-Rab34 and HA-Rab7.
Folliculin directs the formation of a Rab34-RILP complex to control the nutrient dependent dynamic distribution of lysosomes (2016)
Figure 7

Figure 7. Folliculin controls lysosome distribution and dynamics in BHD kidney cancer cells.

(a) Western blot showing endogenous Rab7 and Rab34 expression in UOK257 and UOK257-2 cell extracts and relative levels of the active forms as measured by GST-RILP pull down. HSC70 is used as a loading control. Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments, error bars show S.E.M., * = p<0.05.

(b) (top) Representative confocal immunofluorescence images showing intracellular distribution of LAMP1 in UOK257 and UOK257-2 cells. Dashed line highlights cell periphery in UOK257-2 cells. Scale bar = 10μm. (bottom left) Graph showing cumulative distribution of LAMP1 intensity in UOK257 vs UOK257-2 cells. Error bars show S.E.M. from 30 cells in 3 replicates. (bottom right) Graph showing Lysotracker-Red PCC decay with time from confocal movies of UOK257 and UOK257-2 cells. Mean values from 5 cells. Error bars show S.E.M.

(c) Widefield immunofluorescence images showing early endosomes (EEA1) and Golgi (golgin-97) in UOK257 and UOK257-2 cells. Scale bar = 10μm.

(d) Western blot showing FLCN and FLCNΔDENN expression in stable UOK257 derived cell lines.

(e) Representative widefield fluorescence images showing intracellular distribution of LAMP1 in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Scale bar = 10μm. Graphs showing cumulative distribution of LAMP1 intensity in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells. Error bars show S.E.M from 30 cells in 3 replicates.

(f) (left) Western blot showing endogenous Rab7 and Rab34 expression in UOK257, UOK257-FLCN and UOK257-FLCNΔDENN cells and relative levels of activity as measured by GST-RILP pull down. HSC70 is used as a loading control. (right) Graphs show quantification of bound Rab7/Rab34 from 3 independent experiments (p<0.05, *** = p<0.001).

(g) Western blot showing endogenous RILP expression in the indicated cell lines. Graph shows quantification of RILP expression from 3 independent experiments (* = p<0.05, *** = p<0.001).
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 1

Figure 1. Deposition of α-amylase-resistant polyglucosan deposits in the hind brain and heart of HOIL-1[C458S] mice.

(A-F) Representative PAS-stained sections of the pons (A) and hippocampal (C) regions of the brain and heart (E) of one year old HOIL-1[C458S] and WT mice are shown. Scale bar = 100 μm. Arrow heads indicate α-amylase-resistant PAS-positive polyglucosan deposits. Graphs showing α-amylase resistant PAS scores of the pons (B) and hippocampal (D) regions of the brain and the heart (F) of HOIL-1[C458S] (red) and WT (blue) mice aged 0.5, 1.0 and 1.5 years. The number of biological replicates analysed at each age is indicated. The word zero highlighted in blue indicates that no α-amylase resistant, PAS-positive material could be detected in the WT mice. The error bars show mean + SEM. Statistical significance between the genotypes was calculated by using two-way ANOVA and Šidák's multiple comparison's test. **** denotes p<0.0001.
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 2

Figure 2. HOIL-1 ubiquitylates oligo- and polysaccharides in vitro.

(A) HOIL-1 activity towards purified bovine liver glycogen was measured using Cy5-labelled ubiquitin. Control assays lacking ATP, UBE2L3, HOIL-1 and glycogen are indicated, as are subsequent treatments with 50 μg/mL human salivary α‑amylase and 1.5 M hydroxylamine (NH2OH). The gel has been stained using the periodic acid-Schiff (PAS) method to visualise glycogen (purple-magenta) and Coomassie Blue stain to visualise protein.

(B) Prior to PAS and Coomassie staining, the gel shown in (A) was stained with Flamingo fluorescent protein stain (shown in red) and glycogen ubiquitylation observed by visualising the fluorescent Cy5-ubiquitin signal (shown in green).

(C) Chemical structure of the α-1,4-linked heptasaccharide maltoheptaose.

(D) In vitro ubiquitylation of maltoheptaose by HOIL-1. Assays were treated with α‑amylase or hydroxylamine as indicated. Reaction products were detected by Coomassie staining.

(E) Ubiquitylated maltoheptaose was purified from reaction components and analysed by MALDI-TOF, revealing an additional 1135 Da attached to the ubiquitin. This is consistent with addition of maltoheptaose to ubiquitin via an oxyester linkage (expected additional mass is 1153 for maltoheptaose minus 18 to account for the loss of a water molecule during ester bond formation).
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 3

Figure 3. NMR spectroscopy identifies the hydroxyl groups ubiquitylated by HOIL-1 in maltoheptaose and maltose.

(A) Glycine region from the 1H-15N HSQC spectrum of [15N,13C] Ubiquitin-maltoheptaose (blue) overlayed with the same region for [15N,13C] Ubiquitin (red). Visible amide assignments are labelled. Arrows indicate the two major chemical shift changes for Gly75 and Gly76 due to conjugation of maltoheptaose to the C-terminus.

(B) 1D 1H NMR overlay for the anomeric region of maltoheptaose (red) and [15N,13C] Ub-maltoheptaose (blue) with the 1H-13C HSQC of the latter shown above. Key anomeric assignments are labelled, and arrows indicate the chemical shift perturbations upon conjugation. The full aliphatic region of the 1D 1H NMR spectra is shown in Fig. EV3A.

(C) 1H-13C HMBC NMR spectrum (left) showing a 3-bond connectivity from the C-terminal Gly76 carbonyl 13C to the 6-CH2 proton chemical shifts. Extracted 2D 15N plane (107.9 ppm) from the 3D HN(CA)CO spectrum showing the 1HN amide to 13C carbonyl correlation for Gly76 of ubiquitin.

(D) Overlay of the 1H-13C HSQC spectra for free ubiquitin (red) and ubiquitylated-maltose (blue). Assignments for ubiquitin CHα proton are labelled together with the assignments for the ligated and free 6-CH2 positions of the disaccharide labelled. Asterisk indicates a buffer component. The full aliphatic region of the 2D 1H-13C HSQC spectra is shown in Fig. EV3B.

(E) Chemical structure of cyclomaltoheptaose (β-cyclodextrin).

(F) HOIL-1 activity against the indicated maltosaccharides (2 mM) was assayed and visualised by staining with Coomassie protein stain. Where indicated, assays were treated with 1.5 M hydroxylamine. Met1-linked ubiquitin tetramer was also included in these assays.

(G) Chemical structure of 6‑mercapto-cyclomaltoheptaose (heptakis-(6‑deoxy-6‑mercapto)-β‑cyclodextrin).

(H) In vitro ubiquitylation of the indicated cyclodextrins (2 mM) by HOIL-1, visualised by Coomassie staining. Met1-linked ubiquitin tetramer was also included in these assays.
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 4

Figure 4. HOIP and Sharpin bind starch-like glucans in vitro.

(A) 1 μg of each of the indicated recombinant proteins was incubated with amylose resin for 60 min, the beads were pelleted by centrifugation, and proteins in the pellet (P) and supernatant (S) were compared to the protein input (I) by means of western blotting with the appropriate antibodies.

(B) Bovine liver glycogen and potato starch-derived amylopectin were incubated for 60 min with the indicated recombinant proteins, centrifuged for 90 min at 100,000 × g, and protein in the resulting supernatants (S) and pellets (P) analysed by western blotting with the appropriate antibodies. Protein without polysaccharide was used as a negative control (Buffer).

(C) Amylose binding assays of full-length (1-1072) and truncated HOIP protein, visualised by immunoblotting. Shown on the right are schematic representations of the truncated HOIP constructs used. Images not drawn to scale. PUB, peptide N-glycanase and UBA- and UBX-containing protein domain; ZF, zinc finger domain; NZF, Npl4 zinc finger domain; UBA, ubiquitin-associated domain; RBR, RING-in-between-RING domain; LDD, linear ubiquitin chain determining domain.

(D) Amylose binding assays of full-length (1-387) and truncated Sharpin mutants along with schematic representations of the Sharpin constructs used. Images not shown to scale. PH, pleckstrin homology domain; LTM, LUBAC-tethering motif; UBL, ubiquitin-like domain.

(E) Diagrams of amylose, amylopectin, and glycogen, indicating differences in polymer chain-length and degree of branching.
HOIL-1 ubiquitin ligase activity targets unbranched glucosaccharides and is required to prevent polyglucosan accumulation (2022)
Figure 5

Figure 5. HOIL-1 is allosterically activated by Met1- and Lys63-linked ubiquitin chains. (A) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin dimers for the indicated times and visualised by Coomassie staining.

(B) Maltoheptaose ubiquitylation by HOIL-1 was assayed at 30°C in the presence of Met1- or Lys63-linked ubiquitin tetramers for the indicated times and visualised by Coomassie staining.

(C) HOIL-1[T203A/F204A/R210A] was assayed at 30°C in the absence or presence of the indicated ubiquitin dimers and tetramers. These mutations have been reported to impair the binding of ubiquitin to the NZF domain (Sato et al., 2011; Gomez-Diaz et al., 2021) and their approximate location in HOIL-1 is indicated (schematic not to scale).

(D) Assay of HOIL-1[Δ194-222] lacking the NZF core domain at 30°C in the absence or presence of the indicated ubiquitin oligomers.

(E) Allosteric activation of HOIL-1[233-510] (lacking the LTM, UBL and NZF domains at the N‑terminus) by ubiquitin oligomers.