EMBO/soda-nel huggingface dataset:
The antiobesity factor WDTC1 mediates adipogenic suppression via the CRL4WDTC1 E3 ligase (2016)
Figure 1

Figure 1. WDTC1 is a substrate receptor of CRL4 E3 complexes.

A. Domain structure of human WDTC1 with locations of the H-box and WDXR motifs indicated. Alignment of WDTC1 H-box motifs in different species (boxed in grey) and bolded orange represents key residues contacting DDB1 [13]. Alignment of DWD boxes in different DWD proteins (bottom right) with WDXR submotif indicated; tandem DWD boxes are shown for WDTC1 only.

B. Endogenous CUL4A and CUL4B complexes were isolated from 293T cell lysates by immunoprecipitation (IP) and associated proteins were detected by immunoblotting (IB). Mock includes beads only control.

C. Flag-tagged WT and mutant WDTC1 proteins were transiently expressed in 293T cells. Flag-WDTC1 complexes were immunoprecipitated with anti-FLAG and their associated proteins were detected by immunoblotting as indicated; EV, empty vector control.

D. Top, the modeled structure of WDTC1 in complex with CRL4 (PDB 4A0K); WDTC1 superimposition was based on its H-box (PDB 317N). WDTC1 domains are rendered in same colors as linear domain structure in A; WDXR arginines were rendered in dark blue ball-and-sticks. Bottom, schematic summarizing results presented in C.

E. 293T cells were transfected with HA-ubiquitin along with various combinations of plasmid and siRNA as indicated. Transfected cells were lysed under denaturing conditions to obtain whole cell extracts (WCE) and immunoprecipitated with anti-FLAG. WDTC1 ubiquitylation was evaluated by immunoblotting as indicated.

F. 293T cells transiently expressing Flag-WDTC1 were transfected with scramble (scrm) siRNA or siRNAs against DDB1, CUL4A and CUL4B. Flag-WDTC1 levels and knockdown efficiency were assessed by immunoblotting as indicated.
The antiobesity factor WDTC1 mediates adipogenic suppression via the CRL4WDTC1 E3 ligase (2016)
Figure 2

Figure 2. WDTC1 suppresses adipogenesis in a CRL4-dependent manner.

A. 3T3-L1 preadipocytes were adipogenically induced and lysates were prepared from cells collected at indicated days. Endogenous WDTC1 interaction with CRL4 subunits was evaluated by immunoprecipitation with anti-WDTC1 followed by immunoblot analyses with indicated antibodies. Fatty acid synthase (FAS) levels were monitored to evaluate induction.

B-D. 3T3-L1 stable cells expressing vector control (EV), Flag-tagged WT or mutant WDTC1 proteins were adipogenically induced. Adipogenesis was assessed by Oil Red O staining (ORO) (B); plate (top) and microscopic (bottom) views, 0.1 mm scale bar. Triglyceride levels were quantified by an enzymatic assay (C). Adipogenic gene expression was evaluated by RT-qPCR analysis (D).

Data information: in C-D, error bars represent the mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.005, significant change compared to EV control. Statistical significance was analyzed by paired two-tailed Student’s t-tests.
The antiobesity factor WDTC1 mediates adipogenic suppression via the CRL4WDTC1 E3 ligase (2016)
Figure 3

Figure 3. CRL4 binding mutants of WDTC1 cannot rescue WDTC1 knockdown phenotype.

A. Schematic of WDTC1 knockdown-rescue lentiviral vector (top). 3T3-L1 preadipocytes were lentivirally transduced to generate stable cells expressing a non-specific (NS) shRNA control, shWDTC1 targeting endogenous Wdtc1 mRNA, or cells simultaneously expressing shWDTC1 and shRNA-resistant WDTC1; bottom, confirmation of knockdown and WDTC1 ectopic expression.

B-D. 3T3-L1 stable cells described in (A) were adipogenically induced. Their adipogenic potential was assessed by ORO (B), triglyceride quantitation (C) and adipogenic gene expression (D).

Data information: in C-D, error bars represent the mean ± SEM from 3 independent experiments. *P < 0.05, **P < 0.005, significant change compared to NS control. Statistical significance was analyzed by paired two-tailed Student’s t-tests.
The antiobesity factor WDTC1 mediates adipogenic suppression via the CRL4WDTC1 E3 ligase (2016)
Figure 4

Figure 4. CRL4WDTC1 E3 ligase promotes H2AK119ub ubiquitylation.

A. Induced 3T3-L1 cells stably expressing Flag-WDTC1 were subjected to subcellular fractionation. Aliquots of total cell extract (TCE) and subcellular fractions were analyzed by immunoblot as indicated; fraction purity was assessed by α-Tubulin and histone H3 antibodies.

B. Chromatin enriched nuclear extracts from 293T cells cotransfected with Flag-WDTC1 and HA tagged core histones were immunoprecipitated (IP) with anti-FLAG and immunoblotted (IB) as indicated. The probable H2AK119ub1 band detected in the nuclear extract is indicated by an asterisk.

C. Soluble chromatin extracts were prepared from preadipocyte or induced 3T3-L1 cells stably expressing control (EV) and Flag-WDTC1 proteins. H2A monoubiquitylation was detected by H2AK119ub1-specific antibody and total H2A served as loading control. Relative intensity represents ratio of H2AK119ub1 over total H2A signal normalized to EV control.

D. Affinity purified Flag-WDTC1 wildtype and mutant ΔH complexes from 293T cells were the source of E3 ligase in in vitro ubiquitylation assays. Copurification of CRL4 E3 ligase proteins was confirmed by immunoblotting and Coomassie Blue staining (left panel). In vitro ubiquitylation assays of recombinant H2A were performed in the presence of indicated proteins. Reaction products were resolved by SDS-PAGE, and ubiquitylated H2A was detected by anti-H2AK119ub1 (right panel).

E. In vitro ubiquitylation assays were performed with purified wild-type and mutant histone proteins as substrates. H2A ubiquitylation was assessed by immunoblotting with anti-ubiquitin and various antibodies as indicated.

Data information: in C, error bars represent the mean ± SD from 2 (preadipocyte) or 3 (induced) independent experiments. *P < 0.05, significant change compared to EV control. Statistical significance (induced only) was analyzed by paired two-tailed Student’s t-tests.
Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 1

Figure 1. Phagolysosomal dysfunction in the RPE of miR-211-/- mice.

(A) Confocal images of representative eye cryosections immunostained with anti-Lamp1 antibody from 2-month-old miR-211-/- and control mice sacrificed 3h after light on, at 10 AM (diurnal condition). Enlarged boxes highlight Lamp1-positive structures (white arrowheads) in the RPE. Autofluorescence from lipofuscin granules are visible in all cryosections of miR-211-/- compared to control littermates. Nuclei were counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 10 μm.

(B) RPE were isolated at 10 AM (diurnal condition) from 2-month-old WT and miR-211-/- mice. Representative Western blot analysis of the Lamp1, Cln5, Trpml1, Sqstm1/p62, Cathepsin D (CtsD) and LC3 proteins from WT and miR-211-/- mice. Note a decrease of both proCtsD and its maturation CtsD heavy chain (hc). The plots show the quantification of the indicated proteins normalized to the Gapdh loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05, **p ≤ 0.01.

(C) Cathepsin B (CtsB) activity in RPE lysates from 2-month-old miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). CtsB was reduced in miR-211-/- compared to control mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (miR-211-/- vs WT), *p ≤ 0.05.

(D) Representative images of conventional TEM analysis of RPE of both 2-month-old and 3-month-old WT and miR-211-/- mice. miR-211-/- mice show accumulation of phagolysosomes (red arrows) in the RPE. Scale bar 2 μm. Enlarged boxes highlight phagolysosome-like structures containing poorly processed POS. Scale bar 1.5 μm. Representative images of conventional TEM analysis of RPE shown at higher magnification also highlighted accumulation of lipofuscin-like granules (blue arrows) in 3-month-old miR-211-/- compared to control WT mice. Melanosomes are indicated by white asterisks. Scale bar 500 nm (n=3 mice).

(E) Graphs showing number of lipofuscin-like granules (n/100 μm2) from the RPE of 3-month-old miR-211-/- mice as in (D). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- vs WT) ***p ≤ 0.005 (n=6 mice).

Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.
Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 3

Figure 3. Pharmacological inhibition of Ezrin results in autophagy induction in ARPE-19 cells.

(A) Representative images from ARPE-19 cells treated with DMSO and NSC668394. All cells were fixed and stained with anti-LC3 (green) and anti-LAMP1 (red) antibodies and nuclei were counterstained with DAPI (blue). Scale bar 5 μm.

(B) The plot shows the quantification of the numbers of LC3/LAMP1-positive vesicles per cell (n ≥ 100). Values represent means ± s.e.m from 3 independent experiments. Student's t-test (NSC668394 vs DMSO) ***p ≤ 0.005.

(C) Representative Western blots of LAMP1, EZRIN, EZRIN-pT567, CLN5, TRPML1, SQSTM1/p62, CTSD and LC3 proteins from DMSO or NSC668394 treated cells. Note an increase of both proCTSD and its maturation CTSD heavy chain (hc). The plot shows the quantification of the indicated proteins normalized to the GAPDH loading control. Bar graphs represent mean values ± s.e.m. of at least n = 6 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05, ***p ≤ 0.005.

(D) Representative images of RFP-GFP-LC3 assay in ARPE-19 cells transiently transfected with RFP-GFP-LC3 and treated with DMSO or NSC668394. Nuclei were counterstained with DAPI (blue). Scale bars: 5 μm.

(E) Box plots showing quantitative analysis of RFP+GFP+ puncta (Autophagosome) and RFP+GFP- puncta (Autolysosome) (n ≥ 100 cells) from 3 independent experiments. Box limits represent 25th percentile and 75th percentile; horizontal lines represent medians; whiskers display min. to max. values. Student's t-test (NSC668394 vs DMSO) *p ≤ 0.05.

(F) Endolysosomal Cathepsin activity was revealed by incubation with cathepsin B (CTSB) Magic Red substrate for 20 min and images of the living cell collected on the HC analysis. Scale bar 10 μm. (G) Graph showing the mean values of intensity of cresyl violet fluorescence ± s.e.m. of representative data (n ≥ 100 cells) from 3 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.

(H) Cathepsin B (CTSB) activity in lysates from NSC668394-treated and DMSO-control ARPE-19 cells. Graph showing the percentage of values ± s.e.m. of CTSB activity from 4 independent experiments. Mann and Whitney test (NSC668394 vs DMSO) *p ≤ 0.05.
Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 4

Figure 4. Ezrin inhibition results in TFEB nuclear translocation.

(A) Representative images from HC assay of HeLaTFEB-GFP cells transfected with control (siCTRL) or siPPP3CB, and subjected to the indicated conditions. Analysis of TFEB nuclear translocation in HeLaTFEB-GFP cells transfected with siCTRL or siPPP3CB and treated with DMSO or NSC668394, silenced for EZRIN (siEZR) or serum-starved (stv). Both pharmacological inhibition and silencing of Ezrin induced TFEB nuclear localization in stable HeLaTFEB-GFP cells. Starvation (stv) was used as control. Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is rescue after silencing of PPP3CB. Scale bar 5 μm.

(B) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared to control; n=3 independent experiments were performed. Mann and Whitney test (NSC668394 and stv vs DMSO; siEZR vs siCTRL) *p ≤ 0.05.

(C) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells and subjected to the silencing of PPP3CB. At least 3 independent experiments were performed. Mann and Whitney test (siPPP3CB vs siCTRL) *p ≤ 0.05.

(D) qRT-PCR assay for PPP3CB from HeLaTFEB-GFP cells transfected with siPPP3CB and siCTRL. The graph shows the reduction in PPP3CB expression level normalized to the Hprt. Bar graphs represent mean values ± s.e.m. Mann and Whitney test (siPPP3CB vs siCTRL), ***p ≤ 0.005 (n=6 independent experiments).

(E) Both pharmacological inhibition and silencing of EZRIN induce downshift of endogenous TFEB electrophoretic mobility in Western blot analysis. Torin treatment was used as control.

(F) Representative traces of ML1-GCaMP3 normalized fluorescence recorded in transiently transfected ARPE-19 cells. During time-lapse recording, cells were stimulated with ML-SA1 (20 µM) after addition (arrowhead) of DMSO or NSC668394. Where indicated, the specific ML1 inhibitor ML-SI3 (10 µM) was added. Bar graph reports the mean values ± s.e.m. of the time required by fluorescence to decay to half of the ML-SA1-induced peak. n = 50 cells from 3 independent experiments. ***p<0.005 by Mann and Whitney test.

(G) Nuclear translocation of TFEB in stable HeLaTFEB-GFP cells subjected to the indicated conditions is reduced after Ca2+ chelator BAPTA treatment. Scale bar 5 μm.

(H) The graph shows the mean ± s.e.m. of the percentage of nuclear TFEB translocation in Ezrin-inhibited cells compared with DMSO under Ca2+ chelator BAPTA treatment. At least 3 independent experiments were performed. Mann and Whitney test (stv, stv+BAPTA, BAPTA, NSC668394, NSC668394+BAPTA vs DMSO) *p ≤ 0.05.

(I) qRT-PCR analysis for TFEB target genes (TRPML1, BECLIN, MAPLC3B and LAMP1) was performed on ARPE-19 cells treated with DMSO or NSC668394. Bar graphs represent mean values ± s.e.m. of at least 3 independent experiments. Mann and Whitney *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.005.
Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 5

Figure 5. NSC668394 rescues Lysosomal biogenesis in miR-211-/- mice.

(A) Confocal images of representative eye cryosections immunostained with anti-Ezrin (green) /anti-ZO-1 (red), anti-Ezrin-pT567 (green) /anti-ZO-1 (red), anti-Lamp1 (green) and anti-Sqstm1/p62 (green) antibodies from 6-month-old miR-211-/- sacrificed five months after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm.

(B) RPE was isolated 5 months after DMSO or NSC668394 treatment from 6-month-old miR-211-/- mice. Representative Western blots proteins from these tissues were performed to determine the expression levels of Lamp1, Ezrin, Ezrin-pT567, Sqstm1/p62 and LC3. The plots show the quantification of the indicated proteins normalized to the β-Actin loading control. Bar graphs represent mean values ± s.e.m. of independent experiments (n=6 mice). Mann and Whitney test (DMSO vs WT; NSC668394 vs DMSO), *p ≤ 0.05, ***p≤0.005.

(C) Cathepsin B (CtsB) activity in RPE lysates from miR-211-/- and WT mice sacrificed 3h after light on at 10 AM (diurnal condition). RPE was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. CtsB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of CtsB activity ± s.e.m. of independent experiments (n=3 mice) Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-), *p ≤ 0.05.

(D) GUSB activity from retina from miR-211-/- and control mice sacrificed 3h after light on at 10 AM (diurnal condition). Retina was isolated 1 week after DMSO or NSC668394 treatment from 2-month-old miR-211-/- mice. GUSB was rescued in NSC668394-treated miR-211-/- compared to DMSO-control miR-211-/- mice. Bar graphs represent percentage of GUSB activity ± s.e.m. of independent experiments (n=3 mice). Mann and Whitney test (DMSO-miR-211-/- vs WT; NSC668394-miR-211-/- vs DMSO-miR-211-/-).

Data information: (RPE) Retinal Pigment Epithelium; (OS) outer segment; (ONL) outer nuclear layer.
Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance (2020)
Figure 6

Figure 6. Pharmacological inhibition of Ezrin rescues the miR-211-/- phenotype.

(A) Representative images of conventional TEM analysis of RPE of WT, DMSO-treated miR-211-/- mice and NSC668394-treated miR-211-/- three-month-old mice. NSC668394-treated miR-211-/- mice show rescue of accumulation of double-membrane phagolysosome-like structures containing poorly processed POS (red arrows) compared to DMSO-treated miR-211-/- mice. Scale bar 1 mm.

(B) Confocal images of representative eye cryosections immunostained with anti-Rhodopsin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment, which are sacrificed 3h after light on at 10 AM (diurnal condition). Nuclei are counterstained with DAPI (blue). At least n = 6 mice per group. Scale bar 100 μm. The enlarged box highlights rhodopsin accumulation in the RPE (green spots). Scale bar 5 μm. Autofluorescence from lipofuscin granules from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Representative images of retina cryosections immunostained with anti-Cone Arrestin (green) antibody from WT and miR-211-/- mice at three months of age after DMSO or NSC668394 treatment. Nuclei are counterstained with DAPI (blue). N = at least 6 mice per group. Scale bar 100 μm.

(C) Graphs showing number of lipofuscin granules from the RPE of mice as in (B). Bar graphs represent mean values ± s.e.m. Mann and Whitney test (miR-211-/- DMSO vs WT and miR-211-/- NSC668394 vs DMSO treated mice) ***p ≤ 0.005. *p ≤ 0.05 (n=6 mice).

(D) Graphs show cone percentage (cones/area) from the retina of mice treated as in (B). Error bars represent s.e.m. Mann and Whitney test (DMSO vs WT and NSC668394 vs DMSO treated mice) ***p ≤ 0.005 (n=6 mice).

(E) Representative flicker traces at three months of age show the rescue of flicker responses of NSC668394-treated miR-211-/- mice (green lines) compared to DMSO-treated miR-211-/- control mice (red lines). WT mice were used as a control (black lines). Flicker recordings were performed with light intensities ranging from 10−4 to 15 cd s/m2 in steps of 0.6 logarithmic units at 6 Hz frequency.

(F) Flicker responses, plotted as a function of stimulus intensity, from WT (black lines), DMSO-treated miR-211-/- (red lines) and NSC668394-treated miR-211-/- (green lines) mice, at three months of age. The amplitude of the recordings from NSC668394 miR-211-/- treated mice is significantly rescued compared to DMSO-treated miR-211-/- mice. WT mice were used as a control. Error bars represent s.e.m. ANOVA test (DMSO vs WT and NSC668394 vs DMSO treated mice) **p ≤ 0.01, ***p ≤ 0.005.

Data information: (OS) outer segment; (ONL) outer nuclear layer; (RPE) Retinal Pigment Epithelium.
LncPSCA in the 8q24.3 risk locus drives gastric cancer through destabilizing DDX5 (2021)
Figure 2

Figure 2. lncPSCA suppresses gastric cancer cell proliferation in vitro and in vivo.

(A) A diagram of sgRNA locations for development of the CRISPR/Cas9-engineered lncPSCA-knockout gastric cancer cells.

(B) The detailed information of deleted sequences in various lncPSCA-knockout clones (MGC80-3: KO-C1 and KO-C3; HGC-27: KO-C14 and KO-C40).

(C) In MGC80-3 and HGC-27 cells, lncPSCA-knockout resulted in significantly enhanced cell proliferation comparing with the control cells. Stably lncPSCA overexpression could obviously inhibit viability of gastric cancer cells. Data show one representative example of three biological replicates.

(D) Colony formation assays. Upper subpanel: photos of cell clones in each well; lower subpanel: numbers of cell clones in each well. Data show one representative example of three biological replicates.

(E) lncPSCA-knockout significantly promoted growth of MGC80-3 (n = 8) or HGC-27 (n = 7) xenografts compared with control xenografts after 27 or 18 days.

(F) HE and Ki67 staining of MGC80-3 or HGC-27 xenografts. Scale bar = 100 μm.

Data information: Each value represents mean±SD. The difference between two groups was calculated using Student's t test. *P<0.05; **P<0.01, ***P<0.001.
LncPSCA in the 8q24.3 risk locus drives gastric cancer through destabilizing DDX5 (2021)
Figure 3

Figure 3. lncPSCA reduces migration, invasion and metastasis capabilities of gastric cancer cells.

(A) In MGC80-3 and HGC-27 cells, lncPSCA-knockout accelerates wound-healing and the stably enforced lncPSCA expression inhibits wound-healing. The dashed lines indicate the edges of the cell layers.

(B) lncPSCA inhibits invasion abilities of MGC80-3 and HGC-27 cells. Cells on the lower surface of the chamber were stained by crystal violet. Scale bar = 100 μm.

(C) In MGC80-3 and HGC-27 cells, expression changes of different markers (β-catenin, ZO-1, SNAIL and VIMENTIN) of epithelial-to-mesenchymal transition were examined after overexpression or knockout of lncPSCA.

(D) Fluorescent images of tumors in nude mice with tail vein injected MGC80-3 and HGC-27 cells with or without knockout of lncPSCA.

(E) Representative images of hematoxylin and eosin-stained slides of lung metastatic nodules of MGC80-3 and HGC-27 with or without knockout of lncPSCA. Scale bar = 100 μm.

(F) Incidence of distant metastases in nude mice (n = 8 per group).