EMBO/soda-nel huggingface dataset:
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 6

Figure 6. USP36 promotes Nop58 and Nhp2 binding to snoRNAs.

(A, B). USP36 promotes the binding of Nop58 to box C/D snoRNAs. H1299 cells transfected with Flag-Nop58 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box C/D snoRNAs (A). The expression of Nop58 and USP36 proteins is shown in (B). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05, compared to Nop58 alone (Student's t-test).

(C, D). USP36 promotes the binding of Nhp2 to snoRNAs. H1299 cells transfected with Flag-Nhp2 and V5-USP36 individually or together were assayed by RNA-IP with anti-Flag or control mouse IgG, followed by RT-qPCR detection of the indicated box H/ACA snoRNAs (C). The expression of Nhp2 and USP36 proteins is shown in (D). Shown is one representative experiment of three independent experiments. Data were presented as mean ± SD, n=3 technical replicates. *P<0.05; **P<0.01, compared to Nhp2 alone (Student's t-test).
The Deubiquitinase USP36 Promotes snoRNP Group SUMOylation and is Essential for Ribosome Biogenesis (2021)
Figure 7

Figure 7. USP36 is critical for rRNA processing and translation.

(A). Diagram of the pre-rRNA processing showing the pre-rRNA processing intermediate products and the location of probes (5'-ETS, ITS1 and ITS2) used in Northern blot analysis (left) as well as the major (black) and minor (grey) processing pathways (right). (B). USP36 is required for rRNA processing. HeLa cells transfected with scr or USP36 siRNA (left panels) or infected with scr or USP36 shRNA lentiviruses (right panels) were assayed for rRNA processing by Northern blot using 5'- ETS, ITS1 and ITS2 probes as indicated.

(C, D). Knockdown of USP36 inhibits translation. HeLa cells infected with scr or USP36 shRNA lentiviruses were incubated with O-propargyl-puromycin (OPP) followed by Click-iT OPP protein synthesis assays. Representative images (C) and quantification from three independent experiments (D). Data were presented as mean ± SD, n=3 biological replicates. ***P<0.001, compared to scr control (Student's t-test). Scale bar = 50 μM.

Expanded View Figure Legends
Drug screening identifies tazarotene and bexarotene as therapeutic agents in Multiple Sulfatase Deficiency (2023)
Figure 3

Fig. 3. Tazarotene and bexarotene reverse lysosomal pathophysiology in MSD primary fibroblasts.

(A) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). Scale bar = 20 µm.

(B) Representative confocal images of control and MSD fibroblasts with either tazarotene/bexarotene (10/20 µM, 6 days). Labelling with anti-LAMP1 antibody (green fluorescence) and DAPI (nuclei, blue). Scale bar = 10 µm.

(C) Quantification of total intensity of LAMP1- green fluorescence. N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), # p<0.05 (MSD DMSO vs MSD treated). RFU: relative fluorescence units. See details on p-values in appendix table S18.

(D) Quantification of LAMP1- green fluorescence vesicle size (µm). N = 20 images and 13 z-series optical sections per condition with a step-size of 0.26 microns, displayed at maximum extension and automated equalization of brightness. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. *** p<0.001 (DMSO treated MSD cells compared to DMSO treated control cells), ## p<0.01 (MSD DMSO vs MSD treated). See details on p-values in appendix table S19.

(E) Representative spectra of the heparansulfate derived oligosaccharide HN-UA(1S) as GAG marker analyzed via mass-spectrometry in DMSO (control) treated MSD primary fibroblasts (variant FGE Gly247Arg homozygous, upper panel) and in the same cell line simultaneously treated with 10 and 20 µM tazarotene and bexarotene for 21 days (lower panel). Integrated peak areas correspond to the amount of HN-UA(1S).

(F) Quantification of specific oligosaccharide markers for GAG species in three different MSD primary fibroblast lines with FGE variants as indicated and one control fibroblast line after 21-day treatment with tazarotene/bexarotene. 10/20 µM. Data represent mean ±SD of 3-8 independent experiments (biological replicates). Unpaired t-test comparing DMSO conditions of control and MSD fibroblasts and treated and DMSO control conditions in every MSD cell line for every marker. Details on significance levels for each marker are summarized in table EV1. The lowest significance levels among individual markers for treatment and DMSO condition in MSD fibroblast lines are displayed. * p<0.05, ** p<0.01.
Drug screening identifies tazarotene and bexarotene as therapeutic agents in Multiple Sulfatase Deficiency (2023)
Figure 4

Fig. 4. Tazarotene and bexarotene work through retinoid receptors in MSD fibroblasts.

(A) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RAR receptor antagonist AGN 193109 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 3-11 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01, ### p<0.001. Difference against 0 µM DMSO control: ** p<0.01, *** p<0.001, **** p<0.0001. See details on p-values in appendix table S20.

(B) ARSA activity quantification upon treatment of MSDi cells with 10 µM tazarotene, 20 µM bexarotene, and 10 µM tazarotene and 20 µM bexarotene in combination with increasing concentrations of the pan-RXR receptor antagonist HX 531 (24 h pretreatment) for 72 hours. Data represent mean ±SD of 4-14 independent experiments (biological replicates). Two-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations/conditions. # p<0.05, ## p<0.01. Difference against 0 µM DMSO control: **** p<0.0001. See details on p-values in appendix table S21.
Drug screening identifies tazarotene and bexarotene as therapeutic agents in Multiple Sulfatase Deficiency (2023)
Figure 6

Fig. 6. Tazarotene and bexarotene treatment response in MSD fibroblasts requires residual FGE function.

(A) ARSA protein amount quantification after treatment of MSD primary fibroblasts (variant FGE Gly247Arg homozygous) with tazarotene, bexarotene, and tazarotene/bexarotene in combination for 6 days referred to β- actin amounts and normalization of ARSA activity based on ARSA protein amount (specific ARSA activity). Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. # p<0.05. Difference against 0/0 µM control: * p<0.05, *** p<0.001. See details on p-values in appendix table S24.

(B) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSDi) after 6 days of simultaneous treatment with increasing concentration of tazarotene. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 µM control: *** p<0.001, **** p<0.0001. See details on p-values in appendix table S25.

(C) Quantification of ARSA activities in CRISPR/Cas9 generated ARPE19 SUMF1 -/- cells and appropriate controls (ARPE19 wild type, MSD primary fibroblasts (variant FGE Gly247Arg homozygous)) after 6 days of simultaneous treatment with tazarotene/bexarotene 10/20 µM for up to 21 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. Displayed are significance levels for the next significant difference between adjacent concentrations. ## p<0.01. Difference against 0 days control: **** p<0.0001. See details on p-values in appendix table S26.

(D) ARSA activity quantification after simultaneous treatment of MSD primary fibroblasts (variants FGE Gly247Arg homozygous, FGE Ser64Ter homozygous) with tazarotene and bexarotene. Treatment time 6 days. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. * p<0.05. See details on p-values in appendix table S27.
Drug screening identifies tazarotene and bexarotene as therapeutic agents in Multiple Sulfatase Deficiency (2023)
Figure 7

Fig. 7. Improved FGE variant protein stability after tazarotene and bexarotene treatment.

(A) Pulse-chase-experiment in HT1080 FGE Ser155Pro cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (C) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. ** p<0.01, *** p<0.001. See details on p-values in appendix table S28.

(B) Pulse-chase-experiment in HT 1080 FGE Gly247Arg cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (full length FGE, C) and cleaved and secreted (Δ72 FGE, M) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular and secreted FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. * p<0.05, *** p<0.001. See details on p-values in appendix table S29.

(C) Pulse-chase-experiment in HT 1080 FGE Ala279Val cells after pre-treatment with tazarotene/bexarotene and DMSO (control) for 3 days. Upper panel: representative autoradiogram of intracellular (full length FGE, C) and cleaved and secreted (Δ72 FGE, M) 35S isotope labelled FGE protein in either condition with a chase time of 4 hours. Lower panel: quantification of 35S isotope labelled intracellular and secreted FGE protein amounts. Data represent mean ±SD of 3 independent experiments (biological replicates). One-way ANOVA followed by Tukey's test for multiple comparisons. See details on p-values in appendix table S30.

Expanded View Figure legends
Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes (2018)
Figure 1

Figure 1. Selected endosomal Rab RNAi screen identified Rab22A as a regulator of RE dynamics.

A IFM images of KIF13A-YFP transfected control and Rab-knockdown HeLa cells. TN: average tubule number (mean±SEM, n=3).

B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 1A (mean± SEM). n=3. nc: total number of cells. nt: total number of tubules. *P≤0.05, **P≤0.01, ***P≤0.001 and ns=not significant.

D IFM images of KIF13A-YFP and mCherry-Rab7A/11A/22A transfected HeLa cells.

E Live cell imaging of GFP/mCherry-Rab22A with respect to mCherry-Rab11A or GFP-Rab7A in HeLa cells. Magnified view of insets (at 0, 20, 40 seconds) are shown separately.

F Pull-down of different His-KIF13A domains using HeLa cell lysate and then probed with indicated Rab proteins. The bead bound His-KIF13A in each pull-down was shown as coomassie gel. *, non-specific bands. Note, part of this experiment was shown in Fig 5F.

Data information: In (A, D, E), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.
Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes (2018)
Figure 2

Figure 2. Rab22A localizes to the E/SE and REs and regulates RE dynamics

A IFM images of KIF13A-YFP and mCherry-Rab22AWT/Q64L/S19N cotransfected HeLa cells. Arrowheads and arrows point to the KIF13A-positive tubular REs and E/SEs respectively. The colocalization coefficient (r, in mean±SEM) between two markers was indicated separately. Scale bars: 10 μm.

B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 2A. n=3. nc: total number of cells. nt: total number of tubules. **P≤0.01, ***P≤0.001 and ns=not significant.

D Subcellular fractionation of HeLa cells to probe the localization of Rab22A (red box) with respect to other organelle specific proteins. *, non-specific bands.
Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes (2018)
Figure 3

Figure 3. Rab22A functions upstream in the pathway of BLOC-1 and BLOC-2 and regulates RE dynamics.

A IFM images of KIF13A-YFP transfected control and Rab22A-, BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells. Cells were stained for AP-1 (γ) or internalized with Tf-Alexa fluor 594.

B, C Graphs represent the measurement of KIF13A-positive TN (B) and TL (C) in HeLa cells of Fig 3A. n=3. nc: total number of cells. nt: total number of tubules. *P≤0.05 and ***P≤0.001.

D Immunoblotting analysis of proteins in control and Rab22A-, BLOC-1-, BLOC-2-depleted HeLa cells. *, non-specific bands. Protein band intensities were quantified and indicated on the gels.

E IFM images of mCherry-Rab22A and KIF13A-YFP cotransfected control and BLOC-1Mu-, BLOC-2HPS6- knockdown HeLa cells.

Data information: In (A, E), arrowheads and arrows point to the KIF13A-/Rab22A-positive tubular REs and E/SEs respectively. Scale bars: 10 μm.
Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes (2018)
Figure 4

Figure 4. Rab22A regulates melanocyte pigmentation, cargo transport and functions upstream of BLOC-1.

A BF and IFM images of control and Rab22A-depleted melanocytes. The colocalization coefficient (r, in mean±SEM) between the two markers indicated separately. Nuclei are stained with Hoechst33258.

B Graph represents the quantified melanin content in control sh, Rab22A sh and BLOC-1- melanocytes. n=3. The fold-change in melanin content (mean±SEM) indicated separately. ***P≤0.001.

C Immunoblotting analysis of proteins in Control sh and Rab22A sh melanocytes.

D EM of control and Rab22A-knockdown MNT-1 melanocytes. Arrowheads indicate PMEL fibrils. Scale bars: 1 μm. I, II, III, IV: stages of melanosomes. M: mitochondria. Magnified view of insets are shown separately and they emphasize the stage I melanosomes in both conditions. Note: the presence of hybrid stage I/II melanosomes in Rab22A-knockdown cells. Insets (b)-(d) and (g)-(h) are from different cells of respective condition. Scale bars: 200 nm.

E BF and IFM images of GFP-Rab22A transfected BLOC-1-(Mu) melanocytes.

F Immunoblotting analysis of proteins in BLOC-1- and BLOC-1R melanocytes.

Data information: In (A, E), arrows indicate the melanocyte pigmentation and arrowheads point to the localization of TYRP1/GFP-Rab22A. Scale bars: 10 μm. In (C, F), protein band intensities were quantified and indicated on the gels. *, non-specific bands.
Rab22A recruits BLOC-1 and BLOC-2 to promote the biogenesis of recycling endosomes (2018)
Figure 5

Figure 5. Rab22A regulates membrane association of BLOC-1 and BLOC-2, and forms a complex with BLOC-1-BLOC-2-KIF13A.

A Subcellular membrane fractionation of control and Rab22A-, BLOC-1-, BLOC-2-knockdown HeLa cells and probed the fractions for pallidin, dysbindin and muted. Red colored box emphasizes BLOC-1 membrane association in the respective cell types.

B, C Cytosol-membrane fractionation of homogenates from HeLa cells (B) or melanocytes (C) as indicated. Protein band intensities were quantified and indicated the percentage membrane association on the gels.

D, E Pull-down of His-Rab22AWT using HeLa (D) or melanocyte (E) lysate. In E, the beads were preloaded with GTPγS or GDP.

F Pull-down of different His-KIF13A domains using HeLa cell lysate.

G Model illustrating the recruitment (left) and association (middle) of Rab22A, BLOC-1 and BLOC-2 in a sequential manner onto the membrane buds of E/SEs followed by interaction with KIF13A motor. Rab22A-BLOC-1-BLOC-2 complex possibly extend the membrane buds into RE tubules with KIF13A motor (right) movement on microtubules.

Data information: In (A-F), * indicates non-specific bands. In (D-F), the bead bound His-Rab22A/His-KIF13A domains were shown as coomassie stained gel separately or in Fig 1F.

Expanded view figure legends
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 1

Figure 1. Bacterial dysregulation promotes the secretion of LIF by IECs, which differentially affects immune cells and epithelial cells

A Quantitative expression of Lif mRNA in the colon of DSS-challenged and control mice (n=6 per group).

B LIF measured by ELISA in mouse colon explant supernatants from DSS-challenged and control mice (n=4 per group).

C Quantitative expression of Lif mRNA in LPLs and IECs from DSS-challenged and control mice (n=4 per group).

D Quantitative expression of Lif mRNA in the colon of SPF mice treated with or without antibiotics (Abx) at the indicated time points during colitis induction (n=3 per group).

E Quantitative expression of Lif mRNA in LPLs and IECs from mice treated with or without Abx at the indicated time points during colitis induction (n=3 per group).

F mRNA expression of the indicated genes in IECs stimulated by LPS (2.5μg/ml), PGN (3μg/ml) or LTA (2μg/ml) for 2 hours.

G, H Serum concentrations of LIF in IEC supernatants untreated (G) or pretreated with monensin (H) followed by LPS (2.5μg/ml) or LTA (2μg/ml) for 12hours, as determined by ELISA. The data are representative of three independent experiments.

I, J Immunoblot analysis of the expression and modification of the indicated proteins in the total cell lysate of CD4+ T cells (I) and IECs (J) treated with LIF (20 ng/ml) at different time points. The lower panel shows the intensity analysis of the bands from three independent experiments. The data are representative of three independent experiments.

K Stat4 mRNA expression in LPLs and IECs (n=6).

L, M The proliferation rate of CD4+ T cells (L) and DLD-1 cells (M) was determined in the presence or absence of LIF. The data are representative of two independent experiments. *p< 0.05, **p< 0.01, ***p< 0.001 and ns: not significant (Student's t-test). Error bars represent the SEM.
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 2

Figure 2. LIF administration promotes the amelioration of intestinal colitis.

A Weight loss of wild-type (WT) (n=6) or Stat4 knockout(Stat4-KO) (n=6) mice receiving intraperitoneal injection of PBS or LIF during challenge with 3% DSS.

B Macroscopic changes in the colon of the mice on day 10 of colitis induction as described in (A).

C Comparison of the colon length in the mice on day 10 of colitis induction as described in (A).

D DAI (stool consistency and bleeding score) of colitis mice treated as described in (A).

E H&E histology of representative colons from colitis mice. Scale bar, 20μm.

F Weight loss of Rag1-/- (n=6) mice transferred with CD45RBhi CD4+ T cells and intraperitoneally injected with PBS or LIF for 8 weeks.

G Macroscopic changes in the colon of Rag1-/- mice treated as described in (F).

H Comparison of colon lengths in the Rag1-/- mice at week 8 (n=5).

I H&E histology of representative mouse colons on day 54 of colitis induction as described in (F). Scale bar, 100μm.*p< 0.05, **p< 0.01 and ***p< 0.001 (Student's t-test). Error bars represent the SEM.
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 3

Figure 3. LIF activates STAT4 to block Th17 cell differentiation in the mouse colitis model and in vitro.

A Colons obtained from WT or Stat4-KO mice on day10 of the colitis model induced as in Figure 2A were immunostained with an antibody against phosphorylated STAT4. Scale bar, 50μm.

B Immunoblot analysis of STAT4 phosphorylation in spleen tissue from colitis mice.

C FACS staining of LPLs isolated on day10 from the colon of WT or Stat4-KO colitis mice receiving PBS or LIF (n=4 per group). The percentage of IL-17A+ and/or IFNγ+ CD4+ T cells in vivo was analyzed.

D Quantitative expression of Il17a mRNA in MLNs and LNs from colitis mice treated as described in Figure 2A (n=3 per group).

E FACS staining of WT and Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into Th17 cell subsets.

F qPCR analysis of Th17-related genes in WT or Stat4-KO naïve CD4+ T cells on day 4 of induction into Th17 cell subsets in the absence or presence of LIF (n=3 per group).

G FACS staining of WT or Stat4-KO naïve CD4+ T cells treated with LIF (50 ng/ml) on day 4 of induction into nonpathogenic or pathogenic Th17 cells. The data are representative of three independent experiments. *p< 0.05, **p< 0.01 and ***p< 0.001 (Student's t-test). Error bars represent the SEM.
STAT4 activation by leukemia inhibitory factor confers a therapeutic effect on intestinal inflammation (2019)
Figure 4

Figure 4. LIF-activated STAT4 is phosphorylated on SPXX motifs within the C-terminal transcription regulation domain.

A Immunoblot analysis of STAT4 phosphorylation in CD4+ T cells pretreated with PBS or LY2784544 (1μM) for 12 hours followed by LIF for the indicated number of minutes.

B Immunoblot analysis of CD4+ T cells treated with LIF for the indicated number of minutes followed by immunoprecipitation with an anti-LIFR antibody.

C Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs. The red asterisks denote phosphorylated STAT bands.

D Sequence alignment showing that the C terminus of STAT4 and LIFR contains four SP repeats in both humans and mice. The SPXX motifs are displayed and underlined in red. The red asterisks point out the potential phosphorylated serine residues.

E The mass spectrometry analysis of purified STAT4 protein revealed the phosphorylation of STAT4 at serine residues in the C terminus.

F Immunoblot analysis of the indicated protein modifications in the total lysate of CD4+ T cells treated with LIF (20 ng/ml) at different time points.

G-I Coimmunoprecipitation and immunoblot analysis of LIF-treated HEK293T cells cotransfected with the indicated constructs.

J Immunoblot analysis of the distribution of the indicated proteins in the CD4+ T cell fraction.

K Cultured HeLa cells transfected with GFP-tagged STAT constructs were exposed to LIF and imaged 30 minutes later. Scale bar, 50μm.