EMBO/soda-nel huggingface dataset:
HDAC1 Dysregulation Induces Aberrant Cell Cycle and DNA Damage in Progress of TDP-43 Proteinopathies (2020)
Figure 8

Figure 8. Deregulation of HDAC1 is involved in aberrant cell cycle activity and DNA damage in the frontal cortices from patients with FTLD-TDP.

Representative IF staining of TDP-43 and HDAC1 in the frontal cortices from normal individuals and patients with FTLD-TDP. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.

Quantification of cells with co-mislocalized HDAC1 and TDP-43 from each view of microscope. N = 5 per group.

Linear regression analysis of cells with HDAC1 mislocalization and TDP-43 proteinopathies. Total cell counts: 3000 per samples. P = 0.0001 by Pearson correlation analysis.

Representative IF staining of γH2AX and Ki67 in the frontal cortices from normal individuals and patients with FTLD-TDP from each view of microscope. Scale bar: 50 μm. The circled area is emphasized for showing the distribution of immunoreactivity in cell subregions. Scale bar: 15 μm.

Quantification of cells with γH2AX and Ki67 immunoreactivity. N = 5 per group.

Linear regression analysis of cells with DNA damage and aberrant cell cycle activity. Cell counts: 40 -113 (normal) and 418-887 (FTLD) per samples. p < 0.0001 by Pearson correlation analysis.

Dot-blot of HDAC1 and TDP-43 in urea-soluble fractions from the frontal cortex of normal individuals and patients with FTLD-TDP.

Quantification of HDAC1 and TDP-43 expression levels in urea-soluble fractions. N = 5 per group.

Data information: All data represent mean ± SEM, **** p < 0.0001 by t-test.

Expanded View Figure Legends
TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 1

Fig 1. TRIM27 binds directly to ULK1 and this binding is regulated by nutritional starvation

Venn diagram of FIP200 binding proteins identified in MEF following FIP200 co-immunoprecipitation analysis under normal versus starvation conditions.

Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and the indicated combinations of empty HA-vector, HA-tagged FIP200, ULK1, or ATG13 before immunoprecipitation with anti-HA antibodies. Immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to Western blotting against anti-HA or Flag.

C-F. WT (C), FIP200 knockout (KO) (D), ULK1/2 knockout (E) MEF cells or ATG13 knockout 4T1 cells (F) were subjected to starvation conditions before immunoprecipitation against control Immunoglobulin G (IgG) or anti-TRIM27 before blotting against the indicated endogenous forms of TRIM27, FIP200, ULK1, ATG13, and USP7.

G. Co-immunoprecipitation assays in HEK293T cells after co-transfection with Flag-TRIM27 and either full length ULK (HA-ULK1 1-1051) or the indicated various truncated forms of ULK1 designated HA-ULK1 1-828, HA-ULK1 1-600, and HA-ULK1 1-500; AA, amino acids.

H. Co-immunoprecipitation assays in HeLa cells stably expressing HA-ULK1 and immunoprecipitation against IgG or HA followed by Western blotting as indicated.

HeLa cells stably expressing Flag-TRIM27 were starved as indicated or starved for 2h before replenishing with complete medium. Anti-Flag immunoprecipitates (top panel) and input samples (bottom panel) were then subjected to blotting against Flag and ULK1.

Purified recombinant GST or GST-TRIM27 was incubated with recombinant HA-ULK1 and bound ULK1 analyzed by Western blotting.

Schematic diagram of TRIM27 binding to ULK1-ATG13-FIP200 complex in normal or starvation conditions.

Data information: For B-J, the experiments were performed at least three times independently with similar results.
TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 2

Fig 2. TRIM27 promotes ULK1 degradation via K48-linked ubiquitination of ULK1 at K568/K571

A-B. Ubiquitination assays in HEK293T cells after co-transfection with HA-ULK1 and His-Ub along with Flag-TRIM27 (A) or after knockdown of TRIM27 using independent shRNAs (shTRIM27-1 and -2) (B). Samples recovered with Ni-NTA (top panel) and input samples (bottom panel) were then subjected to blotting against anti-HA, Flag or His as indicated.

C. In vitro ubiquitination assays performed with the indicated combinations of recombinant GST, GST-TRIM27, affinity-purified HA-ULK1 and His-Ub in the presence or absence of E1, E2, and ATP. The reactions were analyzed with anti-Ub, anti-HA, or anti-GST.

D-E. Western blot analysis of ULK1 levels in HeLa cells stably expressing control or TRIM27 shRNAs (D), or HEK293T cells transfected with empty Flag vector (-) or Flag-TRIM27 after treatment with or without 10 μM MG132 for 4 hours (E).

F. Fluorescence microscopy image of MEFs with stable expression of mCherry-ULK1 and GFP-TRIM27 after treating cells with DMSO (MOCK) or 10 μM MG132 for 4 hours. Scale bar, 10 μm.

G-J. HA-ULK1 levels (G, H), or endogenous ULK1 levels (I, J) in 50 μg/mL cycloheximide (CHX)-treated HEK293T cells with or without co-expression of Flag-TRIM27 (G, H) or TRIM27 RNAi (I, J). Western blots against HA-ULK1 and actin control (G, I) were subjected to densitometric quantitation (H, J).

K-L. Ubiquitination assays were performed in HEK293T cells as per (A, B) after co-transfection with full length HA-ULK1 or the indicated truncated constructs (K) or double (K568/K571R) or single (K568R, K571R) lysine substitution mutants (L) in the presence or absence of Flag-TRIM27.

M. Ubiquitination of HA-ULK1 measured as per (A, B) in cells co-expressing Flag-TRIM27 with either His-Ub WT, His-Ub K48, or His-Ub K63, respectively.

N. In vitro ubiquitination assays performed as per (C) against affinity-purified HA-ULK1 WT or the HA-ULK1 K568/K571R mutant in the presence or absence of GST-TRIM27.

O. ULK1/2 knockout MEFs stably expressing the indicated HA-ULK1 constructs were subjected to Western blot analysis against HA or actin before (0h) and after treatment with CHX for 12 hours.

Data information: For (A-O), the experiments were performed at least three times independently with similar results. All the statistical data are presented as mean ± s.d. of three independent experiments. Statistical analysis was performed using two-tailed unpaired Student's t-tests (H and J), **P < 0.01.
TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 3

Fig 3. TRIM27 is a negative regulator of autophagy

A. Western blot analysis of autophagy marker proteins (p62 and Ser318 phosphorylated ATG13) in WT HeLa cells and two independent TRIM27 KO clones. ULK1 and TRIM27 levels were measured, along with actin as a loading control.

B. TRIM27 KO HeLa cells were reconstituted with Flag-TRIM27 and Western blotting was performed as per (A).

C. LC3 conversion was measured by Western blot in WT or TRIM27 KO HeLa cells with or without pretreatment with 200 nM bafilomycin A1 (BAF A1).

D. Western blot analysis of indicated proteins in MEFs generated from Trim27 +/+ or Trim27 -/- mice.

E-H. Immunofluorescence analysis of endogenous LC3 in MEFs generated from Trim27+/+ or Trim27-/- mice cultured in fed (E) or starved conditions (G). Quantification of LC3B puncta Fed conditions (F), starved conditions (H) (n = 50 cells per condition). Data are mean ± s.d. in F and H. Scale bar, 10 μm.

I-J. Western blot analysis of autophagy markers in the kidneys of three-month-old Trim27 +/+ and Trim27-/- mice. Each lane represents a different mouse (I). Quantification of p62, LC3-II/LC3-I ratio, and LC3-II/actin ratio in I (J). Data are mean ± s.e.m. for three mice per genotype.

K. Microscopy analysis of GFP-LC3 puncta in MEFs generated from indicated genotype mice in normal or starvation conditions. Scale bar, 10 μm.

L-M. Quantitation of data presented in (K) shows mean number of GFP-LC3 puncta per cell ± s.d. (n = 50 cells per condition).

Data information: (F, H, J, L and M) Statistical analysis was performed using two-tailed unpaired Student's t-tests, *P< 0.05,***P<0.001.
TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 4

Fig 4. STK38L negatively regulates starvation-induced autophagy via promoting binding between TRIM27 and ULK1.

A. Ubiquitination analysis of HA-ULK1 in HeLa derivatives generated in Fig 1(H) co-expressing His-Ub with or without TRIM27 knockout, the cells were treated with starvation for the indicated time.

B. Western blot analysis of ULK1 levels in HeLa cells with or without TRIM27 knockout. The cells were treated with starvation for the indicated time. The lysates were analyzed using anti-ULK1, anti-TRIM27, or anti-Actin.

C. HA-ULK1, HA-ULK1 K568/K571R were stably expressed in ULK1 knockout HeLa cells, the cells were treated with starvation, the lysates were analyzed by western blot using anti-HA, or anti-Actin.

D. List of potential Top 10 proteins that bind to TRIM27 under starvation conditions identified by mass spectrometry.

E. Co-immunoprecipitation of Flag-TRIM27 with HA-ULK1 in HEK293T cells with or without STK38L knockdown, the cells were treated with starvation. Flag-TRIM27 was immunoprecipitated using anti-Flag, the bound HA-ULK1 was detected using anti-HA.

F. Co-immunoprecipitation of endogenous TRIM27 with endogenous STK38L in HeLa cells.

G. Co-immunoprecipitation of Flag-STK38L with ULK1, or TRIM27 in HEK293T cells with or without starvation treatment. Flag-STK38L was immunoprecipitated using anti-Flag, the immunoprecipitates and lysates were analyzed with anti-ULK1, anti-TRIM27.

H. Co-immunoprecipitation of endogenous TRIM27 with endogenous ULK1 in HeLa cells with or without STK38L knockout. The cells were treated with starvation before lysis.

I. Western blot analysis of ULK1 levels in starved HeLa cells expressing either Myc-STK38L, or Flag-TRIM27, or both. The lysates were analyzed using anti-ULK1, anti-Flag, anti-Myc, or anti-actin.

J. Ubiquitination analysis of HA-ULK1 in HEK293T cells co-expressing Flag-TRIM27 and His-Ub, or together with Myc-STK38L.

K. Western blot analysis of ULK1 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time.

L. Western blot analysis of p62 or LC3 levels in HeLa cells with or without STK38L knockout, the cells were treated with starvation for the indicated time. Bafilomycin A1 (BAF A1) was used to inhibit the fusion between autophagosomes and lysosomes.
TRIM27 cooperates with STK38L to inhibit ULK1-mediated autophagy and promote tumorigenesis (2022)
Figure 6

Fig 6. Knockout of Trim27 inhibits breast tumorigenesis but promotes lung metastasis in PyMT mice.

A. Comparison of the relative mRNA levels of TRIM27 in normal breast tissues (Normal, n=113) and indicated breast tumors (Basal，n=143；Her2，n=67；LumA，n=427；LumB，n=190) in the TCGA database. The number of biological replicates are shown as indicated. P < 0.0001 (one-way ANOVA).

B. Analysis of the correlation between ULK1 and TRIM27 expression in the CPTAC proteomics database. P = 0.0027 (Pearson's correlation method).

C-D. Western blot analysis of ULK1 and TRIM27 levels from tumor tissues (T), Pericarcinomatous tissues (P), and their matched surrounding normal tissues (N). GAPDH was used as a loading control (C). A total of 34 samples of human breast cancer were classified into nine groups based on the expression of ULK1 and TRIM27 in tumor tissues compared with surrounding normal tissues (D).

E. Schematic diagram of Trim27-/- PyMT and Trim27+/- PyMT mice breeding, Trim27+/- PyMT mice as a littermate control.

F. Representative tumors in MMTV-PyMT mice of the indicated Trim27 genotypes. Scale bar, 5mm.

G. Western blot analysis of indicated proteins in Trim27-/- PyMT or Trim27+/- PyMT mice in primary breast tumors. Actin was used as a loading control.

H. Kaplan-Meier tumor-free survival curves of Trim27+/- PyMT (median = 75 d, number = 20), or Trim27 -/- PyMT mice (median = 95 d, number = 16). P < 0.0001 (Mantel-Cox test).

I-K. Pulmonary surface nodules and hematoxylin and eosin (H&E) staining in Trim27+/- PyMT mice and Trim27-/- PyMT mice (I and J). Scale bar, 1mm. (K). Bar chart showing quantification for the number of metastatic nodules per lung lope (Trim27+/-, n=6 mice; Trim27-/- n=6 mice), when compared at the same final timepoint. Each symbol represents a mouse; Data are presented as mean ± s.d. from three independent experiments; ***P<0.001 (unpaired student's t-test).

Expanded View Figure Legends
A novel platform for attenuating immune hyperactivity using EXO-CD24 in Covid-19 and beyond (2022)
Figure 1

Figure 1. Characterization of CD24-displaying exosomes (EXO-CD24).

(A) Representative clones screened for CD24 expression under regulation of tetracycline. The cells were exposed to 1 µg/ml tetracycline for 48 h. 20 µg from each sample were subjected to Western blot analysis for CD24 and analyzed with anti-CD24 SWA11 mAb. The membrane was reprobed with anti-tubulin antibody to assess the uniformity of sample loading.

(B) The effect of tetracycline concentration on CD24 expression levels was examined by flow cytometry.

(C) Exosome-based ELISA was used to measure expression of CD24 in the purified exosomes (D) Western blot analysis of EXO-CD24 exosomes. Tetracycline (Tet) was used to induce CD24 expression in the cell line from which exosomes are derived. HSP70 was used as an exosomal marker.

(E) Flow cytometry analysis of EXO-CD24. Left panel: Cells were gated based on cell size (forward scatter [FSC] versus side scatter [SSC]); Beads of known sizes of 100nm, 200nm and 500nm were used for size reference; Middle-left panel: EXO‑CD24 labeled with anti-CD24 FITC antibody and gated to the exosome population; Middle-right panel: EXO‑CD24 labeled with anti-CD81 APC antibody and gated by to the exosome population; Right panel: EXO‑CD24 double labeled with anti-CD24 FITC and anti-CD81 APC antibodies and gated by to the exosome population.

(F) Cryo-TEM images of isolated CD24‑exosomes. On a number of different devices, many experiments were conducted and representative images are shown. Arrows point to double-membraned vesicles (exosomes). Scale bars are 100 nm (left panel) and 200 nm (right panel).
A novel platform for attenuating immune hyperactivity using EXO-CD24 in Covid-19 and beyond (2022)
Figure 4

Figure 4. Characterization of mCD24-displaying exosomes (EXO-mCD24).

(A) Exosome-based ELISA was used to measure expression of mCD24 in the purified exosomes. The expression level was compared to mCD24-negative exosomes and to mouse recombinant protein. The data are represented as average of three technical replicates ± SEM.

(B) Flow cytometry analysis of EXO-mCD24. EXO‑mCD24 labeled with APC-conjugated anti-mCD24 antibody and gated to the exosome population.

(C) Western blot analysis of EXO-mCD24 exosomes. HSP70 was used as an exosomal marker.
A novel platform for attenuating immune hyperactivity using EXO-CD24 in Covid-19 and beyond (2022)
Figure 8

Figure 8: patients' systemic inflammatory indices

Systemic CRP levels. Values are normalized against baseline value (Day 0). Descriptive statistics (minimum, maximum, median, first quartile, third quartile) were calculated for CRP levels. these are presented graphically via box plots before and after treatment (n=35 patients)

(B) Systemic cytokine levels. Cytokine levels were measured during (Day 3/4) or after (Day 7/8) treatment with EXO-CD24 using Quantibody Multiplex ELISA Array. Values are normalized against baseline value (Day 0). Samples from 11 patients were analysed. Results from 8 patients are shown (results for 3 patients are not shown because one or more baseline results were below detection level). Box plot of Systemic cytokine levels by days after treatment (minimum, maximum, median, first quartile, third quartile). (C) During the course of EXO-CD24 treatment and follow-up, cytokine levels were measured for the rest of the patients (n=24 patients). Values represent the fold change from day 0, mean±SEM. (D) Monitoring systemic IL-6 cytokine level following EXO-CD24 treatment. Samples from 17 patients, representing the 4 experimental groups, were analysed using the BD™ Cytometric Bead Array (CBA). Values represent mean±SD.
Attenuation of SARS-CoV-2 replication and associated inflammation by concomitant targeting of viral and host cap 2'-O-ribose methyltransferases (2022)
Figure 4

Figure 4 | DZNep treatment modulates tissue and immune processes.

(a-d) Mass spectrometry based analysis of cells treated with DZNep and infected with SARS-CoV-2 and SARS-CoV. (a) Schematic representation of LC-MS/MS experiments. A549-ACE2s and NHBEs were pretreated for 6 h with 0.75 and 1.5 μM DZNep, respectively, or vehicle (PBS), and infected with SARS-CoV-2 or SARS-CoV at MOI 3 for 24 h (A549-ACE2) or 36 h (NHBEs). Changes in protein abundances were analyzed according to the depicted scheme using LASSO-based linear model followed by fixed LASSO inference based p-value estimation as described in the methods section. (b) Number of significantly up- or downregulated proteins in indicated comparisons according to (a). (c) Donor-normalized LFQ abundances of viral nucleoprotein (N) and spike (S) in the indicated conditions. Error bars represent mean +/- sd of n=4 donors (NHBE) or n=4 independently infected A549-ACE2 cultures. Statistics were calculated using Student's two-sided t-test as indicated. (d) Expression patterns according to (a) of a selection of genes related to the disease-relevant pathways as annotated.

(e) NHBEs (6 independent donors) were pretreated for 6 h with 0.75 μM DZNep or vehicle and infected with SARS-CoV or SARS-CoV-2 at MOI 3. 24 h later, accumulation of IL-6 and IP-10 was measured in the supernatant by ELISA. Donor-wise IL-6 and IP-10 secretion, normalized to vehicle treated uninfected controls (as further described in materials and methods) is shown.

(f) Schematic representation of the proposed disease-relevant functions of DZNep in the context of COVID-19 alongside the model of their molecular origin.
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 1

Figure 1. Hair follicle progenitor cells express Gata6.

(A) Overview of hair follicle stem and progenitor cell dynamics with transcription factors known to regulate each stage. (B) Lineage tracing of the anagen matrix from -actin-CreER:Confetti, 7 days after induction with either a high dose of tamoxifen (TM) (top) or low dose (bottom) for single cell clonal lineages. (C) Images of skin sections at various stages of the hair cycle, immunostained for Gata6 (red), CD34 or K14 (green) and DNA (blue). (D) TM induction scheme (D) and images (D’) for Gata6 inducible knockout (iKO) and wild type (WT) mice over time, showing the loss of Gata6 (red) from the hair follicle. Scale bars: 30 m.
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 3

Figure 3. Gata6 is not necessary for terminal differentiation of matrix cells.

(A) Scheme for BrdU pulse chase experiment to track matrix cell differentiation into the upper inner lineages during anagen. The plus sign marks the starting point for TM or BrdU. (B-G) Gata6 WT and iKO skin after 3 days after TM induction and 12 hours after BrdU labeling or 5 days after TM induction and 2 days after BrdU labeling stained with markers as indicated in corresponding color.
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 4

Figure 4. Hair follicle progenitor show impaired proliferation and increased DNA damage and apoptosis following loss of Gata6.

(A) Hair follicles exhibit immunostaining signal for DNA damage marker H2A.X (red) and apoptosis marker Caspase3 (green) as early as one day after Gata6 iKO induction. Top panel shows a section of UV irradiated epidermis as a positive control for DNA damage. Note that Caspase 3 is not expressed in the H2A.X cells of the epidermis but is found in some iKO hair follicle matrix cells. Right panels show single color images as indicated at top (B) Quantification (average SD) of the percentage of apoptotic cells per matrix as determined by counting of Caspase3+ cells (n=50 follicles from 3 mice per group) (C) Quantification (average SD) of the percentage of DNA damaged cells per matrix as determined by counting of H2A.X+Caspase3- cells (n=50 follicles from 3 mice per group). (D) Regional distribution of apoptosis was analyzed by dividing the matrix into four compartments as shown in the legend (left) and counting the fraction of Caspase3+ cells per each matrix compartments (right; n=50 follicles from 3 mice per group). (E) Proliferation in the matrix compartments shown in (D) was assessed by BrdU labeling for 1 hour, followed by staining, and quantification (F) (interior cells labeled with solid bars, exterior cells labeled with striped bars). Unpaired t-test: *P-value < 0.05. n=20 follicles from 2 mice per group.
Gata6 promotes hair follicle progenitor cell renewal by genome maintenance during proliferation (2016)
Figure 5

Figure 5. Proliferation-induced DNA damage in keratinocytes results in cell cycle arrest and cell death.

(A) Colony formation of WT and TM induced Gata6 iKO keratinocytes stained with hematoxylin. Primary keratinocytes were isolated from induced iKO newborn mice, plated on feeder cells, and stained with hematoxylin at day 7 after plating (top). Phase contrast images of cells are shown below. Scale bars: 30 m. (B) Quantification of colony formation from (A), average SD from n=3 mice in each genotype. (C) Western blot of WT and iKO cultured keratinocytes after 0 or 6 hours of tamoxifen induction, blotted with actin and Gata6 antibodies, show a decrease in Gata6 protein levels after 6 hours. (D) Phase contrast images of WT and Gata6 iKO keratinocytes 3 days after induction with TM (n > 3 per genotype). (E-H) Images and quantification of DNA damage in cultured keratinocytes based on staining for DNA damage marker H2A.X (E and G) and comet assay (F and H). Arrowheads in (F) indicate DNA damaged cell nuclei with comet shape. Quantification of H2A.X (G) represents average SD from at least 300 cells per condition from 3 experiments using different cell lines . *Unpaired t-test P-values: 6h=0.01 and 24h=0.004. (H) Quantification of comet assay represents average SD (n=3). (I-J) FACS cell cycle profiles and quantification show that Gata6 iKO keratinocytes show decreased proliferation and G2-M arrest. (J) Quantification of FACS cell cycle analysis represent average SD (n=3 per genotype). *Unpaired t-test P-values: G1 = 0.03, S = 5x10-4, G2-M = 7x10-4. (K) Quantification of the frequency of H2A.X+ staining in keratinocytes within S phase (EdU+) or another cell cycle stage (EdU-) following a 20 minute EdU pulse labeling (average SD, n=5). *Unpaired t-test P-values WT=0.1, iKO= 0.05. Mann-Whitney U test P-values: WT=0.09, iKO=0.03. (L-M) Cultured keratinocytes were made quiescent by gradual serum depletion. (L) Quantification of EdU+ cells in proliferative and serum depleted quiescent cells (average SD, n=4). (M) Quantification of H2A.X+ keratinocytes during proliferative and quiescent conditions (average SD, n=4). *Unpaired t-test P-value = 0.04. Mann-Whitney U test P-value=0.06.