EMBO/soda-nel huggingface dataset:
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 3

Fig 3. Deletion of COP9/COP9 constitutive photomorphogenic homolog subunit 8 (COPS8) in IECs leads to the loss of the Paneth cell lineage in distal ileum crypts.

(A) Representative hematoxylin and eosin (HE) staining of ileum. Paneth cells with typical eosinophilic granules (black arrows) on HE-stained sections at the base of crypts in Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ), but not COPS8 knockout (KO) (COPS8ΔIEC) epithelium. Scale bar 50 μm, n=7 biological replicate per genotype.

(B) The granule protein, lysozyme, was examined by immunofluorescence (Red) and counted in the ileum of COPS8fl/fl and COPS8ΔIEC mice (scale bar 100 μm). The column graph represents number of lysozyme+ cells presented as mean ± SEM from seven biological replicates. **P<0.01 using Student's t-test.

(C) Transmission electron microscopy (TEM) of crypts of COPS8fl/fl and COPS8ΔIEC mice. The base of the crypt in COPS8ΔIEC mice is occupied by poorly differentiated columnar epithelial cells that lack secretory granules, rudimentary electron-dense granules (black arrows), granules in the lumen (blue arrows) and have a contracted endoplasmic reticulum (ER) (white arrows). Scale bar 5μm, data are from seven biological replicates per genotype.

(D) Goblet cells were stained by Alcian blue and counted in COPS8fl/fl and COPS8ΔIEC epithelia of duodenum and jejunum (scale bar 100 μm). The column graph represents number of Alcian blue+ cells presented as mean ± SEM from seven biological replicates. *P<0.05 using Student's t-test.

(E) The marker for enteroendocrine cells (between two dashed line), chromogranin, was detected by immunofluorescence and counted in the ileum of COPS8fl/fl and COPS8ΔIEC mice (scale bar 100 μm). The column graph represents number of chromogranin A+ cells per crypt-villus presented as mean ± SEM from seven biological replicates. *P<0.05 using Student's t-test.

(F) RNA sequencing-based measurements of transcripts comprising antimicrobial peptides (AMPs)-related genes in crypts isolated from COPS8fl/fl and COPS8ΔIEC mice. Representative data from three biological replicates per genotype.

(G-H) Real-time quantitative reverse transcription Polymerase chain reaction (PCR) (qRT-PCR) analysis of the expression of genes encoding AMPs in the ileum (G) and colon (H) in COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates. *P<0.05, **P<0.01, ***P<0.001 using Student's t-test.
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 4

Fig 4. COP9/COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8) deficiency increases susceptibility to DSS-induced colitis.

(A) Real-time quantitative reverse transcription polymerase chain reaction (PCR) (qRT-PCR) analysis of the differential abundance of selected bacterial taxa in stool samples of ileum and colon of Villin-Cre and COPS8-lox alleles expressing (COPS8fl/fl ) and COPS8 knockout (KO) (COPS8ΔIEC) mice.

Error bars indicate the mean ± SD from three biological replicates per genotype. **P<0.01, NS- nonsignificant using Student's t-test.

(B) qRT-PCR analysis of the abundance of luminal and mucosal bacteria in ileum (top) and cecum (bottom) of COPS8fl/fl and COPS8ΔIEC mice. Error bars indicate the mean ± SD from three biological replicates per genotype. *P<0.05, NS- nonsignificant using Student's t-test.

(C) Scanning electron microscopy of ileum of COPS8fl/fl and COPS8ΔIEC mice. Scale bar 50 μm, representative data from three biological replicates per genotype.

(D-J) Mice were treated by 2% dextran sodium sulfate (DSS) in drinking water for 7 days.

(D) Survival curves of COPS8fl/fl and COPS8ΔIEC mice during DSS-induced colitis. Difference in survival was determined with Kaplan-Meier analysis, **P<0.01. Representative data from 20 biological replicates per genotype.

(E) Changes in body weight are presented as percent of initial weight was determined using Mann-Whitney test, *P<0.05. Representative data from 20 biological replicates per genotype.

(F) Representative hematoxylin and eosin (HE) stained sections of colon and cecum from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 9). Scale bar 100μm, representative data from five biological replicates per genotype.

(G) Histopathologically scored sections of distal colon; cecum and colon length were analyzed from COPS8fl/fl and COPS8ΔIEC mice after DSS-indcued colitis (day 7). Data are represented as mean ± SEM from five biological replicates per genotype. **P<0.01 using Student's t-test.

(H) Cytokine levels (interleukin (IL)-1β, IL-6 and tumor necrosis factor-α (TNF-α)) in the colon collected on day 7 in colitis induced COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from five biological replicates per genotype. *P<0.05, **P<0.01, NS- non-significant using Student's t-test.

(I) The frequency of CD11b+Ly6C+ and CD11b+Ly6G+ cells in colonic lamina propria (cLP) of COPS8fl/fl and or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Ly6C+ and Ly6G+ cells, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test.

(J) Representative FACS plots and percentage of intracellular staining of Forkhead box protein P3 (FOXP3), Interferon gamma (IFN-γ) and IL-17A in CD3+CD4+ T cells from colonic lamina propria of COPS8fl/fl or COPS8ΔIEC mice with DSS-induced colitis. The column graph represents percentage of Treg+ and ratio of Th1/Th17 cells, represented as mean ± SEM from three biological replicates per genotype. *P<0.05 using Student's t-test.
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 5

Fig 5. COP9/COP9 Constitutive Photomorphogenic Homolog Subunit 8 (COPS8) deficiency modifies gut microbiota composition and bacterial diversity that contributes to intestinal inflammation.

(A) Principal components analysis of 16S rRNA gene-sequencing analysis of gut microbes obtained from Villin-Cre and COPS8-lox alleles expressing (COPS8fl/f ) and COPS8 knockout (KO) (COPS8ΔIEC) mice. PC1 and PC2 explain the 64.47% and 27.95% variation, respectively. Representative data from five biological replicates per genotype.

(B) Heatmap of differentially represented bacterial species in feces between COPS8fl/fl and COPS8ΔIEC mice. The top 30 genera that are shared by all of the samples shown. The scale was the percentage composition (log2) based on the 16S rRNA gene sequences analyzed using the Greengene database (gg_otus2013). The hierarchical clustering based on Spearman Rank correlation (average linkage method) was performed using Gene Cluster 3.0 and images generated on Java Treeview. Representative data from five biological replicates per genotype.

(C) The relative abundance of bacteria phylum in the feces of COPS8fl/fl and COPS8ΔIEC mice. Representative data from five biological replicates per genotype.

(D) LEfSe analysis was applied to identify high-dimensional biomarkers that discriminate between feces from COPS8fl/fl and COPS8ΔIEC mice. Representative data from five biological replicates per genotype.

(E) Histologic severity scores of COPS8fl/fl mice and COPS8ΔIEC after fecal transplantation. Data represents the histological score of mice analyzed on day 160. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01 using one-way ANOVA.

(F) Inflammation scores in the colon of 5-month-old COPS8fl/fl and COPS8ΔIEC mice. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.

(G) Percentage of rectal prolapse of 5-month-old mice COPS8fl/fl and COPS8ΔIEC. Data are represented as mean ± SEM from seven biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.

(H) Flow cytometric analysis of CD45+ infiltrating immune cells in the lamina propria of the ileum. Data are represented as mean ± SEM from three biological replicates per genotype. **P<0.01, NS- non-significant using one-way ANOVA.

(I) Real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the expression of genes encoding inflammatory cytokines in the ileum. COPS8fl/fl and COPS8ΔIEC mice were either untreated (phosphate buffered saline (PBS)) or treated with the antibiotic cocktail (Abx) for 3 months. Data are mean ± SEM from three biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 6

Fig 6. Intestinal epithelial COPS8 is required for MBELN-mediated protection against mouse colitis.

(A) Graph showing loss of weight following oral dextran sodium sulfate (DSS) (2%) treatment with and without mulberry bark derived exosome-like nanoparticles (MBELNs) for 7 days (10 x 109 particles/dose/day/mouse) once daily for 7 days in Villin-Cre and COP9/COP9 constitutive photomorphogenic homolog subunit 8 (COPS8)-lox alleles expressing (COPS8fl/fl) and COPS8 knockout (KO) (COPS8ΔIEC) mice. Changes in body weight are presented as percent of initial weight determined using Mann-Whitney test, *P<0.05, **P<0.01, NS- non-significant. Representative data from seven biological replicates per genotype.

(B) Representative image showing changes in colon morphology and length following DSS-induced colitis with concurrent treatment with MBELNs COPS8fl/fl and COPS8ΔIEC mice. Representative data from seven biological replicates per genotype.

(C) Column graph showing changes in colon length. Data are mean ± SEM from seven biological replicates. **P<0.01, NS- non-significant using one-way ANOVA.

(D-E) Hematoxylin and eosin staining (HE) to show histological changes (D) and graph showing severity scores based on histological data (E). Scale bar 200μm, column graph represents colon severity score, presented as mean ± SEM from three biological replicates per genotype. *P<0.05, NS- non-significant using Student's t-test.

(F) Expression of anti-microbial peptide (AMPs) in the colon of COPS8fl/fl and COPS8ΔIEC mice while being treated with MBELNs by oral administration. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA. (G) Protein expression tight junction protein Zonula occludens-1 (ZO-1) using Western blotting in colonic intestine epithelial cells. Data are mean ± SEM from three biological replicates per group. *P<0.05, **P<0.01, NS- non-significant using one-way ANOVA.

(H) Measurement of serum fluorescein isothiocyanate (FITC)-dextran to evaluate the permeability of the intestines. Data are mean ± SEM from five biological replicates per group. *P<0.05, **P<0.01 using one-way ANOVA.

(I-J) Transcriptional expression of nuclear factor kappa B (NF-κB) (I) and inhibitor of nuclear factor kappa-β kinase (IKK-β) (J) mRNA using Real-time quantitative reverse transcription Polymerase chain reaction (qRT-PCR) in colonic epithelial cells upon treatment with MBELNs in addition to DSS. Data are mean ± SEM from three biological replicates per group. **P<0.01, NS- non-significant using one-way ANOVA.
Exosome-like nanoparticles from Mulberry bark prevent DSS-induced colitis via the AhR/COPS8 (2021)
Figure 7

Fig 7. MBELN-derived HSPA8 protein binds to hAhR and activates AhR.

(A) Western blots showing expression of AhR in MC38 cells induced by different fractions (protein, lipid and RNA) derived from Mulberry bark derived exosome-like nanoparticles (MBELNs). Data shown are from three biological replicates.

(B) The effect of different size protein fractions of mulberry bark derived exosome-like nanoparticles (MBELNs) on the expression of aryl hydrocarbon receptor (AhR) demonstrated in Western blots. Data shown are from three biological replicates.

(C) Surface plasmon resonance (SPR) experiment showing MBELN-derived protein binding to recombinant human His-AhR (hAhR) and mass spectrometry (MS) analysis of proteins binding to hAhR on a SPR chip. Data shown are from three biological replicates.

(D) Computational analysis showing docking of Heat Shock Protein Family A (Hsp70) Member 8 (HSPA8; represented in red color) to hAhR (represented in blue color). Data shown are from three biological replicates.

(E) MC38 cells treated with MBELN-HSPA8 showing activation of AhR by Western blot. Data shown are from three biological replicates.

(F) SPR experiment showing recombinant HSPA8 protein interaction with recombinant hAhR on a SPR chip. Data shown are from three biological replicates.

Expandable view figure legends:
DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages (2017)
Figure 2

Figure 2

SAMHD1 inhibits HIV-1 at a post RT step following DNA damage

MDM were transfected with control or pool of SAMHD1 (KD) siRNA and 3 days later treated with 5μM ETO and infected in the presence of ETO with VSV‐G pseudotyped HIV‐1 GFP 18h later. Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).

MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP) or deleted for Vpx (SIV VLP del Vpx) or deleted for Vpr (SIV VLP del Vpr). Cells from a representative donor were used for immunoblotting. The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. (n = 4, mean ± s.e.m.; **P‐value ≤ 0.01; (ns) non‐significant, paired t‐test).

D-F. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIVmac virus‐like particles containing Vpx/Vpr (SIV VLP). DNA was isolated 18 h post‐infection for qPCR quantification of F. Late RT products; G. 2LTR circles; H integrated viral DNA.

Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant, paired t‐test).

2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).

Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).

The percentage of infected cells was quantified by Hermes WiScan and ImageJ 48h post-infection. (n = 3, mean ± s.e.m.; (ns) non‐significant; **P‐value ≤ 0.01, paired t‐test).
DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages (2017)
Figure 3

Figure 3

Vpx Q76A rescues DNA damage induced block to HIV-1 infection

MDM were differentiated and cultured in Human serum instead of FCS.

MDM were differentiated and cultured in FCS. A standard culture condition used in all experiments. See Materials and Methods. .

A-B. MDM were treated with 5μM ETO for 18h and co‐infected in the presence of ETO with VSV‐G HIV‐1 GFP and SIVmac virus‐like particles containing Vpx wild type (WT)/Vpr or Vpx Q76A mutant/Vpr (SIV VLP Q76A). (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).

C-F. MDM we treated with 5μM ETO for 18h and co‐infected in the presence of ETO with HIV-1 BaL and SIV VLP Q76A. DNA was isolated 18 h post‐infection for qPCR quantification of D. Late RT products; E. 2LTR circles; F. integrated viral DNA.

The percentage of infected cells was quantified by the automated cell‐imaging system Hermes WiScan and ImageJ 48h post-infection. Cells from a representative donor were used for immunoblotting. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).

Late viral RT products. AZT: MDM treated with 20μM AZT, a reverse-transcriptase inhibitor, were used as control. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).

2LTR circles. (n = 3, mean ± s.e.m.; (ns) non‐significant; *P‐value ≤ 0.05, paired t‐test).

Integrated copies of viral DNA, Alu-Gag qPCR. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; **P‐value ≤ 0.01, paired t‐test).
DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages (2017)
Figure 4

Figure 4

ETO induced DNA damage does not activate type I IFN response in MDM

MDM were treated with 5μM ETO for 18h, 3μg/ml cGAMP and 10ng/ml IFNβfor 18h. RNA was isolated and qPCR performed for selected genes using TaqMan assays. Expression levels of target genes were normalised to GAPDH. (n = 3, mean ± s.e.m.; *P‐value ≤ 0.05; (ns) non‐significant, paired t‐test).

MDM were treated with 5μM ETO or cGAMP for 18h or 100ng/ml LPS for 2h. Cells were stained and analysed for IRF3 translocation into nucleus. Scale bars: 10μm.

Quantification of nuclei positive for IRF3 staining. (n = 3, mean ± s.e.m.).
DNA damage induced by topoisomerase inhibitors activates SAMHD1 and blocks HIV-1 infection of macrophages (2017)
Figure 5

Figure 5

ETO regulates SAMHD1 phosphorylation through the p53, p21 pathway

MDM were treated with increasing concentrations of ETO and CTH. Cells were stained for MCM2 expression, acquired and analysed using the automated cell‐imaging system Hermes WiScan and ImageJ. On average 104 cells were acquired. (n = 3, mean ± s.e.m.).

B-D. A representative example of 3 independent experiments.

MDM were treated with 5μM ETO, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.

Quantification of specific proteins band intensities from immunoblot in panel B using a CCD camera. Intensities of protein bands were normalized to intensity of actin protein band.

MDM were treated with 5μM ETO or 0.01μM CTH for 18h, lysed and immunoblotting performed to detect cell cycle/cell cycle arrest and DNA damage associated proteins.
BI-ALLELIC PATHOGENIC VARIANTS IN NDUFC2 CAUSE EARLY-ONSET LEIGH SYNDROME AND STALLED BIOGENESIS OF COMPLEX I (2020)
Figure 1

Figure 1. Genetic analyses of subjects with homozygous NDUFC2 variants from two unrelated families.

A Family pedigrees of Subjects 1, 2 and 3 show segregation of identified homozygous NDUFC2 variants within each family.

B Sequencing chromatograms depicting the identified 22 nucleotide deletion (c.346_*7del) identified in Subject 1 (S1), the heterozygous state of the deletion in the father, and the wild-type NDUFC2 sequence in Control DNA.

C Sequencing chromatograms depicting the identified missense variant (c.173A>T; p.(His58Leu)) in Subject 3 (S3) and the wild-type NDUFC2 sequence in Control DNA.

D Clustal Omega sequence alignment shows evolutionary conservation of the p.His58 residue (highlighted) along with its neighbouring residues (human NDUFC2: NP_004540.1).
BI-ALLELIC PATHOGENIC VARIANTS IN NDUFC2 CAUSE EARLY-ONSET LEIGH SYNDROME AND STALLED BIOGENESIS OF COMPLEX I (2020)
Figure 2

Figure 2. Biochemical studies and expression of mRNA transcript level in subject fibroblasts.

A NDUFC2 mRNA transcript levels in fibroblast cell lines of controls (white), Subject 1 (grey) and Subject 3 (black). Quantification of NDUFC2 mRNA transcript levels (normalised to ACTB) showed diminished transcript levels in Subject 1 while transcript levels were unaffected in Subject 3. Bars correspond to standard deviations of two independent experiments, performed in duplicate and analysed by Student's t-test; *p<0.05.

B Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in fibroblast cell lines from controls (white), Subject 1 (grey) and Subject 3 (black). Mean residual enzyme activities of control fibroblasts (n = 8) are set at 100% and error bars represent 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.

C Assessment of respiratory chain enzyme activities, normalised to citrate synthase (CS), in skeletal muscle from controls (white) and Subject 3 (black). Mean residual enzyme activities of control skeletal muscle (n>100 samples) are set at 100% and bars are 1 standard deviation of control activities. Asterisks (*) denote enzyme activity below control range.

D Respiration of Subject 3 and Subject 1 derived fibroblasts (black and grey, respectively) compared to control fibroblasts (white) using high-resolution respirometry. ROX-corrected analysis of basal respiration, Oligomycin inhibited LEAK respiration and maximum uncoupled respiration (ETS). Both subject fibroblasts exhibited a severe mitochondrial respiration defect as measured by oxygen consumption. Data are mean ± SD from at least 3 experiments and analysed by Student's t-test. **p<0.01, ***p<0.001.
BI-ALLELIC PATHOGENIC VARIANTS IN NDUFC2 CAUSE EARLY-ONSET LEIGH SYNDROME AND STALLED BIOGENESIS OF COMPLEX I (2020)
Figure 3

Figure 3. Functional investigation of subject fibroblasts and lentiviral- transduced subject fibroblasts.

A Western blot analysis of various structural subunits of complex I in fibroblasts from Subjects 1 and 3 (S1 and S3 respectively) with three age-matched controls (C1, C2, and C3).

B BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria from subject and control fibroblasts solubilised using DDM. Immunoblotting was performed using antibodies to structural subunits from each OXPHOS complex (CI [NDUFB8], CII [SDHA], CIII [UQCRC2], CIV [MT-CO1], and CV [ATP5A]).

C Western blot analysis of whole cell lysates from controls (C1 and C2) and subject cell lines (S1 and S3) that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 100 or 200 ng/ml doxycycline (for 48 and 96 hours respectively) where indicated.

D BN-PAGE analysis of OXPHOS complex assembly in enriched mitochondria isolated from controls (C1 and C2) and subject cell lines that were either untransduced (-) or transduced with the lentiviral vector (pLVX) containing wild-type NDUFC2 (+). Transduced cell lines were either uninduced or induced with 200 ng/ml doxycycline for 72 hours.