EMBO/soda-nel huggingface dataset:
Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy (2014)
f3

(a) Intracellular ROS production was assessed using DCFH-DA. (b) Plasma membrane calcium influx was measured by analysing the quench of Fura-2 fluorescence upon addition of extracellular Mn2+. (a,b) Were conducted in enzymatically digested single FDBs from WT, mdx and p47−/−-mdx mice. Bars represent average±s.e.m. from n=15 individual fibres for each condition. (c) Immunoblot analysis of Src protein with anti-P-Src or anti-Src antibodies. (d) Autophagy marker proteins were analysed by immunoblotting with antibodies as indicated (c) and (d) were conducted in enzymatically isolated fibres from FDBs of WT, mdx and p47−/−-mdx mice. Representative images are shown. GAPDH was detected as a loading control. Bars represent average±s.e.m. from n=3 independent biological experiments. (e) LC3-LAMP1 colocalization in single FDBs from mdx and p47−/−-mdx mice was analysed using confocal microscopy. Representative images from n=3 independent biological experiments are shown (scale bar=100 and 40 μm for red box areas). (f) Immunofluorescence assay to detect lysosomes (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. White arrows indicate lysosome and yellow arrows indicate nucleus. (g) Immunohistochemistry to detect lysosome (LAMP1) in TA muscle from WT, mdx and p47−/−-mdx mice. Black arrows indicate LAMP1 positive (brown) structures. Histogram plot quantifying the number of immunopositive LAMP1 structures per fibre. Representative images from n=3 independent biological experiments are shown. Scale bar represents 100 μm for f and 140 μm for g. Statistical differences between groups were determined using ANOVA with Tukey’s post hoc test. *P0.05 and **P0.01.
Src-dependent impairment of autophagy by oxidative stress in a mouse model of Duchenne muscular dystrophy (2014)
f4

(a) Haematoxylin and eosin staining of cross-sections from Dia showing central nuclei (arrow head) and smaller fibres (arrow). (b) Immunoblot analysis of macrophage content (CD68). (c) Immunofluorescent and bright field images of Dia cross-sections showing fibre-type distribution. Type I (red), IIA (green), IIB/IIX ( white x, unstained and viewed from bright field overlay). (d) Serum creatine kinase activity. (e) Force–frequency relationship in Dia muscle strips from WT (black), mdx (red) and p47−/−-mdx (blue) mice. Scale bars represent 55 μm. For e, #P0.01 p47−/−-mdx versus mdx.##P0.01 p47−/−-mdx versus WT and mdx. Mdx was statistically different than WT at all frequencies of stimulation. Statistical differences between groups were determined using ANOVA with Tukey’s post hoc test. *P0.05 and **P0.01.
Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation (2019)
Figure 4

Figure 4: Schistosomal-EVs harbor miRNAs.

Schistosomal- miRNA was extracted from either EVs that were isolated from schistosomal-growing medium or control pellets that were isolated from unused schistosomal-medium as described in the Materials and Methods (Schistosomal-EVs purification section). Equal aliquots were subjected to qRT-PCR with specific primers to the schistosomal-miR-10, Bantam and miR-125; the expression is presented as ∆Ct from background Ct that was set as 40 cycles. The mean +/- SEM was calculated from 3 independent experiments. P-value was calculated by t-test. * p<0.05, **p<0.01.
Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation (2019)
Figure 5

Figure 5: Schistosomal-EV-enclosed miRNAs penetrate Th cells.

A-C) The trans-well system was performed as in Fig. 1. Schistosome-exposed Th cells or control Th cells were subjected to qRT-PCR with specific schistosomal-Taqman primers to (A) miR-10-5p, (B) Bantam, and (C) miR-125b.

D-F) The trans-well system was performed as in Fig. 1. The Control wells contained only unused schistosomal-medium; Supernatant wells contained 0.1 μm of the filtered schistosomal-growing medium; Th wells contained sorted CD4+CD11c- from spleens; Th+APCs contained sorted CD4+CD11c- and CD4-CD11c+ in 5:1 ratio, respectively. In Data information: In (A-E) the expression is presented as arbitrary units calculated as 2∆∆CT of miRNA relative to snRNA U6 and in (F) mRNA relative to β2 microglobulin. The mean +/- SEM was calculated from at least 3 independent experiments (except in F of 2 independent experiments). P-value was calculated by Wilcoxon signed-rank t-test. *p<0.05, **p<0.01.
Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation (2019)
Figure 6

Figure 6: Schistosomal-miRNAs are found in lymph node-derived Th cells.

A) Image of a schistosome-infected mouse. The lymph nodes, from which the Th cells were isolated, are indicated.

B) Th cells were isolated from the indicated lymph nodes and spleen. In the case of uninfected mice, the Th cells from different lymph nodes and the spleen were mixed. qRT-PCR was performed with specific Taqman primers to either schistosomal-miR-10 or -Bantam. The presence of the miRNAs is presented as arbitrary units calculated as 2∆∆CT of the miRNA divided by snRNA U6. The mean +/- SEM was calculated from 4 independent experiments. P-value was calculated by Mann Whitney t-test. *p<0.05, **p<0.01.
Schistosomal extracellular vesicle-enclosed miRNAs modulate host T helper cell differentiation (2019)
Figure 7

Figure 7: Live Schistosoma and its miR-10 affect the expression of MAP3K7.

A) The putative binding sites of miR-10 in the MAP3K7 3'UTR (taken from http://www.targetscan.org/vert_72/). Above the wild type seed sequence, in red, the sequences that were placed in the mutated plasmid.

B) HEK-293 cells were transfected with a plasmid expressing schistosomal-miR-10 at the indicated concentration together with either psiCHECK-II vector, psiCHECK-WT-MAP3K7-3'UTR or psiCHECK-mut-MAP3K7. After 24h, the cells lysates were subjected to luciferase assay. The results are presented as the ratio of renilla/luciferase expression that was normalized relative to cells transfected only with psiCHECK-II. Values are expressed as the mean+/-SD of at least 3 independent experiments. Statistics were performed using t-test *p<0.05, ***p < 0.005.

C) A representative experiment in which protein extracted from Jurkat-T cells stably over expressing stably control vector or miR-10 expressing vector were subjected to WB analysis with anti-MAP3K7 or anti-GAPDH antibodies.

D) The graph presents densitometry analysis of 3 WBs analyses performed as in (C). The mean +/- SD was calculated from 4 independent experiments. Statistics were performed using t-test (*p<0.05).

E) Jurkat-T cells stably expressing either control vector or a vector containing schistosomal-miR-10 were co-transfected with a control Luciferase plasmid or NF-κB luciferase reporter vector. In all transfections, a plasmid expressing Renilla vector as an internal control was added. 48h post-transfection, cells were harvested and were analyzed by luciferase reporter assay. Values were normalized to the Renilla activity. Data are represented as mean +/-SEM from 5 independent experiments. Statistics were performed using t-test, **p<0.01.

F-I) Representative WB experiment demonstrating that MAP3K7 is downregulated in schistosomal-exposed Th cells (F) or to Jurkat (H) in comparison to the control unexposed cells. (G, I) The graphs present densitometry analysis of 3 WB analyses performed as in (F and H), respectively. The mean +/- SD was calculated from 3 independent experiments. Statistics were performed using paired one-tailed t-test (*p<0.05).
Hedgehog signaling can enhance glycolytic ATP production in the Drosophila wing disc (2022)
Figure 3

Figure 3. Hh activity promotes glycolytic ATP production upon OxPhos inhibition.

(A-B) Schematics indicating where the RNAi for Ptc was induced (A) and where Ptc and Hh are normally expressed (B). Dark and light magenta indicate high vs low Ptc expression, respectively. Sub-compartments enclosed with dashed lines indicate regions analyzed in (D-G). V=Ventral, D=Dorsal, A=Anterior, P=Posterior. (C) Timelapse of ATP sensor FRET efficiency in the apGalts>PtcRNAi wing pouch after 10 μM antimycin A (ant.A) addition. Red dashed line indicates the position of the AP boundary. (D, F) Mean FRET efficiency analyzed over time in the (D) dorsal anterior (DA) and ventral anterior (VA) sub-compartments or the (F) dorsal anterior (DA) and dorsal posterior (DP) sub-compartments of apGalts>PtcRNAi wing discs. Shaded regions indicate SD; dots represent the mean per timepoint; solid lines illustrate a fit of the mean data. (E, G) Kinetic parameters of the fit line for ATP decline in the DA and VA compartments (E) or DA and DP compartments (G). Lines connect the corresponding regions of the same disc; dots represent the average per timepoint; box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median. * = p-value < 0.05, ns = not significant p-value using a Kruskal-Wallis test (n=11 discs). (H) Schematic showing the dorsal expression of the dominant negative Ci allele (CiDN). (I) Timelapse of ATP sensor FRET efficiency in apGalts > CiDN wing discs after 10 μM antimycin A addition. (J, K) (J) Mean FRET efficiency measured over time in the dorsal and ventral compartments in apGalts > CiDN wings. Shaded regions indicate SD; dots represent the average per timepoint; solid lines illustrate a fit of the mean data. (K) Kinetic parameters of the fit line for ATP decline in dorsal and ventral compartments. Lines connect the corresponding regions of the same disc; dots represent the average per timepoint; box plots summarize the data: boxes encompass the 2nd-3rd quartiles, with whiskers indicating the 1st and 4th quartiles and the red line indicating the median ** = p-value < 0.01, *** = p-value < 0.001 using a Kruskal-Wallis test (n=13 discs). Black arrows indicate the addition of the drug.
βH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination (2020)
Figure 1

Figure 1. βH-spectrin co-localizes with myosin-II to medio-apical fibers in mesodermal cells during ventral furrow formation.

(A, B) Schematic illustration of a constricting cell undergoing a cycle of ratcheted pulsatile constriction. Upregulation of myosin-II (blue) causes coalescence of actin fibers (red) in a radially polarized manner. Contraction of medio-apical actomyosin fibers, which are anchored to the plasma membrane (black) through adherens junctions (purple dots) and are mechanically coupled to a junctional actomyosin belt (red), causes constriction of the cell surface. Disassembly of medial actomyosin is accompanied by surface relaxation, which is contained by a ratcheting mechanism that stabilizes the constricted state. Repeated cycles of actmyosin-mediated ratcheted contraction result in an incremental shrinkage of the cell surface. (B) Schematic illustrating medio-apical (cyan) and junctional (purple) actomyosin fibers.

(C) Confocal image of a constricting cell during ventral furrow formation showing an overlay between the myosin-II probe Sqh::GFP and the plasma membrane marker GAP43::mCherry. Scale bar, 5 μm

(D) Movie (still frames) of a gastrulating Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin imaged in a cross section using two-photon microscopy. βH-spectrin is enriched at the apical surface of ventral mesodermal cells during tissue invagination (arrowheads). Scale bar, 50 μm.

(E) Z-Projections of confocal image stacks showing the apical cell surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry at 5 min (top), 7 min (middle) and 9 min (bottom) after initial medio-apical accumulation of βH-spectrin. Scale bar, 20 μm

(F, G) Immunostaining of βH-spectrin (green) visualized by STED nanoscopy revealed medio-apical βH-spectrin supra-cellular fibers in mesodermal cells (F). In ectodermal cells, βH-spectrin localizes to apical cell junctions (G). Note, in (F) the junctional βH-spectrin signal is shown in magenta as a proxy for cell membranes. Scale bars, 2.5 μm.

(H-J) Confocal images of the ventral surface of a Drosophila embryo expressing endogenously tagged mVenus::βH-spectrin (H), the myosin-II marker Sqh::mCherry (I) and a merge of the two (J) with βH-spectrin in green and Sqh::mCherry in magenta. Scale bars: 20 μm.

(K-L) Co-staining of phalloidin (F-actin reporter; magenta) and βH-spectrin (green) of the apical surface of a mesodermal cell at the onset of ventral furrow formation (K) and at a later time point (L) visualized by confocal microscopy. White dashed lines indicate the cell boundaries segmented based on the phalloidin-staining of sub-apical confocal sections. Scale bars: 5 μm.
βH-spectrin is required for ratcheting apical pulsatile constrictions during tissue invagination (2020)
Figure 2

Figure 2. Knock-down of βH-spectrin impairs apical constriction and tissue invagination.

(A) Z-Projection of confocal image stacks spanning 5 μm of a control embryo (left) co-expressing endogenously tagged mVenus::βH-spectrin (green) and the plasma membrane marker GAP43::mCherry (magenta) and of an embryo additionally expressing a maternally driven anti-GFP nanobody KD module to knock-down βH-spectrin (right). Co-expression of the anti-GFP nanobody KD system resulted in a drastic decrease of βH-spectrin protein levels as assessed by the fluorescent signal intensity which dropped below the detection limit.

(B) Pie diagrams quantifying the percentage of control embryos (top, n=50) and βH-spectrin knock-down embryos (bottom, n=40) that formed ventral furrow either normally or with minor phenotypes (green) and the percentage of embryos that displayed severe abnormalities or did not at all undergo tissue invagination (red). The assessment of phenotypes was done in an unbiased randomized approach.

(C-J) Confocal images of a control embryo (C,D) and βH-spectrin knock-down embryos with a minor phenotype (E,F), with a severe phenotype that underwent tissue invagination (G, H) and with a severe phenotype that did not undergo tissue invagination (I, J). Surface view is shown in (C,E,G,I) and the cross section through the tissue at two different time points in (D,F,H,J). All embryos expressed the plasma membrane marker GAP43::mCherry. Scale bars, 20 μm.

(K, L) Quantification of apical surface area (K) and anisotropy (L) in control embryos (green, nControl: 6 embryos) and in βH-spectrin knock-down embryos (red, nβH-spectrin KD: 7 embryos). Solid lines indicate the mean values and the semi-transparent regions the corresponding standard deviation.
TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation (2022)
Figure 1

Figure 1. TRIM33 is a novel protein interactor of AR

A, MS-ARC graphs showing AR-associated proteins with DHT stimulation in LNCaP cells (left) and VCaP cells (right). The proteins were clustered according to their molecular function. The length of each line represents the ratio of normalized LFQ intensity between the AR pull-down and the IgG pull-down.

B, Peptide coverage of TRIM33 for the corresponding RIME experiment.

C, Validation of the AR and TRIM33 interaction by co-immunoprecipitation assays. Endogenous AR protein in LNCaP cells (left) or VCaP cells (right) were immunoprecipitated and detected for TRIM33 by immunoblotting. IgG served as a negative control.

D, In vitro GST pull-down assays were performed with bacterially expressed and purified GST or GST-tagged AR immobilized onto GSH beads. Immobilized GST protein beads were used to pull down purified his-MBP (left) or his-MBP-tagged TRIM33 (right).
TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation (2022)
Figure 3

Figure 3. TRIM33 enhances the AR transcriptional program

A-C, AR-dependent luciferase assays were performed on LNCaP cells transfected with siTRIM33 or negative control (NC) (A), increasing amounts of TRIM33 plasmid (B), or TRIM33 plasmid and/or siAR (C). Western blot analysis shows the protein level of TRIM33 in LNCaP cells after TRIM33 knockdown (A) or overexpression (B).

D, LNCaP cells (left) and VCaP cells (right) were transfected with the indicated siRNAs and treated with EtOH or 10 nM DHT for 12 h. Gene expression was measured by RT-qPCR after 48 h. Data were normalized against GAPDH.

E, LNCaP (left) or VCaP (right) cells were transfected with the indicated amount of TRIM33 plasmid and treated with EtOH or 0.1 nM DHT for 12 h. Gene expression was measured by RT-qPCR after 48 h. Data were normalized against GAPDH.

F, GSEA was performed to determine the enrichment of androgen-induced (upper panel) or -repressed gene set (lower panel) in siTRIM33-regulated gene set identified by RNA-seq in LNCaP cells treated with DHT.

G, Gene expression of LNCaP cells treated with siTRIM33 or NC were profiled by RNA-seq. A total of 414 co-induced genes (androgen-induced and siTRIM33 suppressed) and 536 co-repressed genes (androgen-suppressed and siTRIM33 induced) were identified and clustered across all samples.

H, Functional enrichment analysis showing the main biological processes and pathways significantly enriched by AR-TRIM33 coactivated genes (p < 0.05, DAVID 6.8).

Data information: In (A-E), data are presented as mean ± SD (n = 3 independent experiments). *P < 0.05; **P < 0.01; ns=not significant (two-way ANOVA).
TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation (2022)
Figure 5

Figure 5. TRIM33 prevents Skp2-mediated proteasomal degradation of AR

A-D, AR transcript levels were measured by RT-qPCR on LNCaP cells (A and C) or VCaP cells (B and D) transfected with siTRIM33 or the TRIM33 expression construct and treated with EtOH or DHT. Protein lysates were harvested at 72 h after transfection and subjected to western blot analysis with the indicated antibodies.

E and F, LNCaP cells transfected with siTRIM33 or NC were treated with cycloheximide (CHX). Protein lysates were harvested at the indicated time points for western blot analysis. AR protein levels were quantified and normalized to β-actin and then plotted relative to 0 h.

G, LNCaP cells transfected with siTRIM33 or NC were treated with DMSO or MG132 for 8 h. Protein lysates were extracted and subjected to western blot analysis with the indicated antibodies.

H and I, HA-Ub was expressed in LNCaP cells co-transfected with siTRIM33 (H) or the TRIM33 expression construct (I). The cells were treated with DMSO or MG132 for 4 h before harvesting. Endogenous AR was immunoprecipitated from cell lysates, and interacting proteins were detected by immunoblotting with the indicated antibodies.

J and K, LNCaP cells transiently expressing either myc-tagged Skp2 or myc-tagged SPOP with Flag-TRIM33 were harvested and subject to immunoblot analysis with the indicated antibodies.

L, shTRIM33- or shCtrl-LNCaP cell lines transfected with NC or siSkp2 were harvested 72 h after transfection and subjected to immunoblot analysis with the indicated antibodies.

M, HEK293T cells were co-transfected with flag-TRIM33 and various myc-tagged AR constructs. AR-FL and AR truncated mutants were immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.

N, HEK293T cells were co-transfected with myc-AR-N and various flag-tagged TRIM33 constructs. AR-N was immunoprecipitated with an anti-myc antibody and then subjected to western blot analysis with the indicated antibodies.

O, LNCaP cells were transfected with various flag-tagged TRIM33 constructs. AR-associated proteins were immunoprecipitated with an anti-AR antibody and then subjected to western blot analysis with the indicated antibodies.

P, LNCaP cells were co-transfected with myc-Skp2 and various flag-TRIM33 constructs. AR and other proteins were detected by western blot analysis with the indicated antibodies.

Q, AR pull-down was performed on protein extracts from MG132-treated HEK293T cells overexpressing myc-AR, HA-Ub, myc-Skp2, and various flag-tagged TRIM33 constructs. AR ubiquitination and specific protein levels were detected by western blot analysis with the indicated antibodies.

R-S, LNCaP cells were transfected with various flag-TRIM33 constructs. AR binding was detected by ChIP-qPCR (R), while the expression of AR target genes was detected by RT-qPCR (S).

Data information: In (A-D, F, R, and S), data are presented as mean ± SD (n = 3 independent experiments). * P < 0.05, **P < 0.01, ns=not significant (two-way ANOVA).
TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation (2022)
Figure 6

Figure 6. TRIM33 is overexpressed in PCa

A, TRIM33 mRNA expression levels were analyzed in normal and tumor samples from multiple publicly available patient cohorts. A two-sided Wilcoxon signed-rank test was used for the TCGA Prostate, Ding 2016, and Jing 2020 datasets, while a two-sided Wilcoxon rank-sum test was used for the other datasets. Measurements shown in box plots are the first and third quartiles and are split by the medians, whiskers extending a 1.5-fold interquartile range beyond the box.

B and C, A human PCa progression tissue microarray was used for immunohistochemistry staining. Representative staining images are shown in B. TRIM33 staining by Gleason Scores is summarized in the histogram. Scale bar: 100 μm.
TRIM33 drives prostate tumor growth by stabilizing androgen receptor from Skp2-mediated degradation (2022)
Figure 8

Figure 8. TRIM33 is required for prostate tumor cell growth in vitro and in vivo.

A and B, AlamarBlue assays were performed on LNCaP cells (A) and VCaP cells (B) treated with siTRIM33 or NC at the indicated times (n = 3 independent experiments).

C-F, Flow cytometry analyses were performed on LNCaP cells transfected with siTRIM33 or NC for detecting cell cycle (C) and cell apoptosis (E). The bar charts show the distribution of cells in each cell cycle phase from C (D) or the percentage of apoptotic cells from E (F) under indicated conditions (n = 3 independent experiments).

G-I, TRIM33 depletion reduced prostate xenograft tumor growth. LNCaP cells stably expressing shCtrl or shTRIM33 were inoculated in the right flanks of male nude mice. Tumor growth was measured twice per week, as shown in G. Tumors were excised at the endpoint (H) and weighed (I). (n = 4 mice/group).

J, Western blot analysis of AR and TRIM3 protein levels in tumor tissues from H.

K, AlamarBlue assay was used to measure the viability of shTRIM33- or shCtrl-LNCaP cell lines with 72 h of treatment of different amounts of Casodex (upper panel) or MDV3100 (lower panel). Cell viability values of cells under specific conditions were normalized to the cell viability values of untreated cells. Cytotoxicity plots (left panels) represent n = 3 independent experiments with 6 replicates per drug concentration for each experiment. Bar graphs (right panels) show the IC50 value.

L, A model depicting the role of TRIM33 on AR signaling in PCa. TRIM33 is relatively lowly expressed in normal conditions, allowing Skp2 to ubiquitinate and degrade AR. In PCa where TRIM33 is highly expressed, TRIM33 binds to AR and prevents AR from Skp2-mediated ubiquitination and subsequent proteasomal degradation, thereby enhancing AR signaling and driving PC tumor growth. The model was created with BioRender.com.

Data information: In (A, B, E, F, G, I, and K), data represents the mean ± SD.*P < 0.05; **P < 0.01 (two-way ANOVA).

Legends for EV figure