EMBO/soda-nel huggingface dataset:
TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment (2022)
Figure 4

Figure 4. Deletion of Tert leads to decreased ERV expression and compromised interferon response in mice.

A Expression of murine ERVs in blood of WT and G1 Tert-/- mice. n = 5 mice per group.

B Expression of MMERGLN_LTR and MMERGLN-int in livers of WT and G1 Tert-/- mice treated with ENU or saline (control). n = 3 mice per group.

C Enriched biological processes (BP) for WT versus G1 Tert-/- mice. GO was analysed for upregulated genes in WT mice compared to G1 Tert-/- mice under control or ENU treatment.

D Heat map showing log2 fold change differences of interferon-related genes expression between ENU treatment and control (saline) in WT and G1 Tert-/- mice by RNA-seq. n = 3 mice per group.

E Heat map showing log2 fold change differences of cytokine/chemokine between ENU treatment and control (saline) in WT and G1 Tert-/- mice by cytokine/chemokine array. n = 5 mice per group.

F ELISA of CXCL10 in WT and G1 Tert-/- mice treated with poly(I:C) or saline (control). n = 5 mice per group.

Data information: Data represent mean ± SEM (A, B and F). *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. Unpaired two-tailed Student's t-tests were used for A, and two-way ANOVA with Fisher's LSD tests were used for B and F.
TERT activates endogenous retroviruses to promote an immunosuppressive tumour microenvironment (2022)
Figure 5

Figure 5. TERT promotes tumour immune suppression.

A Scatterplot representing differences in ssGSEA of cellular pathways in TERThigh versus TERTlow tumours (n = 4,632 tumours in each group) across TCGA.

B TCGA fragments per kilobase million (FPKM) values of TERT, CXCL10, ISG15, and OASL in tumours grouped in TERThigh (n = 500) and TERTlow groups (n = 500). Data represent mean ± SD. P-values were analysed using unpaired two-tailed Student's t-tests.

C TCGA FPKM values of interferon-related genes in tumours grouped in TERThigh (n = 500) and TERTlow (n = 500) groups. Heat map showing mean FPKM values of each gene.

D and E Expression of TERT (D) and DDX58, IFIH1, CXCL10, IFI44, ISG15, and OASL (E) in TERThigh and TERTlow cancer types (6 types in each). ACC (adrenocortical carcinoma), n = 79. KICH (kidney chromophobe), n = 66. KIRP (kidney renal papillary cell carcinoma), n = 291. LGG (brain lower grade glioma), n = 532. PRAD, n = 502. THCA, n = 513. BLCA (bladder urothelial carcinoma), n = 414. CESC (cervical squamous cell carcinoma and endocervical adenocarcinoma), n = 306. DLBC (lymphoid neoplasm diffuse large B-cell lymphoma), n = 48. LUSC (lung squamous cell carcinoma), n = 502. OV (ovarian serous cystadenocarcinoma), n = 430. All boxplots include the median line, box indicates the interquartile range (IQR) and whiskers denote the 1.5 × IQR. P-values were analysed using two-tailed Mann-Whitney U test.

F Regression analysis of the correlation between TERT expression and percentage of CD4+ cells in colon tumours. n = 36.

G Representative sections of immunohistochemical staining of CD4 and FOXP3 in TERT-ERVshigh and TERT-ERVslow colon tumours. Red arrowheads indicate FOXP3+ cells. Scale bar = 100 μm.

H Schematic model showing TERT contributing to immunosuppressive tumour microenvironment by activating ERVs.

Expanded View Figure legends
Protection of Rhesus Macaque from SARS-Coronavirus challenge by recombinant adenovirus vaccine (2020)
Figure 1.

Immune response and physiology of monkeys immunized with recombinant adenovirus vaccine. a. Time course of antibody response. Serum samples were diluted 40-fold prior to ELISA analysis. Animals in the high-dose and low-dose group were immunized with 1×109 and 1×108 pfu virus each time, respectively. Arrows indicate the time point for immunization. b. Determination of virus neutralization antibody titer using 96-well microneutralization assay. An arbitrate titer of 2 was assigned to animals in the placebo and control group since there was no detectable neutralization activity in these samples. Statistical significance between different data sets was calculated using t-test. c. ELISPOT analysis of IFN-γ response in PBMC samples. Pool A and B contain peptides corresponding to S1-orf8 fusion and N, respectively. Data represent two animals from each group. d. Average food consumption after live virus challenge.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 1

Fig 1. Loss of glycolytic enzymes or Ecdysoneless lowers ATP levels

A Schematic illustration of the working principle of the ATP sensor AT1.03NL. Binding of ATP to the sensing domain (ε subunit of ATP synthase) leads to a conformational change that brings the donor (mse-CFP) and the acceptor (cp173-Venus) closer, resulting in an increase in FRET efficiency (Tsuyama et al., 2013).

B Cartoon to illustrate the experimental setup for inhibiting oxidative phosphorylation (OxPhos) in late third-instar wing discs expressing AT1.03NL. To inhibit OxPhos, dissected wing discs are treated with Antimycin A for 2 h. A reduction in ATP is revealed by a reduced FRET efficiency. The gray frame marks the region of the wing disc shown in FRET efficiency and immunofluorescence (IF) images.

C Cartoon of late third-instar wing disc showing the expression pattern of ap-GAL4, tub-GAL80ts (apGalts) driver. Dashed yellow and green lines indicate the anterior-posterior (A/P) and dorsal-ventral (D/V) compartment boundaries, respectively. The expression pattern of apGalts driver in the dorsal compartment is shown in light red.

D, E Images of FRET efficiency in mock-treated (- ant.A, D) and Antimycin A-treated (+ ant. A., E) ubi-AT1.03NL wing discs at time point 2 h. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars= 50 µm.

F Line graphs showing the FRET efficiency in individual mock-treated (- ant. A, n=26) and ant. A-treated (+ ant. A., n=19) ubi-AT1.03NL wing discs before (0 h) and after 2 h of treatment. Paired t-test, ns= not significant, ***p≤0.001.

G-J Time-controlled knock-down of metabolic enzymes in the dorsal compartment of ubi-AT1.03NL wing discs using the apGalts driver. Images of FRET efficiency in control (G), apGalts>PfkRNAi (H), apGalts>GapdhRNAi (I), and apGalts>Glo1RNAi (J) wing discs after RNAi induction for 48 h (G), 120 h (H, J), and 93 hours (I). A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm.

K Line graphs showing the FRET efficiency in the ventral and dorsal compartment of individual control (n=27), apGalts>PfkRNAi (n=7), apGalts>GapdhRNAi (n=10), and apGalts>Glo1RNAi (n=6) wing discs. Loss of Pfk, Gapdh or Glo1 in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ns= not significant, **p≤0.01, ***p≤0.001.

L Time-controlled knock-down of ecdysoneless (ecd) in the dorsal compartment of ubi-AT1.03NL wing discs using apGalts>ecdRNAi. Image of FRET efficiency in apGalts>ecdRNAi wing disc 48 h after RNAi induction. A rainbow colormap is used to indicate the FRET efficiency levels. Scale bars = 50 µm.

M Line graph showing the FRET in the dorsal and ventral compartment of apGalts>ecdRNAi (n=28) wing discs. Loss of Ecd in the dorsal compartment of the wing disc reduces the levels of ATP. Paired t-test, ***p≤0.001.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 2

Fig 2. Knock-down of Gapdh and over-expression of dominant-negative Ecd result in smaller, rounder adult wings

A-D Adult wing phenotype of knock-down of Gapdh (C765>GapdhRNAi), over-expression of full-length Ecd (C765>ecd), over-expression of C-terminally truncated form of Ecd (C765>ecdDN), and knock-down of ecd (C765>ecdRNAi) in the whole wing disc (see Appendix Fig S3A for the expression pattern of C765-Gal4). Images of adult wings of control (A), C765>GapdhRNAi (A'), C765>ecd (B), C765>ecdDN (C), and C765>ecdRNAi (D) male flies. (A'') Overlay of the wings shown in A and A'. (C') Overlay of control wing and wing shown in C and C'. Loss of Ecd throughout the wing discs produces adults with vestigal wings (D). In contrast, the effects of ecdDN over-expression or Gapdh knock-down are less severe on tissue survival, giving rise to smaller wings with altered proportions. Images consist of a series of tiles that were stitched together by the microscope software. Scale bar= 500 μm.

E, F Tukey box-and-whiskers plot showing quantifications of the area (E) and shape (aspect ratio: major/minor axis, F) of control, C765>GapdhRNAi, C765>ecd, and C765>ecdDN wings. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to ± 1.5 times the interquartile range. Sample size is indicated for each perturbation. Loss of Gapdh or over-expression of EcdDN leads to smaller wings that are increased along the anterior-posterior (A/P) axis compared to the proximal-distal (P/D) axis. Statistical analysis was performed using one-way ANOVA, followed by Dunnett's multiple comparisons test. ***p≤0.001.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 3

Fig 3. Glycolytic activity regulates Smoothened membrane accumulation

A-B' Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A, A') and apGalts>ecdRNAi (B, B') wing discs, stained for the Hedgehog (Hh) pathway components Smoothened (Smo, A, B), and Ci155 (A', B'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=5) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>ecdRNAi (Smo: p≤0.001, Ci155: p≤0.001) wing discs. Thus, loss of Ecd elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.

C-E' Time-controlled knock-down of metabolic enzymes in the dorsal compartment of the wing disc. IF of apGalts>PfkRNAi, apGalts>GapdhRNAi, and apGalts>Glo1RNAi wing discs, stained for Smo (C, D, E) and Ci155 (C', D', E'). Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of apGalts>PfkRNAi (n=5), apGalts>GapdhRNAi (n=5), and apGalts>Glo1RNAi (n=6) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed significant differences in Smo and Ci155 expression in the dorsal compartment between control and apGalts>PfkRNAi (Smo: p≤0.001, Ci155: p≤0.01), apGalts>GapdhRNAi (Smo, Ci155: p≤0.05), or apGalts>Glo1RNAi (Smo: p≤0.001, Ci155: p≤0.05) wing discs. Thus, loss of Pfk, Gapdh or Glo1 elevates Smo levels on the plasma membrane and increases the range of Ci155 stabilization in the anterior compartment. Wing discs were analyzed 120 h after RNAi induction. Scale bars= 50 μm.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 4

Fig 4. Glycolytic activity regulates Smo localization to the primary cilium in mammalian cells

A-D''' IF of NIH3T3 cells stably expressing Smo-mEos2 treated with mock (control, A-A'''), the Smo agonist (+ SAG, B-B''') or the glycolytic inhibitors 3-bromopyruvate (+3-BP, C-C''') and 2-deoxyglucose (+ 2-DG, D-D'''). DAPI staining, shown in gray, was used to visualize cell nuclei. Acetylated tubulin (AcTub), used as a ciliary marker, is shown in magenta, Smo-mEos2 is shown in green. Zoomed-in images of cilia marked by white arrows in A'', B'', C'', and D'' are shown in A''', B''', C''', and D'''. Scale bars= 5 μm, but 2 μm in A''', B''', C''', and D'''.

E, F Box plot showing quantification of Smo-mEos2 ciliary localization in NIH3T3 cells upon 3-BP (E) and 2-DG (F) treatment, respectively. Lower and upper hinges correspond to the first and third quartiles, vertical lines extend to the minimum and maximum values. Sample size is indicated for each perturbation. Treatment with glycolytic inhibitors induces Smo-mEos2 localization to the primary cilium. t-test, ***p≤0.001.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 5

Fig 5. Loss of Ecd sensitizes tissue to Hh by stabilizing Smo

A-B Time-controlled knock-down of ecd in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). IF of control (A) and apGalts>ecdRNAi (B) wing discs, stained for Hh. Next to the images are quantifications of the respective staining in the dorsal versus ventral compartment of control (n=4) and apGalts>ecdRNAi (n=5) wing discs. Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analysis (t-test) revealed no significant differences in Hh expression in the dorsal compartment between control and apGalts>ecdRNAi wing discs. Thus, loss of Ecd does not affect Hh production and spreading. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.

C-F Time-controlled knock-down of ecd in the dorsal compartment of dispatched mutant wing discs (dispS037707, apGalts>ecdRNAi, see Fig 1C for the expression pattern of apGalts). IF of control (C), apGalts>ecdRNAi (D), dispS037707 (E) and dispS037707, apGalts>ecdRNAi (F) wing discs expressing the decapentaplegic (dpp) reporter gene dpp-lacZ (dppZ), stained for dppZ. Next to the images are quantifications of the respective stainings (n=4 (C, F), n=5 (D), n=3 (E)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Statistical analyses (t-test) revealed a significant difference in dppZ expression in the dorsal compartment between control and apGalts>ecdRNAi (p≤0.01) (C vs D), and dispS037707 and dispS037707, apGalts>ecdRNAi (p≤0.01) wing discs (E vs F). Loss of Ecd elevates the expression of dppZ in a broader than normal stripe. However, lack of Ecd function alone is not sufficient to trigger dpp expression, which instead depends on the presence of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.

G-J IF of dispS037707 (G, I) and dispS037707, apGalts>ecdRNAi (H, J) wing discs, stained for Smo (G, H) and Ci155 (I, J). Next to the images are quantifications of the respective stainings (n=2 (G, H), n=3 (I), n=4 (J)). Graphs show mean (thick line) ± SD (thin lines). Dashed yellow lines indicate the position of the A/P boundary. Although statistical analyses (t-test) revealed no significant differences in Smo and Ci155 expression in the dorsal compartment between control and dispS037707, apGalts>ecdRNAi wing discs, loss of Ecd slightly induces an increased Smo accumulation and Ci155 stabilization independently of Hh. Wing discs were analyzed 48 h after RNAi induction. Scale bars= 50 μm.
Glycolysis regulates Hedgehog signaling via the plasma membrane potential (2020)
Figure 6

Fig 6. Loss of Ecd or Gapdh depolarizes the plasma membrane and reduces the uptake of the N-acylethanolamide analogue PAC-NAE

A-A''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). The plasma membrane potential was detected using DiBAC4(3). The fluorescence intensity of DiBAC4(3) increases upon plasma membrane depolarization. (A) The change in plasma membrane potential upon genetic perturbation was quantified as the fold change in the ratio of DiBAC4(3) signal intensity in the dorsal to ventral compartment (control (n=19), apGalts>ecdRNAi (n=36) and apGalts>GapdhRNAi (n=18) wing discs). Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. (A'-A''') Representative images of DiBAC4(3) assay in control (A'), apGalts>ecdRNAi (A''), and apGalts>GapdhRNAi (A''') wing discs 48 h or 120 h after RNAi induction, respectively. V/D indicates the boundary between the ventral and dorsal compartments. Next to the maximal projections are shown the sum projections of cross-sections along the A/P axis. Loss of Ecd or Gapdh alters the plasma membrane potential. Scale bars= 50 μm.

B Structure of the N-acylethanolamide analogue PAC-NAE. PAC-NAE contains a C15 fatty acid featuring diazirine and alkyne (red) modifications.

C-C''' Time-controlled knock-down of ecd or Gapdh in the dorsal compartment of the wing discs (see Fig 1C for the expression pattern of apGalts). PAC-NAE uptake was quantified as the ratio of PAC-NAE signal intensity in the dorsal to ventral compartments. Shown is the fold change from control in apGalts>ecdRNAi and apGalts>GapdhRNAi wing discs (C). For each condition, dorsal/ventral PAC-NAE signal intensity was quantified for 5 wing discs. Error bars indicate ± SD. test-test, **p≤0.01, ***p≤0.001. (C'-C''') Representative images of PAC-NAE uptake in control (C'), apGalts>ecdRNAi (C'') and apGalts>GapdhRNAi (C''') wing discs at 48 h or 120 h after RNAi induction, respectively. Loss of Ecd or Gapdh blocks the uptake of PAC-NAE. Dashed green lines mark the D/V boundary. Scale bars= 50 μm.

D-E'' Dissipation of the plasma membrane potential upon treatment of wing discs with gramicidin A (gA). (D) Quantification of DiBAC4(3) signal intensity in mock-treated (- gA, n=7) and gA-treated (+ gA, n=5) wing discs. (D', D'') Representative images of DiBAC4(3) assay in mock- (D') and gA-treated (D'') wing discs. Next to the maximal projections are shown sum projections of cross-sections along the A/P axis. (E) Quantification of PAC-NAE signal intensity in mock- (n=7) and gA-treated (n=9) wing discs, shown as fold change from control (mock-treated). (E'-E'') Representative images of PAC-NAE uptake in mock- (E') and gA-treated (E'') wing discs. GA treatment strongly reduces the uptake of PAC-NAE. Error bars indicate ± SD. t-test, *p≤0.05, ***p≤0.001. Scale bars= 50 μm.

EXPANDED VIEW FIGURE LEGENDS
USP17 is required for peripheral trafficking of lysosomes (2021)
Figure 1

Figure 1- USP17 is necessary for peripheral lysosome trafficking.

A HeLa cells were transfected with constructs for a non-targeting shRNA (NT), USP17 shRNA1, or USP17 shRNA2. Cells were stained using an anti-LAMP1 antibody (green) and the nuclei counterstained with DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line.

B HeLa and MDA-MB-231 cells were transfected as in A and the distribution of LAMP1 positive vesicles was plotted as vesicle relative position (see C).

C Vesicle relative position is the ratio between vesicle distance from the nucleus border and the sum of the vesicle's distances from the nucleus and cell border. If the vesicle is located within the nucleus the value is set to 0. If the value is 1 then the vesicle is located at the border of cell.

D HeLa cells transfected as in A, in conjunction with a construct coding GFP tagged CD63. The lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line.

E HeLa cells transfected as in A, in conjunction with constructs for either GFP tagged LAMP1 or CD63. The distribution of GFP positive vesicles plotted as vesicle relative position.

F HeLa cells were transfected with empty vector, or expression constructs for USP17, or USP17CS (inactive mutant). 48 hours post-transfection lysates and cell growth media were harvested and immunoblotted for GAPDH, USP17 and CatD, as indicated.

G HeLa cells were transfected as in 1F. 48 hours post-transfection lysates were harvested and 5µg of protein used in a CatD/E activity assay. Relative RFU of triplicate samples at 60 minutes, compared to empty vector control are plotted. The results plotted are representative of results obtained in three separate experiments.

Data information: In (A, C), scale bars 25µm. In (B, D), vesicle position of at least 350 vesicles from a number of cells (n) from a series of confocal images across three separate experiments were analysed using the IMARIS software package and the distribution of vesicles plotted as vesicle relative position (mean value shown as red bar). Error bars represent standard error and **** indicates a p-value less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.
USP17 is required for peripheral trafficking of lysosomes (2021)
Figure 2

Figure 2: USP17 is necessary for EGF-mediated peripheral lysosome trafficking

A HeLa cells were transfected with constructs for a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2. 48 hours post transfection the cells were either serum starved (upper panels), or treated with serum free medium containing 100ug/ml EGF (lower panels) for 16 hours prior to staining for LAMP1 (green) and DAPI (blue). Right hand panels are enlarged images of the indicated area in left panels and the cell membrane is marked by dotted line. Scale bar represents 25μm.

B The distribution of at least 300 LAMP1 positive vesicles from a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicates not significant, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.
USP17 is required for peripheral trafficking of lysosomes (2021)
Figure 3

Figure 3: USP17 is required for plasma membrane repair.

HeLa cells were transfected with constructs encoding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated.

A 48 hours post transfection the cells were washed with Ca2+ and Mg2+ free ice cold HBSS. The different cell populations were split into three aliquots. One aliquot was placed into fresh medium (Left panels). SLO was added to other two aliquots and then the toxin-containing medium was removed and the aliquots were either placed into Ca2+ and Mg2+ free HBSS (Middle panels), or EBSS containing Ca2+ and Mg2+ (Right panels) prior to staining with propidium iodide and analysis by flow cytometry.

B The percentage plasma membrane repair (PMR) was calculated according to the following formula: 1 − (% PI-positive cells with Ca2+ / % PI-positive cells without Ca2+) × 100. Three separate experiments were analysed and plotted. Error bars represent standard error, and * indicates a p-values less than 0.05. One way ANOVA used to determine if statistically significant differences between groups.
USP17 is required for peripheral trafficking of lysosomes (2021)
Figure 4

Figure 4: USP17 opposes the action of RNF26 and deubiquitylates p62.

A HeLa cells were transfected with negative control siRNA and RNF26 siRNA as well as constructs coding a non-targeting (NT) shRNA, USP17 shRNA1 or USP17 shRNA2, as indicated. 48 hours post transfection cells were stained for LAMP-1 (green) and DAPI (blue). Lower panels are enlarged images of indicated area in top panels and the cell membrane is marked by dotted line. Scale bar represents 25μm.

B The distribution of at least 400 LAMP1 positive vesiclesfrom a number of cells (n) from a series of confocal images across three separate experiments was plotted as vesicle relative position (mean value is red bar). Error bars represent standard error, ns indicate not significant, ** indicates a p-values less than 0.01, and **** indicates a p-values less than 0.0001. One way ANOVA used to determine if statistically significant differences between groups.

C HeLa cells were transfected with expression constructs for HA-tagged p62 and FLAG-tagged ubiquitin as well as empty vector, USP17 or USP17CS. After 48hrs cell lysates were prepared and HA tagged proteins were pulled down using anti-HA agarose. Pull downs, and lysates, were immunoblotted with anti-HA, anti-FLAG, and anti-USP17 antibodies, as indicated, to confirm the presence of ubiquitinated p62.