EMBO/soda-nel huggingface dataset:
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 7.

The first 30 amino acids of TFEB are sufficient for binding to active Rag heterodimers. (A) ARPE-19 cells coexpressing active RagB/D heterodimer and the indicated TFEB plasmids were fixed, permeabilized with 0.2% saponin, and stained with antibodies against GST (used to detect Rag proteins). Regions within the dotted boxes are magnified in the insets. Bars, 10 µm. (B) ARPE-19 cells were cotransfected with active RagB/D heterodimers and the indicated TFEB constructs. After 18 h, cell lysates were immunoprecipitated with the anti-HA antibody (used to immunoprecipitate Rag proteins) and immunoblotted with antibodies against GFP and GST (used to detect TFEB-GFP and Rag proteins, respectively). The white line indicates that intervening lanes have been spliced out. (C) Immunofluorescence confocal microscopy showing the subcellular distribution of TFEB-(1–30)-GFP in ARPE-19 cells coexpressing active RagB/D heterodimers (antibodies against GST were used to detect Rags). Bar, 4 µm. (D) Relative RT-PCR analysis of the mRNA expression of autophagy (ATG9B and UVRAG) and lysosomal (MCOLN1) genes in ARPE-19 cells infected with the indicated adenovirus for 48 h. The values are expressed as a ratio to RNA from cells infected with control adenovirus (Ad.-Null). Values are means ± SD of two independent experiments. ***, P < 0.001; **, P < 0.01. (E) Cells were infected with adenovirus as in D and analyzed by immunoblotting with the indicated antibodies. IP, immunoprecipitation.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 8.

Rag GTPases and mTORC1 regulate the function of MITF. (A) Multisequence alignment of the first 30 amino acids of TFEB with MITF isoforms 1, 2, 3, and 7. The green arrows indicate the residues identified as essential for the interaction between TFEB and Rag proteins. Blue letters are to indicate conserved amino acid homology between the different proteins. (B–D) ARPE-19 cells were infected with adenovirus expressing MITF1-FLAG. 16 h later, cells were incubated with DMSO or 250 nM Torin-1 for 1 h and analyzed by immunoblotting (C) or immunofluorescence (B and D) with anti-FLAG antibodies. The region within the dotted box is magnified in the insets. Bars: (B and D, main images) 10 µm; (D, insets) 5 µm. (E) ARPE-19 cells were incubated with 250 nM Torin-1 for 1 h. Cells were then lysed and analyzed by immunoblotting with antibodies against MITF. (F) HEK-293T cells were incubated in medium containing DMSO or Torin-1 for 1 h. Cells were lysed, and nuclei and membrane plus cytosol fractions were obtained by low speed centrifugation. Proteins from the different fractions were subjected to immunoblotting with antibodies against MITF, Lamp1, and Histone H3. (G) ARPE-19 cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were immunoprecipitated with the anti-FLAG antibody and immunoblotted with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). (H) HEK-293T cells expressing active (RagBGTP/RagDGDP) or inactive (RagBGDP/RagDGTP) Rag heterodimers were pulled down using glutathione–Sepharose beads and immunoblotted with antibodies against GST and MITF (used to detect Rag proteins and endogenous MITF, respectively). IP, immunoprecipitation.
Rag GTPases mediate amino acid-dependent recruitment of TFEB and MITF to lysosomes. (2013)
Figure 9.

Rag GTPases regulate recruitment of MITF and TFEB to lysosomes. (A and B) ARPE-19 cells were nucleofected with the indicated Rag- and MITF-expressing plasmids. 12 h later, cells were fixed, permeabilized with 0.2% Triton X-100, and double stained with antibodies against FLAG and GST (used to detect MITF and Rag proteins, respectively). Bars, 10 µm. (C) Model representing the mechanism of TFEB and MITF regulation by Rag GTPases. (top) In nutrient-rich conditions, active Rags promote recruitment of mTORC1 and TFEB to lysosomes, thus facilitating mTORC1-dependent phosphorylation of TFEB. Phosphorylation of TFEB at S211 creates a binding site for 14-3-3 and results in sequestration of TFEB in the cytosol. We suggest that 14-3-3 may mask the Rag-binding domain in TFEB (represented in yellow). (bottom) In the absence of amino acids, Rag GTPases and mTORC1 are inactivated. Dissociation of the TFEB–14-3-3 complex allows transport of TFEB to the nucleus and TFEB-mediated activation of a transcriptional network that promotes autophagy, lysosomal biogenesis, and increased lysosomal degradation. Our model proposes that some MITF isoforms might be regulated in a similar manner. The interaction of MITF-1 with 14-3-3 has been previously described (Bronisz et al., 2006). Please note that mTORC1-dependent phosphorylation of MITF-1 S280 has not been reported and is merely speculative. The representation of the Ragulator–Rag–mTORC1 complex is based on the recent crystal structured described by Gong et al. (2011). P, phosphorylation.
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 1

Figure 1 - Tau inhibits mitophagy in N2a cells

A Immunostaining for the mitochondrial marker ATPβ in N2a cells expressing mito-QC.

B N2a cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control. Treatment with CCCP (8 µM, 17 h) induces mitophagy in control cells, as indicated by a decrease in the GFP/mCherry fluorescence ratio, but not in hTau or hP301L cells. Deconvolution (Classic Maximum Likelihood Estimation) was applied to images for display only.

C Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing significant main effects of CCCP treatment, F (1, 314) = 15.66, p < 0.0001, and tau expression, F (2, 314) = 12.26, p < 0.0001, and a significant interaction effect, F (2, 314) = 6.137, p = 0.0024, n = 40-73 cells/group.

D Cells expressing mito-QC and myc-Parkin together with hTau-V5, hP301L-V5 or an empty vector control, treated with a mixture of antimycin and oligomycin (AO, 15 µM, 17 h).

E Quantification of GFP/mCherry fluorescence intensity per cell. Data were analysed by 2-way ANOVA, showing no significant main effects of AO treatment, F (1, 212) = 0.564, p = 0.4534, or tau expression F (2, 212) = 1.579, p = 0.2087, but a significant interaction effect, F (2, 212) = 9.854, p < 0.0001, n = 16-50 cells/group.

Data information: Data are given as mean and SEM, * = p < 0.05, **= p <0.01, **** = p < 0.0001 for simple effects. Scale bar = 5 µm in (A) and 10 µm in (B, D).
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 2

Figure 2 - CCCP-induced Parkin translocation to mitochondria is impaired in tau expressing cells.

A Illustration of the image analysis process. The inverted "Union Jack" look-up table (LUT) was used to better visualise GFP-Parkin clusters, which were then manually selected as regions of interest (ROIs) and the corresponding mitochondrial fluorescence intensity was measured.

B Representative images of N2a cells expressing GFP-Parkin and hTau, hP301L or empty vector (Control) for 24 h, that were then treated with 10 μM CCCP for 4 h.

C Quantification of Parkin clusters/cell at 24 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 8.18, p = 0.0168, followed by Dunn's post-hoc analysis, n = 33-38 cells/group.

D Quantification of Parkin clusters/cell at 48 h post-transfection. Data were analysed with a Kruskal-Wallis test, χ2 (2) = 19.2, p < 0.0001, followed by Dunn's post-hoc analysis, n = 37-55 cells/group cells/group.

E To assess colocalisation of Parkin clusters and mitochondria at 24 h post-transfection, Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment were calculated. Control r = 0.616, p = 0.0001; hTau r = 0.217, p = 0.2771; hP301L r = 0.548, p = 0.0014 (n = 27-34 cells/ group).

F Pearson correlations between the mean normalised fluorescence intensity of the Parkin clusters and the mitochondrial signal following CCCP treatment at 48 h post-transfection. Control r = 0.434, p = 0.0167; hTau r = 0.128, p = 0.4941; hP301L r = -0.299, p = 0.2794 (n = 15-31 cells/ group). Note the different intensity scales between (E) and (F) reflect different bit depths of the images.

Data information: Data are given as mean and SEM, *= p < 0.05, ***= p < 0.001, ****= p < 0.0001. Scale bar = 10 μm.
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 3

Figure 3 - Mitochondrial membrane potential is unchanged in tau-expressing cells.

A FACS analysis of cells loaded with Rhodamine 123. The geometric mean fluorescence was calculated and expressed as % of the empty vector control. A CCCP-treated cohort was included as a positive control, but not included in the statistical analysis. Data were analysed by one-way ANOVA, F (2, 14) = 0.196, p = 0.8245.

B FACS analysis of cells treated with CCCP after 24 h of tau expression. Data were analysed by one-way ANOVA, F (2, 15) = 0.434, p = 0.6555. The DMSO negative control is shown but was not analysed statistically.

C Representative dot plots of cells loaded with Rhodamine 123. The gates (black line) were set in unstained cells to distinguish Rhodamine-positive cells (right half) from Rhodamine-negative cells (left half).

Data information: Data are given as mean and SEM (n = 5-6 samples/ experimental group from two independent experiments).
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 4

Figure 4. Stabilising the cytoskeleton pharmacologically does not mimic the effect of tau on Parkin translocation.

A Immunostaining for microtubules after transient expression of hTau and hP301L.

B Taxol induces microtubule bundling to a similar extent to tau expression. C Representative images and quantification of Parkin clusters in N2a cells following CCCP and Taxol treatment (Mann-Whitney test, U = 22448, p < 0.0001, n = 224, 273 cells/group for CCCP only and CCCP + Taxol, respectively).

D Immunoblot analysis of acetylated tubulin (Ace-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0432, p = 0.9579).

E Immunoblot analysis of tyrosinated tubulin (Tyr-Tubulin) following expression of hTau and hP301L for 24 h (One-way ANOVA, F(2, 9) = 0.0402, p = 0.9608).

F Representative images and quantification of Parkin clusters in N2a cells following treatment with CCCP and the actin stabiliser Jasplakinolide (JPL) (Mann-Whitney test, U = 16069, p <0.0001, n = 224, 199 cells/group for CCCP only and CCCP + JPL, respectively).

Data information: Data are given as mean and SEM, ****= p < 0.0001. Scale bar = 10 µm.
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 5

Figure 5 - Exogenous tau interacts with Parkin, trapping it in the cytosol.

A Co-immunoprecipitations of V5-tagged tau and GFP-Parkin. The V5 tag was used to pull down tau and the membrane was probed for V5 (using an antibody raised in a different species) and GFP. IP = immunoprecipitation, Flow = flow-through lysate following removal of beads.

B Maximum projection images of proximity ligation assays (PLAs) for tau and endogenous Parkin in cells expressing hTau-GFP, hP301L-GFP or GFP. Inset = negative control (antibodies to tau and histone H3, which are not known binding partners).

C Quantification of PLA signals in maximum projections normalised to the GFP signal. Results were analysed with a Kruskal-Wallis test, χ2 (2) = 27.2, p < 0.0001, followed by Dunn's post hoc analysis to test for differences from the GFP control, n = 17 cells in GFP condition, 14 cells in hTau condition and 27 cells in hP301L condition.

D PLA for Parkin and hP301L in an aged pR5 mice and a littermate control.

E Schematic of tau constructs used in PLA experiments.

F, G PLA for Parkin and ∆Tau, revealing interactions via the projection domain of tau. Data were analysed with a Mann-Whitney test (U = 185, p = 0.9224, n = 21, 18 cells/group for hTau and ∆Tau, respectively).

H, I PLA for Parkin and the microtubule-binding domain construct (MTBD). Data were analysed with a t-test (t = 5.226, p < 0.0001, n = 16, 17 cells/group for hTau and MTBD, respectively).

J, K Cells expressing tau constructs together with GFP-Parkin (24 h) with and without CCCP treatment. Parkin clusters were counted, illustrating that Parkin translocation was impaired by ∆Tau, but not MTBD. Data were analysed with a Kruskal-Wallis test χ2 (2) = 10.4, p = 0.0056, followed by Dunn's post-hoc, n = 67, 42, 53 cells/ group for for hTau, ∆Tau, and MTBD, respectively.

L The V5 fluorescence intensity in each cell analysed in (K) (Mann-Whitney test, U = 953, p = 0.2332, n = 42, 53 cells/group for ∆Tau and MTBD, respectively).

M Cells were transfected with GFP-Parkin and hTau-V5, hP301L-V5 or an empty vector control, and treated with CCCP. PLAs were performed for tau and Parkin. Notably, at Parkin clusters (arrow), no interactions between tau and Parkin were evident.

N 3D reconstructions in IMARIS, illustrating a lack of PLA signals in Parkin clusters.

Data information: Scale bars = 10 μm. Data are given as mean and SEM, ** = p < 0.01, **** = p < 0.0001.
Disease-associated tau impairs mitophagy by inhibiting Parkin translocation to mitochondria (2018)
Figure 6

Figure 6 - Tau inhibits mitophagy in C. elegans neurons.

A Expression pattern of the mito-Rosella biosensor in the nervous system.

B Analysis pipeline of mitochondria in the C. elegans ventral nerve cord. Mitochondria were selected by choosing a threshold in the dsRed channel. This threshold was used to create ROIs around single mitochondria and the corresponding GFP fluorescence intensity was then measured.

C, D Analysis of mitophagy in worms transgenic for mito-Rosella but not tau (WT), that were treated with NaN3 (8 mM, 1 h) or CCCP (15 µM, 2 h). Data were analysed by unpaired t test, NaN3 t = 5.56, p < 0.0001; CCCP t = 3.92 p = 0.0002.

E, F Analysis of mitophagy in worms expressing hTau in response to NaN3 treatment (unpaired t test, t = 1.62, p = 0.1126) and CCCP treatment (Mann-Whitney test, U = 113, p = 0.002).

G, H Analysis of mitophagy in worms expressing hP301L in response to either NaN3 (Mann-Whitney test, U = 126, p = 0.2711) or CCCP (Mann-Whitney test, U = 375, p = 0.3697) treatment.

I Tau expression in worm lysates, as detected using the human tau-specific antibody HT7.

Data information: n = 16-25 worms/group for NaN3 and 23-39 worms/groups for CCCP experiments. Data are given as mean and SEM. *** = p < 0.001, **** = p < 0.0001. Scale bar = 10 µm.­­­­ Images in (C, E and G) are high-magnification of a small number of mitochondria in the ventral cord; examples of the whole ventral cord are displayed in the Appendix, Fig. S1.
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 1

Figure 1. BRLF1 inhibited inflammasome activation induced by herpesviruses.

(a) THP-1 cells were transiently infected with BRLF1-expressing or empty control lentivirus. Twenty-four hours later, the control or BRLF1-expressing THP-1 cells were primed with 40 ng/ml TPA overnight and then were either uninfected or infected with HSV-1 (MOI=1) in serum-free medium for 12 h. The supernatants and whole cell lysates were collected and analyzed by western blotting to detect pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18 and mature IL-18 as indicated.

(b-c) The release of mature IL-1β (b) and IL-18 (c) from THP-1 cells were measured by enzyme-linked immunosorbent assay (ELISA) following HSV-1 infection. Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, **, p< 0.01, Tukey's multiple-comparison test.

(d-f) Ramos cells were infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100). After being transduced with empty control or BZLF1-expressing lentivirus, the supernatants and cell pellets of EBV-WT- or EBV-ΔBRLF1-harboring Ramos cells were collected after 12 h in serum-free culture, and then the levels of EBNA1, BRLF1, BZLF1, pro-caspase-1 (p45), cleaved caspase-1 (p20), pro-IL-1β, mature IL-1β, pro-IL-18, mature IL-18, GSDMD and cleaved N-GSDMD in the supernatants and whole-cell lysates were analyzed as indicated (d). The release of mature IL-1β (e) and IL-18 (f) from Ramos cells were measured by enzyme-linked immunosorbent assay (ELISA). Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, **, p< 0.01, Tukey's multiple-comparison test.

(g) After being transduced with control or BZLF1-expressing lentivirus for 48 h, EBV-WT- and EBV-ΔBRLF1 infected Ramos cells were stained with PI and measured using fluorescent flow cytometry. Representative images of pyroptotic cells are shown and the percentages of pyroptotic cells were calculated in duplicate.
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 2

Figure 2. BRLF1 interacted with RNA polymerase III subunits.

(a) GST-tagged BRLF1-expressing plasmid or control vector was transfected into A549 cells. After HSV-1 infection (MOI=1) for 12 h, the cells were collected, and the whole cell extracts were subjected to immunoprecipitation with GST affinity beads. The immunoprecipitated complexes were then separated by SDS-PAGE, and the image obtained from silver staining is shown.

(b) Scramble (sc) or shRNAs against POLR3F and POLR3G with empty control vector (ctr) or BRLF1-expressing plasmid were transfected into EBV-ΔBRLF1-harboring HNE1 cells and A549 cells, followed by HSV-1 infection. Thirty-six hours after the transfection of the HNE1 cells, and 12 h after HSV-1 infection (MOI=1) of the A549 cells, the cell pellets were collected and then the cleavage of caspase-1 and IL-1β were measured as indicated. The release of mature IL-1β and IL-18 from HNE-1 and A549 cells were measured by enzyme-linked immunosorbent assay (ELISA) after ultrafiltration and concentration with Amicon® Ultra-0.5 centrifugal filter devices. Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, Tukey's multiple-comparison test.

(c) GFP-tagged BRLF1 wild type, BRLF1 L578A or empty vector was transfected into 293T cells with GST-tagged POLR3F or POLR3G expressing plasmids for 48 h. Cells were collected and immunoprecipitation with GST-affinity beads was performed, and then whole cell lysates and immunoprecipitated complexes were analyzed as indicated.

(d) GFP-tagged empty vector or GFP-BRLF1- or GFP-BRLF1 L578A-expressing plasmids were transfected into A549 cells, after which the cells were then infected with HSV-1, or transfected into HNE-1-EBV-ΔBRLF1 cells. The cells were then collected and lysed. Immunoprecipitation with GST-affinity beads was subsequently performed, and then whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.

(e) P3HR-1 cells were induced by TPA plus NaB into lytic infection, and the cells were then collected. Immunoprecipitation with anti-mouse IgG or BRLF1 antibody was performed, after which whole-cell lysates and immunoprecipitated complexes were analyzed as indicated.

(f) 3D structure of BRLF1, the BRLF1 TA domain and the BRLF1 NLS572-582 peptide were predicted using I-TASSER. Receptor-ligand interaction mode of the BRLF1 TA domain and the BRLF1 NLS572-582 peptide with RNA Polymerase III open complex (PDB ID 6F40) was conducted using CDOCKER. The yellow sequence in top figure indicates the TA domain in BRLF1, and the red sequence represents the NLS572-582 peptide. The yellow sequence in the middle figure represents the BRLF1 TA domain, and the red helix in middle and bottom figures represents the DNA double helix.
BRLF1 suppresses RNA Pol III-mediated RIG-I inflammasome activation in the early EBV lytic lifecycle (2020)
Figure 3

Figure 3. BRLF1 inhibited the transcription of viral and cellular immunostimulatory RNAs.

(a) After infected with wild type EBV-WT or BRLF1-deficient EBV-ΔBRLF1 virus at a high titer (MOI=100), POLR3-dependent RNA transcripts in Ramos cells were detected by RNA-seq analysis. The relative levels of cellular small RNA transcripts are shown as RNA fold changes in the heatmap in the mock (mock group vs. mock group), EBV-WT (EBV-WT group vs. mock group) and EBV-ΔBRLF1 (EBV-ΔBRLF1 group vs. mock group) comparisons.

(b) EBV-ΔBRLF1-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A in the absence or presence of BZLF1 for 48 h. The total RNAs were extracted and subjected to real-time PCR analysis. Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, **, p< 0.01, Tukey's multiple-comparison test.

(c) A549 cells were transfected with control vector, BRLF1 or BRLF1 L578A for 36 h. After the cells were infected with HSV-1(MOI=1) for 12 h, the total RNAs were extracted and subjected to real-time PCR analysis.

The values are shown as the mean ± standard deviations of triplicate analyses from three independent experiments. Results are presented as the mean ± SD, n = 3 biological replicates, *, p < 0.05. **, p< 0.01, Student's t‐test.

(d) EBER1 5'-pppRNA detection using splint-ligation. EBV-WT- or EBV-ΔBRLF1-harboring HNE1 cells were cotransfected with empty vector, BRLF1 or BRLF1 L578A, BRLF1 plus BZLF1, or BRLF1 L578A plus BZLF1 for 48 h. The total RNAs were extracted, and 5 μg of each RNA was subjected to splint-ligation with FAM-labeled probe to quantify the 5′-monophosphorylated EBER1 and total EBER1 RNA. The ligation products were separated using urea-PAGE, visualized by a fluorescent scanner and analyzed using ImageJ software. The ratio of 5'-pRNA density to total RNA density and the relative 5'-pppRNA level were calculated for three independent experiments. Representative images and the means of relative 5'-pppRNA levels are shown. Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, Tukey's multiple-comparison test.

(e) EBV-WT-harboring HNE1 cells were transfected with control vector, BRLF1 or BRLF1 L578A-expressing plasmid in the presence of GST-tagged RNA polymerase subunit POLR3F for 48 h. The cells were harvested and ChIP assay was performed with anti-GST affinity beads. The POLR3F-binding promoter fragments of BSRF1, BMLF1, EBER1, EBER2, EBNA1, 7SK, RN7SL1 and the GAPDH as negative control in the ChIP samples were detected by real-time PCR assays. The relative levels of DNA binding were normalized to the input and the means of three independent experiments are shown. Results are presented as the mean ± SD, n = 3 biological replicates, *, p<0.05, Tukey's multiple-comparison test.