EMBO/soda-nel huggingface dataset:
Diverse autophagosome membrane sources coalesce in recycling endosomes. (2013)
Figure 7

VAMP3 Is Required for Fusion of Vesicles Carrying mATG9 and ATG16L1

(A) HeLa cells transfected with control, VAMP3 siRNA for 4 days were transfected during the last 20 hr with mStrawberry-ATG16L1 and labeled for mATG9. The correlation between mATG9 and ATG16L1 was quantified (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001.

(B) HeLa cells were transfected with control or VAMP3 siRNA and then with mStrawberry-ATG16, and 5 min movies were recorded. Homotypic fusion events between mStrawberry-ATG16 vesicles were assessed in control and VAMP3 knockdown cells. Error bar, SEM. NS, not significant.

(C) In vitro fusion assay of ATG16L1 and mATG9 vesicles. HeLa cells were transfected with control or VAMP3 siRNA for 4 days separately and, on the fourth day, either transfected with mStrawberry-ATG16L1 or ATG9L1-pEGFP. PNS of cells expressing the two constructs were mixed for 1 hr at 37C° and observed by confocal microscopy. Histogram shows the correlation of vesicles carrying ATG16L1 that fuse with vesicles carrying mATG9 (Pearson’s coefficient). Error bar, SEM. ∗∗∗p < 0.001. The total number of structures per field (ATG16L1 and ATG9L1) is not significantly different in the control and KD samples (control, 37 ± 3; VAMP3 KD, 45 ± 3; ± SEM; p = 0.106).

(D) Schematic diagram of mATG9 and ATG16L1 itineraries pertinent to their heterotypic fusion and autophagosome formation.

See also Figure S5 and Movies S5 and S6.
Strong anion exchange-mediated phosphoproteomics reveals extensive human non-canonical phosphorylation (2019)
Figure 2

Figure 2. UPAX workflow for unbiased canonical and non-canonical phosphoproteomics.

(A) Schematic representation of the UPAX strategy for phosphopeptide enrichment and MS/MS analysis.

(B) siRNA-mediated knockdown of PHPT1 in HeLa cells (24 h) with reference to GAPDH loading control. NT - non-targeting siRNA control.

(C-F) (C) Representative SAX profile of trypsin digested HeLa lysate (Abs280 nm). Base peak chromatograms are shown for select SAX fractions following high-resolution LC-MS/MS using an Orbitrap Fusion mass spectrometer: (D) fraction 3 (green); (E) fraction 6 (red); (F) fraction 10 (grey). Peptides were fragmented by HCD, with neutral loss of 98 amu from the precursor ions triggering EThCD (Ferries et al, 2017). Tandem mass spectra were separated according to fragmentation strategy in Proteome Discoverer prior to searching with Mascot. The ptmRS node was used for phosphosite localisation. Analysis was performed on three independent biological replicates for each condition (NT or PHPT1 siRNA).
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 1

Figure 1. NF45/NF90 is a nucleolar protein complex that positively regulates rDNA transcription.

Confocal images of HeLa cells stained with NF45/NF90 antibodies and antibodies against nucleolin and fibrillarin. Scale bar, 10 μm.

HEK293 cells were infected with lentiviruses containing negative control shRNA (shNC) or NF45/NF90 specific shRNA (shNF45#1, shNF45#2, shNF90#1 and shNF90#2) for 24 h, and then transfected with rDNA-Luc. Luciferase activity was measured after a further 24 h (n = 3). Western blots show the protein levels of NF45 and NF90.

Empty vector (EV), FLAG-NF45 plasmid (NF45) or FLAG-NF90 plasmid (NF90) were transfected into HEK293 cells for 24 h, and then rDNA-Luc was co-transfected into the cells. Luciferase activity was measured after a further 24 h (n=3). Western blots show the protein levels of FLAG-NF45 and FLAG-NF90.

The line indicates the position of qPCR primer on the 45S pre-rRNA.

E-F The qPCR analysis of 45S pre-rRNA from HEK293 cells treated with indicated shRNAs (E) and plasmids (F) for 24 h. Western blots show the expression levels of NF45, NF90, FLAG-NF45 and FLAG-NF90. (n = 3)

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (C, F). One-way ANOVA analysis followed by Tukey's test (B, E). (* p < 0.05, *** p < 0.001). Exact p values are reported in Appendix Table S3.
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 2

Figure 2. NF45/NF90 preferentially binds to the promoter region of the rDNA gene loci by directly recognizing the ARRE2-like sequence.

Schematic illustration of a single human rDNA gene repeat and positions of the primers used for ChIP.

In Jurkat cells, a ChIP assay was performed with control IgG, NF45 and NF90 antibodies, and then the precipitated DNA was analyzed using qPCR with the aforementioned primers. The relative rDNA fold-enrichment was normalized to control IgG treatment. (n=3)

The human rDNA promoter region contains one ARRE2-like sequence. The red letters in gray shaded area indicate the probe sequences used in panel D. Gray shaded area indicates the upstream control element. Blue color indicates the core promoter region.

EMSA assay. Lane 1) Biotin-labled rDNA probe. Lane 2) Biotin-labled rDNA probe + nuclear protein. Lane 3) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled specific competitor. Lane 4) Biotin-labled rDNA probe + nuclear protein + 100-fold molar excess of biotin-unlabeled mutant competitor. Lane 5) Biotin-labled rDNA probe + nuclear protein + NF90 antibody. Lane 6) Biotin-labled rDNA probe + nuclear protein + NF45 antibody. The details were described in Materials and Methods section.

The relative luciferase activity of HEK293T cells transfected with empty vector (EV) or FLAG-NF90 (NF90) in combination with rDNA-Luc wild type (WT) or mutated rDNA-Luc reporter plasmid (Mutant) for 24 h. (n=3) Western blot shows FLAG-NF90 protein level.

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (E). (*** p < 0.001). Exact p values are reported in Appendix Table S3.
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 3

Figure 3. NF45/NF90 affects rDNA transcription as a positive regulatory factor by recruiting and regulating UBF1 activity.

The regulatory mechanism of the rDNA transcription. Firstly, UBF1 binds to the upstream control element (UCE) of rDNA promoter to recruit SL1, then SL1 recruits Pol I for rDNA transcription.

Co-IP analysis of nucleolar proteins with NF90 in HEK293 cells transfected with EV, and the following plasmids: GFP-S5, GFP-L9, GFP-B23, GFP-UBF, GFP-C23. * represents the target protein.

Co-IP analysis of endogenous UBF with NF45 and NF90 in HEK293 cells.

HEK293 cells were infected by lentivirus containing NF45-specfic shRNA or NF90-specfic shRNA, and then stimulated in serum for 0, 3 and 6 h. Cell lysates were prepared to analyze the expression levels of the indicated proteins by western blot.

ChIP analysis was used to determinate the binding ability of NF45 and NF90 to rDNA promoters in HeLa cells stimulated with serum for 0, 3 and 6 h. (n = 3)

ChIP analysis of NF45 and NF90 binding to the H42.9 loci in HeLa cells with the treatment of CX5461 or DMSO for 2 h. (n = 3)

Confocal images of HeLa cells treated with CX5461 for 2 h showing NF45 and NF90 colocalized with fibrillarin. Scale bar, 10 μm.

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (F). One-way ANOVA analysis followed by Tukey's test (E). (** p < 0.01, *** P < 0.001). Exact p values are reported in Appendix Table S3.
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 4

Figure 4. NF45/NF90-mediated rDNA transcription contributes to T cell activation by interacting with NFATc2 in the nucleolus.

ChIP analysis of NF45 and NF90 binding to the H42.9 loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)

qPCR analysis of pre-rRNA in CD3+ T cells treated with P/I for 6 h. (n=3)

CD3+ T cells were first silenced with shNC, shNF45 or shNF90, and then treated with P/I to extract total RNA for qPCR analysis of pre-rRNA. (n=3)

The analysis of rDNA-luc activity in Jurkat cells with or without NF90 silence upon P/I stimulation or not. (n=3)

Confocal images of Jurkat cells to show NF45 (left panel) or NF90 (right panel) (green fluorescence) colocalized with NFATc2 (red fluorescence) in nucleoli. Scale bar, 10 μm.

Co-IP analysis of NFATc2 and NFATc1 with NF90 in Jurkat cells treated with P/I.

ChIP analysis of NFATc2 binding to the rDNA loci in CD3+ T cells stimulated with P/I for 6 h. (n=3)

Jurkat cells co-transfected with rDNA-luc (wild type or mutant plasmid) with NF90 and/or NFATc2 for 24 h, then rDNA-luc activity was detected after P/I sitmulation or not (DMSO). (n=3)

ChIP analysis of NFATc2 binding to the H42.9 loci in NF90-slienced CD3+ T cells stimulated with P/I for 6 h. (n=3)

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (A, B, C, D, G, I). One-way ANOVA analysis followed by Tukey's test (H). (* p < 0.05, *** p <0.001). Exact p values are reported in Appendix Table S3.
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 5

Figure 5. NF45/NF90-mediated rDNA transcription is necessary for cell proliferation and activation.

CD69 expression, an early marker of lymphocyte activation, in CD3+ T cells infected with shNC, shNF45 or shNF90 lentivirus upon stimulation with P/I for 12 h.

B-C PBMC cells with shNC, shNF45 or shNF90 lentivirus infection were stimulated with P/I for 12h, then the freshly replaced medium without P/I after another 24 h culture was harvested for IL-2 and IFN-γ detections by ELISA.(n=3)

The proliferation of shNC, shNF45 or shNF90-infected PBMCs was monitored using a CCK-8 assay. (n = 3)

Confocal images to show the subcellular localization of NFATc2 in CD3+ T cells treated with P/I and FK506 or CX5461. Scale bar, 10 μm.

Flow cytometry analysis of CD69 expression in P/I-stimulated CD3+ T cells treated with or without FK506 or CX5461.

G-H ELISA assays of IL-2 and IFN-γ in CD3+ T cells with the indicated treatments. (n = 3)

Flow cytometry analysis of CFSE-labeled CD3+ T cells in the presence of DMSO, FK506 and CX5461 for 0, 3 and 7 d.

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by One-way ANOVA analysis followed by Tukey's test (E, C, G, H). (* p < 0.05, ** p < 0.01, *** p < 0.001). Exact p values are reported in Appendix Table S3.
NF45/NF90-mediated rDNA transcription provides a novel target for immunosuppressant development (2021)
Figure 6

Figure 6. CX5461 promotes graft survival in skin and cardiac allograft models.

qPCR analysis of pre-rRNA expression from skin- and heart-transplanted mouse spleens. (n = 5)

pre-rRNA expression levels of PBMC from renal transplantation patients. (Stable group, n = 7. ABMR group, n = 8. TCMR group, n = 8).

Therapeutic scheme showing the daily administration of CX5461 or FK506 in skin‐ or cardiac-allografted mice until day 80.

Skin graft survival curve in mice treated with DMSO, 2.0 mg/kg FK506 or 0.5 -2.0 mg/kg CX5461. (n = 5)

Representative images of HE staining of skin graft at seven days post transplantation. Scale bar, 50 μm.

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of skin-graft recipient mice were analyzed by FACS.

G-H ELISA analysis of IL-2 and IFN-γ levels in skin-graft recipient mouse serum at 4th day post transplantation. (n = 5)

The survival time (days) of cardiac graft in mice treated with DMSO, 2.0 mg/kg FK506 or 2.0 mg/kg CX5461. (n=5)

Representative images of HE and CD3-IHC staining of cardiac graft at seven days post transplantation. NC, Scale bar, 100 μm.

CD3+ T cells (upper) or CD4+ and CD8+ T cells (lower) in the spleens of heart-graft recipient mice were analyzed by FACS.

L-M ELISA analysis of IL-2 and IFN-γ levels in heart-graft recipient mouse serum at 7th day post transplantation. (n = 5)

qPCR analysis of pre-rRNA expression from transplanted mouse spleen. (n = 5)

FACS analysis of CD4+, CD25+ and Foxp3+ Treg cells isolated from superficial cervical lymph nodes of heart-transplanted mice.

Data information: In all panels, bars and error bars represent mean ± SD. Statistical analysis by unpaired Student T-test (A). One-way ANOVA analysis followed by Tukey's test (B, G, H, I, L, M, N). (* p < 0.05, ** p < 0.01, *** p < 0.001). Exact p values are reported in Appendix Table S3.
Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity (2022)
Figure 1

Figure 1 | Vaccination with a VSR-deficient mutant virus induces protection against secondary infection in neonatal mice.

A, At 2 days post vaccination with NoVΔB2, UV- inactivated NoVΔB2 (UV- ΔB2) or culture medium DMEM, BALB/c mice were challenged with WT NoV. The survival data were analyzed by the log rank test, **** indicates p<0.0001, n=7 per group.

B, C, NoV levels in the vaccinated mice hindlimb muscle tissue at 2, 3 or 4 days post infection (dpi) with WT NoV were determined by Western blotting to detect the viral coat protein (CP) and B2 (B) or quantitative RT-qPCR to detect the viral genomic RNA1, n=7 per group (C). Staining of β-actin was used as a loading control (B). The viral RNA1 level was normalized by β-actin mRNA levels (C). n=7 per group. Horizontal bars represent for means. * indicates p<0.05, ** indicates p<0.01, and*** indicates p<0.001 by student's t-test.

D, E, Time course analysis of the accumulation of NoVΔB2 RNA1 determined by RT-qPCR (D) and 21-to 23-nt vsiRNAs determined by deep sequencing (E, two independent sets) from BALB/c suckling mice infected with NoVΔB2. Mice, n=3, in each independent analysis. Data are shown as mean±SD. Normalization was done by β-actin mRNA levels for NoVΔB2 RNA1 (D) or by mature miRNAs for vsiRNAs (E). Accumulation levels of NoVΔB2 RNA1 or vsiRNAs at 3 dpi are set as 100%, respectively.

F, Properties of viral siRNAs (per million mature miRNAs) cloned and sequenced from the time course series of NoVΔB2-infected BALB/c suckling mice. Size distribution, 5' terminal nucleotide and duplexes by 22-nt vsiRNAs are indicated. The percentage of 1U vsiRNAs (21- to 23-nt) in each library is shown in parentheses.
Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity (2022)
Figure 2

Figure 2 | Characterization of viral siRNAs in murine EVs.

A, Model of sample preparation for small RNA deep sequencing.

B, Size distribution of the purified EVs analyzed by NTA.

C, TEM images of purified EVs. Scale bar: 100 nm.

D, Western blotting of HSP70, tsg 101, CD81, CD63, and CD9 in EVs, whole blood, and blood clot derived from suckling mice infected with DMEM or NoVΔB2. Staining of β-actin was used as a loading control.

E, Size distribution of total sequenced small RNAs in the libraries constructed from EVs of DMEM challenged BALB/c suckling mice, and EVs, whole blood, blood clot of BALB/c suckling mice infected with NoVΔB2 at 3 dpi.

F, G, Size distribution of 18- to 28-nt vsiRNAs (F) and genomic coverage depth of each nucleotide position by 21- to 23-nt vsiRNAs (G) were analyzed from BALB/c suckling mice libraries infected by NoVΔB2 at 3 dpi.

H, Pie charts of the top ten most abundant miRNAs and vsiRNAs in hindlimb, EVs, whole blood, blood clot from BALB/c suckling mice infected by NoVΔB2 at 3 dpi. Red charts indicate 18- to 28-nt vsiRNAs abundance. The percentage of miRNA reads was shown above. The abundance of total miRNAs and 18- to 28-nt vsiRNAs reads was set as 1.
Mouse circulating extracellular vesicles contain virus-derived siRNAs active in antiviral immunity (2022)
Figure 4

Figure 4 | Passive protection in vitro and in vivo conferred by isolated EV.

A, Scheme for protocol to determine the in vitro antiviral function of EVs from NoVΔB2-infected BALB/c suckling mice.

B, C, 10μl or 50μl EVs stock from naïve [EVs (mock)] or NoVΔB2-infected [EVs (ΔB2), B] or SINV-infected [EVs (SINV), C] mice were added to BHK cell culture medium along with NoV-WT, and then BHK cells were harvested at 24 hpi. Accumulation of NoV RNA1 in BHK cells were measured by RT-qPCR. Normalization was done by β-actin mRNA levels. Data from at least three independent experiments were combined. Data are shown as mean±SD. ** indicates p<0.01, ns indicates no significant difference by student's t-test.

D, Diagram showing the 462-nt sequence in length from NoV∆B2 RNA1 was inserted into the 3' untranslated region (UTR) of firefly luciferase reporter mRNA, to be targeted by antisense vsiRNAs with EVs from NoV∆B2-immunized BALB/c suckling mice. A translation initiation codon-deleted GFP sequence of the same length was inserted into the same site as control.

E, F, Relative luciferase activity of the two reporter constructs after transfection of 293T cells (E) and 293T-Ago2 KO cells (F) and treatment by different pools of EVs, EVs (∆B2) or EVs (Mock). Error bars indicate standard deviation of three independent replicates. *** indicates p<0.001, ns indicates no significant difference by student's t-test.

G, Experimental design for the in vivo functional analysis of EVs. In brief, mice were first immunized with live NoVΔB2, EVs (mock) or EVs (ΔB2), and challenged with WT NoV at 12 hours post-immunization. The NoV-challenged mice were sacrificed at 1 and 3 days after challenge infection for determining the viral load or monitored daily for survival.

H, I, Accumulation of NoV RNA1 was determined by RT-qPCR in NoVWT-infected BALB/c suckling mice at 1 (H) or 3 dpi (I). n=5~7 per group. Data are shown as mean±SD. ****indicates p<0.0001 by student's t test.

J, Total proteins were extracted from the hindlimb muscle tissue of NoVWT-infected BALB/c suckling mice to measure the viral coat protein (CP) and B2 protein by Western blotting. Staining of β-actin was used as a loading control.

K, Survival curve of WT NoV-challenged suckling mice immunized with live NoVΔB2, EVs (ΔB2) or EVs (mock). n=7 per group. *** indicates p<0.001. All data were analyzed by log rank test.
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 2

Figure 2.

MDGI expression promotes aggressive and invasive growth of glioma cells.

A, B Representative whole coronal section micrographs of murine brain injected with GFP (A) or MDGI-GFP (B) expressing U87MG glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.

C-F Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in C and D), MDGI (green in E and F), and CD31 (red in E and F). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). Arrows in D point to the angiotropic tumours. Arrows in F point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.

G. Quantification of the number of U87MG secondary tumours (diameter >300 µm) detected in the whole brain (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. **P < 0.01, two-tailed, nonparametric Mann-Whitney's U-test.

H. Quantification of the number of invasive U87MG cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) (GFP: n = 5, MDGI-GFP: n = 9). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

I, J Representative micrographs of whole murine brain coronal sections injected with GFP (I) or GFP-MDGI (J) expressing LN229 glioma cells. Xenografted human cells were visualised by using anti-human vimentin (hVim) antibodies (red). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm.

K-N Micrographs of consecutive (separating distance: 9 µm) brain sections stained for human glioma cells using antibodies specific for human vimentin (hVim red in K and L), MDGI (green in M and N), and CD31 (red in M and N). Dashed lines separate the primary tumour mass and the normal brain. Nuclei were visualised by using DAPI (blue). ). Arrows in L point to the angiotropic tumours. Arrows in N point to the MDGI-expressing angiotropic tumour cells. Scale bar: 50 µm.

O. Quantification of the number of invasive LN229 cells detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.

P. Quantification of the number of invasive LN229 cells that grew next to brain blood vessels (angiotropic co-opting tumour cells, diameter <300 µm) detected in the brain (GFP: n = 10, MDGI-GFP: n = 10). Data are represented as mean ± SEM. Data are represented as mean ± SEM. ****P < 0.0001, two-tailed, nonparametric Mann-Whitney's U-test.
Vulnerability of invasive glioblastoma cells to lysosomal membrane destabilisation (2019)
Figure 3

Figure 3.

MDGI silencing reduces glioma cell growth and viability both in vitro and in in vivo.

A. Transmitted light micrographs of MDGI-silenced (shMDGI1 and shMDGI2) and control (Scr) patient-derived BT12 and BT13 spheroids 11 days after gene silencing. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.

B. Methylcellulose assay demonstrating the ability for self-renewal of control (Scr) and MDGI-silenced (shMDGI1 and shMDGI2) patient-derived BT12 and BT13 cells after three weeks of culture. Representative images of at least 3 independent experiments are shown. Scale bar: 200 μm.

C. Quantification of the colony size in methylcellulose of MDGI-silenced (sh1 and sh2) cells relative to the controls (Scr) that is set as 100 (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.

D. MTT-proliferation assay of control (Scr) and MDGI-silenced (shMDGI1) BT12 and BT13 cells. The values of each cell line were normalized to the values at day 1 marked with a dashed line (n = 3). Data are represented as mean ± SD. ***P < 0.001, two-tailed, nonparametric Mann-Whitney's U-test.

E, F Representative whole coronal section micrographs of murine brain injected intracranially with control (BT12 Scr, n = 5) and MDGI-silenced (BT12 sh1, n = 5) BT12 cells. After four weeks animals were sacrificed, the brains were excised and prepared for immunohistochemistry. Control (Scr) cells grew very similarly to the parental BT12 cells forming a tumour mass with invasive tumour cells (single cell invasion) that eventually co-opted existing blood vessels (angiotropism) and formed secondary tumours (upper panels). No tumour growth was observed when MDGI-silenced cells were implanted (lower panels). Glioma cells were visualised by using anti-human vimentin (hVim red) and blood vessels using anti-podocalyxin antibodies (green). Nuclei were visualised by using DAPI (white). Scale bar: 1 mm (E) and 50 µm (F).