EMBO/soda-nel huggingface dataset:
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 2

Figure 2. The ER membrane protein Calnexin is required for accumulation of FUNDC1 in the MAM

A. Top: Cartoon representing the topology of the FUNDC1 protein in the mitochondrial membrane. The cytoplasmic domain that interacts with the ER protein Calnexin is shown. Bottom: Illustration of the different FUNDC1 truncation variants.

B. HeLa cells were transfected with FUNDC1-MYC or empty vector for 24 h. Cell lysates were immunoprecipitated by anti-MYC antibody and immunoblotted with indicated antibodies.

C. HeLa cells were transfected with FLAG-FUNDC1. About 24 h post-transfection, cell lysates were immunoprecipitated by anti-Calnexin and immunoblotted with anti-FLAG and anti-Calnexin antibodies.

D and E. HeLa cells were transfected with the indicated FUNDC1-MYC constructs for 24 h. Cell lysates were immunoprecipitated using anti-Calnexin and immunoblotted using anti-MYC and anti-Calnexin antibodies.

F. Immunoblots of subcellular fractions from HeLa cells transfected with scramble siRNA and exposed to hypoxia (1% O2) for 5 h. PNS: post-nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.

G. Immunoblots of subcellular fractions from HeLa cells transfected with Calnexin siRNA (si-CNX) and exposed to hypoxia (1% O2) for 5 h. PNS: post nuclear supernatant; CYTO: cytosol; ER: endoplasmic reticulum; MITO: mitochondria; MAM: mitochondrial-associated membrane. DRP1 (S) and DRP1 (L) indicate short and long exposures, respectively.

H. Quantification of the MAM:PNS ratio of FUNDC1 or DRP1 in HeLa cells treated with scramble siRNA or si-CNX and exposed to hypoxia (1% O2) for 5 h. Data are presented as mean ± s.e.m. from 3 independent experiments; *** P < 0.001.
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 3

Figure 3. FUNDC1 mediates mitochondrial fission in response to hypoxia

A. HeLa cells were transfected with the indicated si-RNAs. 24 h post-transfection, cells were exposed to hypoxia (1% O2) for 12 h and then immunostained by anti-TOM20. Bar = 50 µm. Boxed regions in the left panels are enlarged in the right panels.

B. Electron microscopy analysis of HeLa cells treated with the indicated si-RNAs for 24 h and then exposed to hypoxia (1% O2) for 12 h. Bar = 1 μm. Boxed regions in the left panels are enlarged in the right panels.

C. Quantification of mitochondrial morphology (fragmented, intermediate and elongated mitochondria) in 100 cells from (A). Data represent mean ± s.e.m. (*** P<0.001).
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 4

Figure 4. FUNDC1 recruits DRP1 to facilitate mitochondrial fission under hypoxic conditions

A. HeLa cells were treated with the indicated si-RNAs and exposed to hypoxia for 12 h. Cells were fixed and immunostained by anti-TOM20 and anti-DRP1 antibodies. Bar = 40 µm.

B. Immunogold electron micrographs of control HeLa cells (top) or HeLa cells transfected with si-FUNDC1 (bottom) and then exposed to hypoxia (1% O2) for 12 h. DRP1 was detected with anti-DRP1 (12 nm gold particles). Blue stars indicate mitochondria. Yellow arrows indicate gold particles. Red arrows indicate double-membraned autophagosomes.

C-H. Immunoblots of subcellular fractions from control HeLa cells or HeLa cells treated with the indicated si-RNA and then exposed to hypoxia for 5 h. PNS: post nuclear supernatant; Cyto: cytosol; Mito (crude): crude mitochondrial fraction containing mitochondria and MAMs.
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 5

Figure 5. FUNDC1 binds directly to DRP1 and their role in mediating mitochondrial fission

A. HeLa cells were co-transfected with FLAG-DRP1 and FUNDC1-MYC for 24 h. Cells were lysed and immunoprecipitated using anti-FLAG antibody. FLAG-DRP1 and FUNDC1-MYC were analyzed with the corresponding antibodies.

B. HeLa cells were co-transfected with empty vector, MYC-DRP1 and FLAG-FUNDC1. 24 h after transfection, cell lysates were immunoprecipitated using anti-FLAG antibody and immunoblotted with anti-MYC and anti-FLAG antibodies.

C. MEFs were transfected with FUNDC1-MYC. 24 h post-transfection, cells were fixed and immunostained by anti-MYC and anti-DRP1. Bar = 10 µm.

D and E. HeLa cells were co-transfected with the indicated FUNDC1-MYC and FLAG-DRP1 constructs for 24 h. Cell lysates were analyzed by immunoprecipitation using anti-FLAG and immunoblotted with anti-MYC and anti-FLAG antibodies.

F. HeLa cells were exposed to hypoxia (1% O2) for the indicated times and cell lysates were immunoprecipitated using anti-FUNDC1 antibody. Endogenous DRP1, Calnexin, LC3 and FUNDC1 were detected by anti-DRP1, anti-Calnexin, anti-LC3, and anti-FUNDC1 antibodies.

G. 10-20 g GST-FUNDC1 or GST was immobilized on glutathione sepharose resin (GE Healthcare). 20-50 g DPR1 was incubated with the GST-FUNDC1 or GST bound resin overnight at 4 °C. The resin was then extensively rinsed and eluted. Samples were subjected to SDS-PAGE, and then visualized by coomassie blue staining and western blot, respectively.

H. Full-length FUNDC1-MYC, FUNDC1-MYC (AA 96-155), FL (Δ129-138) or vector control were transfected with Mitodsred (red) into FUNDC1 KD MEFs. 12 h post- transfection, cells were exposed to hypoxia for 12 h and then stained with anti-MYC (blue) and anti-DRP1 (green). Bar = 10 µm. Numbers at the right show the average length of mitochondria in Mitodsred-positive cells.
FUNDC1 regulates mitochondrial dynamics at the ER-mitochondrial contact site under hypoxic conditions (2016)
Figure 6

Figure 6. Calnexin and DRP1 are involved in FUNDC1-mediated mitophagy induced by hypoxia

A. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h or not. Cells were fixed and immunostained by anti-TOM20 and anti-LC3 antibodies. The colocalization of LC3 and mitochondria was quantified and listed on the right side. Bar = 5 µm.

B and C. HeLa cells were transfected with the indicated si-RNAs for 24 h and then exposed to hypoxia for 24 h. Cells were then treated with or without 50 nM BAF A1 (Bafilomycin A1) for 6 h before harvesting. Cell lysates were immunoblotted with the indicated antibodies.

D and E. Quantification of the TIM23:Actin and TOM20:Actin ratios from (B and C). All data are from 3 independent experiments.

F. HeLa cells were transfected by different truncated FUNDCY-MYC. About 24h post-transfection, cell lysates were immunoblotted using indicated anti-bodies.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 2

Figure 2. Inhibitory effects of BO-110 on pro-lymphangiogenic factors.

Luciferase-based imaging of short therm drug response in Vegfr3Luc;Tyr::CreERT2; BrafV600E; Ptenflox/flox mice. Panels labeled as "basal" and "induced" correspond to the bioluminescence of animals prior and 5 weeks after administration of 4OH-tamoxifen (5 mM, topical administration, 3 consecutive days) for the induction of melanomas. (Right panels) images of mice were treated the day before with one dose of the indicated compounds: αPD-L1 antibody (clone 10F.9G2) or the corresponding control IgG (200ug/dose); vemurafenib (Vem, 50 mg/Kg) or BO-110 (BO, 0.8 mg/kg). Scale: p/s/cm2/sr (x106).

Response of xenografts of mCherry-labeled SK-Mel-147 in Vegfr3Luc nu/nu lymphoreporter mice treated with one dose (24 h) or 4 dosis of BO-110 (BO, 0.8 mg/kg). Left panels correspond to Vegfr3-Luciferase (neolymphangiogenesis) and right panels to mCherry fluorescence emission (tumor content). Scale, Vegfr3Luc: p/s/cm2/sr (x106) and mCherry: p/s/cm2/sr (x109).

qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of HLEC with 0.5 ug/ml BO-110 (BO), 10 µM vemurafenib (Vem), or vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by t-test.

qRT-PCR analysis of relative mRNA levels of VEGFR3 16 h after treatment of HLEC with 0.5 or 1 ug/ml BO-110 (VO), 10 µM vemurafenib (Vem), or the corresponding vehicle control (V). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by ANOVA.

Luciferase signal driven by FLT4 (VEGFR3)-promoter transduced into HLEC treated with vehicle (v) or BO-110 (BO) at the indicated doses (ug/ml) as indicated in methods. Results were normalized to vehicle control. N=4 biological replicates. Error bars correspond to mean ± SD. Statistical significance was determined by ANOVA.

Tubulogenic activity of HLECs in the presence of BO110. Images correspond to cells plated in matrigel and imaged at the indicated time points after treatment with 0.5 µg/ml BO-110. Complete time-lapse imaging of this process is shown in Movie EV1 (Appendix).

Analysis of apoptotic cells at the indicated time points. HLEC cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.

Relative mRNA levels of VEGFC and VEGFD in the indicated melanoma cell lines 8 h after treatment with vehicle (V) or 0.5 µg/ml BO-110 (BO), as determined by qRT-PCR. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 3

Figure 3. Inhibitory effects of BO-110 on Midkine.

Inhibitory effect of the indicated doses of BO-110 (in µg/ml) or vehicle (V) on MDK mRNA expression determined by qRT-PCR in SK-Mel-147 (16 h after treatment). Data correspond to average mRNA levels in three experiments with technical replicates normalized to vehicle control ± SD. Statistical significance was determined by ANOVA.

qRT-PCR analysis of relative mRNA levels of MDA5 16 h after treatment of SK-Mel-147 with the indicated doses of BO-110 (in µg/ml) (BO). Data correspond to the mean ± SD of three experiments with three technical replicates. Statistical significance was determined by ANOVA.

Analysis of apoptotic cells at the indicated time points. SK-Mel-147 cells were treated with vehicle (V) or 0.5 µg/ml BO-110 (BO) for the indicated time points. Cells were collected and apoptosis was analyzed by flow cytometry as indicated in methods. Data correspond to the mean ± SD of 3 experiments. Statistical significance was determined by t-test.

Luciferase signal driven by MDK-promoter transduced into SK-Mel-147 cells treated with vehicle (v) or BO-110 (BO) as indicated in Materials and Methods. Results were normalized to vehicle control. Data correspond to the mean ± SD of four biological replicates. Statistical significance was determined by ANOVA.

MDK secretion by ELISA in SK-Mel-147 melanoma cells treated with vehicle (V) or 0.5 µg/ml BO-110 (BO). Data correspond to the mean ± SD of 3 biological replicates. Statistical significance was determined by Two-Way ANOVA.

Immunohistochemical analysis of MDK repression (pink staining) in SK-Mel-147 xenografts and PDX lesions after treatment with BO-110 (BO, 0.8 mg/kg, 2 doses/week). Histological staining in tumors extracted from animals treated with vehicle control (V) are included as a reference. Nuclei were counterstained in with hematoxylin.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 4

Figure 4. High throughput analysis reveals IFN induction as a key component of the BO-110 mechanism of action.

Heatmaps summarizing proteomic analyses (iTRAQ) performed in SK-Mel-28 and SK-Mel-147 after treatment with BO-110 (1µg/ml, 10 h). Shown are Hallmark gene sets with NES>1. See also Datasets EV1,2 for additional information.

REACTOME gene set analysis of protein changes in SK-Mel-28 and SK-Mel-147 cell lines treated with BO-110 (1µg/ml, 10 h).

Type I IFN mRNA induction (IFNA2 and IFNB1) in SK-Mel-147 melanoma cells treated for 16 h with the indicated amounts of BO-110 (in mg/ml). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control. Statistical significance was determined by ANOVA.

Heatmap showing differentially deregulated signaling cascades in SK-Mel-147 and HLEC treated with 1µg/ml B0-110 for 10 h (versus vehicle-treated controls). Data correspond to mRNA expression profiles analyzed by GSEA using the Hallmark genesets. The scale indicates the normalized enrichment score (NES). See also Dataset EV3.

Heatmap depicting expression changes in interferon-related genes (GO:0034340) in SK-Mel-147 melanoma cells (left panel) and HLEC (right panel) treated with vehicle or 0.5 g/ml of BO-110 for 10 hours. CD274, LAG3 and PDCD1 genes were also included as a reference.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 5

Figure 5. Mechanistic analyses of the repressive activity of BO-110 on melanoma-induced neo-lymphangiogenesis.

IFNB1 mRNA induction analyzed by qPCR at the indicated times after BO-110 treatment (0.5 µg/ml) of SK-Mel-147 melanoma cells or HLEC (left and right graphs, respectively). Data correspond to the mean ± SD of three experiments with three technical replicates normalized to vehicle control.

Quantification of the impact of BO-110 as single agent or in the presence of the indicated blocking antibodies for type I interferon (IFNB1 or IFNAR1). Upper graphs show the effect of these agents on MDK mRNA levels in SK-Mel-147 melanoma cells. Similar treatments were performed on HLEC for the analysis of VEGFR3 mRNA (middle graphs) and tube formation capacity (lower graphs). Data correspond to the mean ± SD of 3 biological replicates in triplicate.

BO-110-driven blockade of the tube-forming capacity of HLEC and rescue with anti-IFNAR1 blocking antibodies. Images correspond to cells plated in matrigel and imaged 8 h after treatment with 0.5 µg/ml BO-110. See also Fig. EV4B, for additional results with anti-IFNAR1 and anti-IFNβ blocking antibodies.

Growth of B16 melanoma xenografts in siblings of Ifnar1+/+, Ifnar1+/- or Ifnar1-/- mice. Treatment started 10 days after tumor cell implantation. BO-110 was administered at 0.8 mg/kg, every third day for 2 weeks. N= 6 mice per condition. Graphs show the mean tumor size ± SD at each time point. Statistical significance was determined by Two-Way ANOVA.

Histological analyses of lymphatic vessel density by Lyve1 (blue) and Prox1 (purple) in representative lymph nodes of animals in (D) processed at the endpoint of the experiment (4 doses of BO-110 or vehicle control). N= 6 mice per experimental condition. See Fig. EV4C for a more complete view of these lymph nodes, where dual Prox1-Lyve1 positive cells were pseudocolored in red.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 6

Figure 6. BO-110 induces a systemic interferon response that inhibits Mdk blood levels and tumor induce lymphangiogenesis.

Impact of BO-110 on tumor-bearing mice implanted with syngeneic B16R2L (5 x 105 cells). When tumors have an average size on 100 mm3, mice were randomized into two groups and treated with BO-110 (BO) or vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points.

Quantification of Vegfr3Luc emission in the tumor area in mice in experiment (A).

Vegfr3Luc emission in the inguinal and brachial lymph nodes of mice in experiment (A).

D-F. Quantification of Vegfr3Luc emission in the spleen, liver and lung of mice in experiment (A), respectively.

G. ELISA analysis of Mdk blood levels in mice in experiment (A).

H. ELISA analysis of mouse Ifn-β blood levels in mice in experiment (A).

I Antitumoral effect of BO-110 on xenografts of SK-Mel-147 (1 x 106 cells) implanted in the back of Vegfr3Luc; nu/nu nude mice. When tumors had an average size on 150 mm3, mice were randomized in two groups and treated with BO-110 (BO, 0.8 mg/kg) or Vehicle (V), every second day for 2 weeks. The arrow indicates the start of the treatment. Tumor size was measured with a caliper at the indicated time points and tumor volume was calculated as indicated in Materials and Methods.

J. Quantification of Vegfr3Luc emission in the tumor area in mice in experiment (I).

K-N. Quantification of Vegfr3Luc emission in the Lymph nodes, spleen, liver and lung of mice in experiment (I), respectively.

O. ELISA analysis of Mdk blood levels in mice from (I).

P. ELISA analysis of mouse Ifn-β blood levels in mice from (I).

For all panels in this figure, N= 4 mice per condition. Statistical significance was determined by Two-Way-ANOVA.
Live imaging of neolymphangiogenesis identifies acute antimetastatic roles of dsRNA mimics (2021)
Figure 7

Figure 7. BO-110 adjuvant treatment prevents metastatic melanoma relapse.

Efficacy of BO-110 as an adjuvant (preventing relapse after surgical removal of the primary lesion). Shown are representative examples of Vegfr3Luc mice implanted with mCherry-SK-Mel-147 and imaged for luciferase emission (prior to and after tumor removal. Animals were left to recover from surgery (4 days) and then treated for 2 weeks (4 dosis) with 0.8 mg/Kg BO-110 or vehicle control (n= 8 for control and n=10 for treatment arm). Scale, p/s/cm2/sr x106.

mCherry emission from tumor cells of the animals in (A). Scale, p/s/cm2/sr x109.

Kaplan-Meier survival curves of animals treated as in (A). 8/8 animals treated with vehicle (V) control had to be sacrificed for humane reasons 110 days after surgery. 9/10 animals in the BO-110 arm (BO) remained tumor-free 8 months after stopping treatment. The grey box marks the period of treatment with BO-110. Statistical significance was determined by logrank test.

Expanded View Figure legends.
Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response. (2015)
Figure 1

OVA257-264 -specific T-cell response and cytokine production

A Specific CD8+ T-cell (OT-I) proliferation in response to immature BMDCs incubated with fdOVA, fdOVA/sc-αDEC or OVA257-264 peptide chemically coupled to anti-DEC-205 antibody (NLDC:pOVA). Percentage of CFSE-labeled proliferating T cells was evaluated on CD8+-gated cells after 72 h of DC:T co-culture. The mean ±SD of three independent experiments is reported.

B Proliferation of adoptively transferred OT-I T cells in C57BL/6 mice (n = 3/group) immunized with vehicle (PBS), NLDC:pOVA, NLDC:pOVA plus CpG, fdOVA, fdOVA/sc-αDEC or LPS-free ovalbumin protein. Percentage of CFSE-labeled proliferating OT-I T cells was evaluated after three days on Vα2+ CD8+-gated cells. The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.

C Percentage of IFN-γ-producing CD8+ splenocytes isolated from mice immunized as in (B). The mean ± SD of two independent experiments is reported. Comparative analyses were performed using Student's t-test for unpaired samples.

D–F IL-6 (D), IL-18 (E) and IFN-α (F) release in supernatants of BMDCs obtained from C57BL/6 mice. Supernatants of BMDCs incubated with NLDC145 antibody, wild-type phage particles (fdWT) or scFv αDEC-205 phage particles (fdsc-αDEC) for 20 h were analyzed by ELISA for cytokine production. Unstimulated culture (medium) and LPS-treated culture were, respectively, used as negative and positive controls. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.
Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response. (2015)
Figure 3

fdsc-αDEC induces IL-6 and IFN-α production mediated by MYD88 and TLR9

IL-6 was evaluated by ELISA in supernatants of BMDCs obtained from C57BL6, MYD88, TLR9 or TLR4 KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. IL-6 release from DCs derived from MyD88−/− mice was totally abolished and dramatically reduced in DCs derived from Tlr9−/− mice, but not affected in Tlr4−/− DCs. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.

IFN-α release evaluated by ELISA in supernatants of BMDCs obtained from C57BL/6 or KO mice and incubated for 20 h with wild-type or fdsc-αDEC phage particles. LPS or CpG-ODN were used as controls. MyD88−/− and Tlr9−/− but not Tlr4−/− BMDCs were unable to produce IFN-α after fdsc-αDEC stimulation. Bars represent mean values ± SD. Cumulative results are shown of three independent experiments assayed in duplicate. Comparative analyses were performed using Student's t-test for unpaired samples.

IL-6 mRNA expression by DC cells. C57BL/6, MyD88−/− and Tlr9−/− mice were inoculated intraperitoneally with fdWT or fdsc-αDEC bacteriophages or, as a control, with LPS. Mice were sacrificed 2 h later, and purified spleen dendritic cells were analyzed for IL-6 mRNA levels by quantitative real-time PCR. Bars represent the mean fold increase ± SD. The experiments were performed three times (n = 2 per group). Comparative analyses were performed using Student's t-test for unpaired samples.

Proliferation of OT-I CD8+ T cells after 72 h of co-culture with 1 × 104 DCs isolated from C57BL/6 mice or from MyD88−/− or Tlr9−/− transgenic mice previously injected with fdOVA/sc-αDEC bacteriophage particles. The panel shows the percentage of divided, CFSE-low OT-I CD8+ T cells. As a control, the proliferation of OT-I CD8+ T cells co-cultured with DCs isolated from non-immunized (NI) C57BL/6 mice is reported. The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.

IFN-γ production by OT-I CD8+ T cells co-cultured for 12 h in the presence of 3.3 × 103 DCs isolated from C57BL/6 mice or MyD88−/− and Tlr9−/− transgenic mice injected as in (D).

Data information: The mean ± SD of two independent experiments with n = 3 per group is reported. Comparative analyses were performed using Student's t-test for unpaired samples.
Antigen delivery by filamentous bacteriophage fd displaying an anti-DEC-205 single-chain variable fragment confers adjuvanticity by triggering a TLR9-mediated immune response. (2015)
Figure 4

Confocal microscopy analysis of BMDCs after phage uptake

A–D BMDCs were incubated with TRITC-conjugated fdWT (A, C) or fdsc-αDEC bacteriophages (B, D) for 6 h. Cells were then fixed, permeabilized, immunostained with anti-EEA-1 (A, B) or anti-LAMP-1 (C, D) (green) antibody and analyzed by confocal microscopy. Representative images are shown. Numbers at the bottom of the right panels indicate the percentage of EEA-1 (A, B) or LAMP-1 (C, D) bacteriophage co-localization (mean values ± SD, N = 2, n = 100). Scale bars, 10 mm. White arrows indicate LAMP-1-positive structures that also are positive or contain fdsc-αDEC or fdWT bacteriophages.