Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Panel A: controls. A1 : Mitochondrial Cox IV was detected in whole cell extracts, nuclear and mitochondrial fractions. A2 : lamin A & C markers were detected only in nuclear fractions indicating the absence of nuclear contaminations in the mitochondrial fraction except for cytoplasmic preparations. Panel A3 reflects the content of syncytin 2 (HERV-FRD 1 ) in all sub-cellular preparations with remarkable expression in the mitochondrial fraction of U87 RETO cells. Panel A4 : detection of the receptor for syncytin 2, MFSD2 in subcellular fractions except for cytoplasmic preparations. Panel B minor expression of pro-apoptotic proteins like BAD and BAX in U87 RETO cells. In contrast, anti-apoptotic proteins like Bcl-2 and Bcl-xL were found strongly expressed. (For comparison of cytoplasmic vs. whole cell distributions of BAX in untreated vs. treated cells, showing no difference upon etoposide-incubation, see .) Panel C : controls: isolated mitochondria with mitochondrial markers MFN1 and MFN2 (positive control) almost without extra-mitochondrial membrane proteins like ABCG2 and lamin A+C (negative controls). Panel D1 : expression of different HERV proteins in the mitochondrial fraction of U87 RETO cells. For syncytin 1 (HERV-W E1 , upper bands) the 53 kDa surface protein and an additional splicing variant below is shown. In addition, the 24 kDa band reflects the transmembrane protein of syncytin 1. For syncytin 2 the 24 kDa protein was not detectable. HERV-V 3-1 was detectable as the expected single band at 32 kDa. Panel D2 : analysis of intracellular distribution of syncytin 1 in untreated U87 RETO cells (control, left lanes) vs. treated U87 RETO cells after incubation with 5 μg/ml etoposide for 10 days. HERV proteins were found enhanced in the mitochondrial fraction upon etoposide stress. Comparable results were obtained for syncytin 2, see . Panel E : after incubating U87 RETO cells with 5 μg/ml etoposide for 10 days, receptors for syncytin 1 and 2 were also clearly detectable in mitochondrial fraction (see also panel A4 ). For SCL1A5, the endogenous protein (53 kDa) and various glycosylated forms were detected. The receptor for syncytin 2, MFSD2, was also detectable as a single band. The figure is representative of at least n = 3 independent experiments.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Expressing, Incubation, Isolation, Positive Control, Variant Assay

Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Mitochondrial samples were dual labeled for Cox IV and different HERV-proteins. Panel A depicts the expression of syncytin 1 (HERV-WE 1, A1 ), syncytin 2 (HERV-FRD 1 , A2 ), their respective receptors SLC1A5 (A3) and MFSD2 (A4) in U87 cells, which were used as source of mitochondria. The total HERV-protein expression in whole U87 cells was set to 100 %, and the relative amount of HERV-proteins associated with the mitochondrial fraction was given in relationship to the total HERV-protein amount in whole cells. Panel B depicts the measurement of different HERVs in the mitochondrial fraction of these U87 glioblastoma cells. The Cox IV- positive gated populations represent more than 94 % (picture B2 ). Picture B3 represents the negative control (secondary antibody). Both syncytin 1 and syncytin 2 ( B4 & B5 ) were found to be highly expressed, accounting for more than 98 %. HERV-V 3-1 ( B6 ) was detected in more than 67 % of the Cox IV positive populations. Plots are representative of at least n = 3 experiments.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Labeling, Expressing, Negative Control

Journal: Oncotarget

Article Title: Cytotoxic stress induces transfer of mitochondria-associated human endogenous retroviral RNA and proteins between cancer cells

doi: 10.18632/oncotarget.21606

Figure Lengend Snippet: Panel A 1 & 2 : positive controls, mitochondria were previously isolated from U87 RETO cells after cytotoxic stress with etoposide (as described above) and labeled with red MitoTracker ® . Endogenous mitochondria of the U87 host-cells were stained with green MitoTracker ® . After 24h-incubation without further cytotoxic stress, cellular uptake of prepared “red” mitochondria into cancer cells was clearly detectable. Panels 3-6 : experiments were performed under the same conditions as described for panels A 1 & 2 . Panels A 3 & 4 : mitochondria isolated from U87 RETO were blocked with both anti-syncytin 1 (HERV-W E1 ) and anti-syncytin 2 (HERV-FRD 1 ) blocking antibodies and the uptake of the labeled mitochondria was monitored after 24 hours. Panels A 5 & 6 : mitochondria isolated from U87 RETO were blocked with both anti-SLC1A5 and anti-MFSD2 blocking antibodies and the uptake of the labeled mitochondria was monitored after 24 hours. These results indicate that syncytin 1 and 2 as well as their receptors play a substantial role in cellular uptake of mitochondria, since the uptake of exogenous mitochondria were less perceived after blockage of these proteins. Panel B : cartoon representation of the proposed mechanisms of mitochondria entrance into U87 cells. Mitochondria from U87 RETO etoposide resistant cells were isolated and labeled with MitoTracker© red and with anti syncytin 1 or anti syncytin 2 antibodies and exogenously applied to U87 cells. The blockage of both syncytins impedes the uptake of free mitochondria.

Article Snippet: Primary antibodies were purchased as follows: anti-HERV-WE 1 (syncytin 1) and anti-HERV-FRD 1 (syncytin 2) from Bioss, USA (Cat. No. bs2962R and bs15466R) and Biorbyt, UK (Cat. No. orb111912); anti-lamin A+C from Novus Biologicals, USA (Cat. No. EPR4100); Cox IV (Cat. No. 11967), ABCG2 (Cat. No. 4477), MFN1 (Cat. No. 13196), MFN2 (Cat. No.9482), Bcl-2 (Cat. No. 2870), Bcl-X L (Cat. No.2764), BAD (Cat. No.9239) and BAX (Cat. No.5023) primary antibodies from Cell Signaling Technology, (USA); polyclonal antibodies targeting SLC1A5 (Cat. No. bs-0473R) and MFSD2A (Cat. No. bs-6073R) from Bioss, USA; conjugated secondary antibodies from Cell Signaling (USA) and Jackson ImmunoResearch Europe Ltd. (Suffolk, UK).

Techniques: Isolation, Labeling, Staining, Incubation, Blocking Assay

Journal: Aging (Albany NY)

Article Title: The prognostic value of major facilitator superfamily domain-containing protein 2A in patients with hepatocellular carcinoma

doi: 10.18632/aging.102333

Figure Lengend Snippet: Decreased expression of MFSD2A in HCC. ( A ) The expression of MFSD2A in HCC and normal liver tissues was analyzed in the TCGA and GTEx databases ( P < 0.01). ( B ) RT-qPCR showed that the relative mRNA expression of MFSD2A in HCC tissues was decreased compared with that in the matched adjacent nontumorous tissues (n = 24, *P = 0.016). ( C ) Densitometric analysis of MFSD2A protein levels relative to GAPDH in HCC and corresponding normal liver samples. The expression of MFSD2A was reduced in tumor tissues when compared with that in corresponding nontumorous tissues (n = 11, *P = 0.0472). ( D ) The protein level of MFSD2A in HCC and corresponding nontumorous specimens was tested by western blotting. GAPDH was used as a loading control. RT-qPCR ( E ) and western blotting ( F ) were used to analyze the expression of MFSD2A in several HCC cell lines and one immortalized hepatic cell line LO2.

Article Snippet: The membrane was placed in 5% nonfat milk for 1 h to block nonspecific binding sites and was then incubated with a goat anti-human MFSD2A antibody (ProSci incorporated; Cat.No.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Control

Journal: Aging (Albany NY)

Article Title: The prognostic value of major facilitator superfamily domain-containing protein 2A in patients with hepatocellular carcinoma

doi: 10.18632/aging.102333

Figure Lengend Snippet: MFSD2A expression and clinical values. ( A , B ) High expression level of MFSD2A (200×magnification); ( C ) low expression level of MFSD2A (200×magnification); ( D ) negative expression of MFSD2A (200×magnification). Low expression of MFSD2A was significantly correlated with tumor grade ( E ) and stage ( F ) analyzed with UALCAN database. The lower expression level of MFSD2A represented higher stage and pathological grade. ( G ) Patients expressing higher levels of MFSD2A show significantly better five-year overall survival ( P = 0.021). Survival curves of 79 HCC patients with different MFSD2A expression are shown. Kaplan-Meier survival curves for high expression of MFSD2A group were significantly different ( P =0.021, log-rank test) from low MFSD2A expression group in 79 HCC patients. Diagnostic outcomes for plasma MFSD2A in the diagnosis of HCC ( H ) MFSD2A concentrations in plasma. ( I ) ROC curve for MFSD2A with HCC versus HC and CHB. MFSD2A=Major Facilitator Superfamily Domain Containing 2A, HCC=hepatocellular carcinoma, CHB=chronic hepatitis B virus infection, HC=healthy control.

Article Snippet: The membrane was placed in 5% nonfat milk for 1 h to block nonspecific binding sites and was then incubated with a goat anti-human MFSD2A antibody (ProSci incorporated; Cat.No.

Techniques: Expressing, Diagnostic Assay, Clinical Proteomics, Biomarker Discovery, Virus, Infection, Control

Journal: Aging (Albany NY)

Article Title: The prognostic value of major facilitator superfamily domain-containing protein 2A in patients with hepatocellular carcinoma

doi: 10.18632/aging.102333

Figure Lengend Snippet: Relationship between MFSD2A expression and clinicopathological features of hepatocellular carcinoma patients.

Article Snippet: The membrane was placed in 5% nonfat milk for 1 h to block nonspecific binding sites and was then incubated with a goat anti-human MFSD2A antibody (ProSci incorporated; Cat.No.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: The prognostic value of major facilitator superfamily domain-containing protein 2A in patients with hepatocellular carcinoma

doi: 10.18632/aging.102333

Figure Lengend Snippet: Univariate and multivariate analyses showing the overall survival rate for hepatocellular patients.

Article Snippet: The membrane was placed in 5% nonfat milk for 1 h to block nonspecific binding sites and was then incubated with a goat anti-human MFSD2A antibody (ProSci incorporated; Cat.No.

Techniques: Expressing

Journal: Neuron

Article Title: Gradual suppression of transcytosis governs functional blood-retinal barrier formation

doi: 10.1016/j.neuron.2017.02.043

Figure Lengend Snippet: (A) Transcytosis is increased in Mfsd2a−/&minus mice. Many tracer-filled vesicles (arrows) were observed in adult retinal endothelial cells from Mfsd2a−/&minus but not Mfsd2a+/+ mice. (B) Quantification of HRP-filled vesicles in retinal endothelial cells from adult Mfsd2a+/+ and Mfsd2a−/&minus mice. Data are shown as mean ± s.e.m. (n= 5 mice per genotype; each circle represents the average vesicular density from 18 – 20 vessels per mouse). Statistical significance was determined by unpaired t-test. (C) EM of the adult retina confirms that specialized tight junctions halt tracer product in both Mfsd2a+/+ and Mfsd2a−/&minus adult mice (D) Quantification of functional tight junctions from Mfsd2a+/+ and Mfsd2a−/&minus adult mice (n = 5 mice per genotype; 18-20 vessels analyzed per mouse; number of tight junctions analyzed are displayed in parenthesis). (E) Immunostaining for Claudin-5 (green) and Mfsd2a (white) on P7 and P10 retinas shows the lack of Mfsd2a expression in nascent, distal vessels at P7. The red dash lines indicate the angiogenic front as determined by Claudin-5 expression, and the pink bar indicates the length from the angiogenic front to the appearance of Mfsd2a expression. In contrast to P7, Mfsd2a is expressed in the distal vessels at P10 (n= 5 mice per age). (F) Mfsd2a expression correlates with functional BRB formation. Immunostaining for Claudin-5 and Mfsd2a in retinas from 10-kDa dextran injected P7 and P10 mice shows that at P7, extravasation of tracer (arrowheads) occurs at nascent vessels where Mfsd2a is absent. At P10, tracer is confined (arrows) in distal vessels where Mfsd2a is expressed (n=5 mice per age). (G) Genetic ablation of Mfsd2a results in incomplete formation of the functional BRB. After intravenous injection of 10-kDa dextran in P5 Mfsd2a+/+ and Mfsd2a−/&minus pups, tracer was confined (arrow) in proximal vessels (bottom) but leaked from distal vessels (top) in Mfsd2a+/+ mice (arrowheads). In contrast, tracer leaked into the retinal parenchyma from both proximal and distal vessels in Mfsd2a−/&minus mice (arrowheads). (H) Permeability index from proximal and distal regions of P5 Mfsd2a−/&minus and Mfsd2a+/+ littermates. Data are shown as mean ± s.e.m. (n=5 mice per genotype; each circle represents the average permeability from each mouse). Statistical significance was determined by unpaired t-test. ***, P < 0.001. Scale bars represents 100 nm in (A and C) and 100 μm in (E,F, and G).

Article Snippet: Isolectin GS-IB4 conjugated to Alexa-Fluor® 488 (Thermo Scientific; {"type":"entrez-nucleotide","attrs":{"text":"I21411","term_id":"1601765","term_text":"I21411"}} I21411 ) or the following primary antibodies: rabbit α-ERG1/2/3 (Abcam Cat# ab92513, RRID:AB_2630401, 1:200), rabbit α-Mfsd2a (Cell Signaling Technology still under development, Cat# 14-87 Mfsd2a, RRID:AB_2617168, 1:200) and mouse α-Claudin-5, (Thermo Fisher Scientific Cat# 352588 Lot# RRID:AB_2532189, 1:400) were then incubated in blocking buffer overnight at 4°C.

Techniques: Functional Assay, Immunostaining, Expressing, Injection, Permeability

Journal: Annals of Diagnostic Pathology

Article Title: Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein ☆

doi: 10.1016/j.anndiagpath.2020.151682

Figure Lengend Snippet: Histologic and molecular correlates of COVID-19 in human brains. Panel A shows the microvessels in normal brain. In comparison, many of the capillaries in COVID-19 brain tissues show marked perivascular edema (panel B). Serial section analyses of the COVID-19 brain shows that the endothelial cells of the microvessels contained the spike glycoprotein (panel C), the ACE2 receptor (panel D) and IL 6 (panel F), but not viral RNA (panel E). The fluorescent yellow signal marks co-localization of the spike protein with IL6 (panel G) and caspase 3 (panel H), respectively, in these endothelial cells. Each magnification is 800× with DAB (brown) signal (panels C, E, F) or Fast Red (red) (panel D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used follow; ABCAM, Cambridge MA (IL6, TNFα, NMDAR2, SHIP1, caspase 3, and C5b-9), Enzo Life Sciences (neuronal NOS, MFSD2a), ACE2 (PROSCI, Poway CA) (cat # 3215) and optimal pretreatment in each case was 30 min with the Leica EDTA antigen retrieval solution.

Techniques: Comparison

Journal: Annals of Diagnostic Pathology

Article Title: Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein ☆

doi: 10.1016/j.anndiagpath.2020.151682

Figure Lengend Snippet: Quantification of cell line data after incubation with different subunits of the SARS-CoV2 spike protein.

Article Snippet: The antibodies used follow; ABCAM, Cambridge MA (IL6, TNFα, NMDAR2, SHIP1, caspase 3, and C5b-9), Enzo Life Sciences (neuronal NOS, MFSD2a), ACE2 (PROSCI, Poway CA) (cat # 3215) and optimal pretreatment in each case was 30 min with the Leica EDTA antigen retrieval solution.

Techniques: Incubation

Journal: Annals of Diagnostic Pathology

Article Title: Endothelial cell damage is the central part of COVID-19 and a mouse model induced by injection of the S1 subunit of the spike protein ☆

doi: 10.1016/j.anndiagpath.2020.151682

Figure Lengend Snippet: Cytologic and molecular correlates of the S1 subunit of the spike protein in cell lines. Panel A shows the cytology of untreated HUVEC cells; these endothelial cells strongly express ACE2 (panel B). Treatment with the S1 subunit of spike protein induced cell aggregation and degeneration (panel C). The S1 subunit was not endocytosed by the RAW cells (panel D) and was evident in the HUVEC cells where it co-localized with caspase 3 as seen in panels F-H. Panel F is the isolated spike data (fluorescent green), panel G the isolated caspase 3 data (fluorescent red) and panel H the merged image with fluorescent yellow marking the co-localization of the two protein. The MN cells likewise showed co-localization of the spike protein and caspase 3 (panel E). The magnifications are 600× (panels A-D) and 1000× (panels E -F) with DAB (brown) signal (panel D). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The antibodies used follow; ABCAM, Cambridge MA (IL6, TNFα, NMDAR2, SHIP1, caspase 3, and C5b-9), Enzo Life Sciences (neuronal NOS, MFSD2a), ACE2 (PROSCI, Poway CA) (cat # 3215) and optimal pretreatment in each case was 30 min with the Leica EDTA antigen retrieval solution.

Techniques: Isolation