Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: EPHA2 is expressed on the cell surface of undifferentiated mouse ESCs and downregulated during differentiation. (A) Relative gene expression of Epha2 mRNA in mouse ESCs (D3) cultured without LIF for 0, 3, 5, and 7 days. (B) Immunoblotting of EPHA2 protein of mouse ESCs cultured as in (A). (C) EPHA2 protein levels in (B) normalized to α-Tubulin. (D) Immunofluorescent staining of mouse ESCs. The cells were cultured with or without LIF for 7 days. Bars; 200 μm. (E, F) Flow cytometric analysis of EPHA2 protein on the cell surface of mouse ESCs. Living cells were stained with an EPHA2 antibody. The propidium iodide + dead cells were removed from the analysis. FSC and SSC profiles (E) and the histogram of EPHA2-AF488 levels (F) of the gated cells in (E) were shown. Statistical analyses in (A) and (C) were calculated by Dunnett’s test. Data were graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01; *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Gene Expression, Cell Culture, Western Blot, Staining

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Knock-down of Epha2 induces spontaneous differentiation of mouse ESCs. (A) qRT-PCR analysis of Epha2 mRNA after knock-down in mouse ESCs (D3). The cells were infected with Epha2 shRNA retrovirus and cultured with or without LIF for 7 days. (B) Images of phase contrast and alkaline phosphatase (AP) staining of Epha2 KD mouse ESCs cultured in ES maintenance medium with LIF for 5 days. Bars; 200 μm. (C) qRT-PCR analysis of undifferentiated state-specific marker genes in Epha2 KD mouse ESCs. (D) Immunofluorescent staining of Epha2 KD mouse ESCs cultured with LIF for 7 days. Bars; 100 μm. (E) Culture conditions of Epha2 KD mouse ESCs after infection with Epha2 shRNA retrovirus. One day after infection with Epha2 shRNA virus, mouse ESCs were cultured with or without 2i in the presence of G418 and LIF for 4 days. (F) Phase contrast images of Epha2 KD mouse ESCs cultured as in (E). Bar; 200 μm. (G) qRT-PCR analysis of Epha2 and undifferentiated state-specific marker genes in Epha2 KD mouse ESCs cultured with 2i. All qRT-PCR analyses were performed with 3 independent biological replicates and graphed as means ± SE. The significant differences were calculated by Tukey test in (A and C) and t -test in (G). * P < .05; ** P < .01, *** P < .001.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Knockdown, Quantitative RT-PCR, Infection, shRNA, Cell Culture, Staining, Marker, Virus

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Expression of EPHA2 in heterogenous subpopulation of human PSCs. (A) qRT-PCR analysis of EPHA2 and undifferentiated state-specific marker genes during EB-based random differentiation of human iPSCs (201B7) without basic FGF. Statistical analysis was done by Dunnett’s test comparing to day 0 and graphed as means ± SE of 3 independent experiments. * P < .05; ** P < .01. (B) Representative flow cytometric plots of living human iPSCs stained with EPHA2 antibody-conjugated with AFF488. Gate3 and Gate4 were sorted as EPHA2 − and EPHA2 + cell populations, respectively. See also . (C) Feature plots of EPHA2 and undifferentiated state-specific genes in publicly available undifferentiated human ESC H1 and H9 data subsets from GSE75748 scRNA-seq dataset. Note that EPHA2 expression in hESCs was heterogeneous. Normalized expression levels were plotted. (D, E) Immunofluorescent staining of human iPSC cultured on SyntheMax II-coated plate with StemFit medium. The cells were fixed with paraformaldehyde in PBS and permeabilized. Bars; 200 μm. (F) qRT-PCR analysis of fractioned EPHA2 + and EPHA2 - subpopulations. EPHA2 + cells express higher OCT4 and NANOG , than EPHA2 − cells. Means ± SE of 3 independent experiments were shown. Statistical significance was defined as * P < .05 by t -test.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Quantitative RT-PCR, Marker, Staining, Cell Culture

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Transplantation of EPHA2 + cells into immune-deficient mice formed tumors in vivo. (A) Immunofluorescent staining of mouse EBs differentiated into hepatocyte lineages. Expression of an early hepatocyte marker AFP at day 10 and a mature marker ALB at day 14 were detected. Bars; 200 μm. (B) Depletion of undifferentiated ES colonies after removal of EPHA2 + cells from EBs. Oct4-egfp ESCs were differentiated by EB formation for 10 and 14 days. EPHA2 + cells were removed from EBs using anti-EPHA2 antibody-bound MACS after dissociation with trypsin/EDTA. The residual cells were cultured in ES maintenance medium with LIF for 7 days. The alkaline phosphatase (AP) activity was visualized by incubating with AP substrate. Bar; 2 cm. (C) The number of EGFP + cell colonies in (B). Statistical analysis was done by Tukey test and graphed as means ± SE of 4 independent experiments. (D) Scheme of in vivo transplantation experiment. Mouse ESCs (D3) were differentiated into hepatocyte linages and the EBs were dissociated by EDTA treatment. The cells were transplanted into SCID mice after depletion of EPHA2 + cells by MACS. (E) Decreased teratoma formation after transplantation of EPHA2 − cells. White arrowheads indicate teratomas. (F) H&E staining of teratomas formed in the testicular subcutaneous tissue without MACS procedure. Typical cell types of 3 germ layers were confirmed. Bar; 500 μm. (G) Typical teratoma formation after transplantation of dissociated EB at day 10 through hepatic portal vein. White arrowheads indicate teratomas. (H) Quantification of teratoma formation in (E) and (G). Statistical analysis was done by Chi-square test, * P < .05, ** P < .01.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Transplantation Assay, In Vivo, Staining, Expressing, Marker, Cell Culture, Activity Assay

Journal: Stem Cells Translational Medicine

Article Title: EPHA2 is a novel cell surface marker of OCT4-positive undifferentiated cells during the differentiation of mouse and human pluripotent stem cells

doi: 10.1093/stcltm/szae036

Figure Lengend Snippet: Co-expression of EPHA2 with OCT4 in EBs during human iPSC differentiation into hepatocyte. (A, B) Relative gene expression of undifferentiation and differentiation markers during hepatic induction. Statistical analysis was done by Dunnett’s test against day 0 and graphed as means ± SE of 3 independent biological replicates. * P < .05; ** P < .01, *** P < .001. (C) Immunofluorescent staining of EBs at days 5, 8, and 10. Bars; 200 μm. (D) Enlarged images of EB at day 5 in (C). Bars; 50 μm. (E, F) Quantification of immune-positive cells in . Box plot showing the percentage of EPHA2 + cells among SOX17 + or OCT4 + cells (E). Box plot showing the percentage of EPHA2 + and TRA1-81 + cells among OCT4 + cells (F). Each box represents 1st quartile, median, and 3rd quartile, and whiskers show the minimum and maximum values. Ten images of independent EBs were analyzed. Total count of DAPI + nuclei per image were between 1 × 10 3 and 2 × 10 3 . Statistical significance was defined by Tukey test of SOX17 and OCT4, respectively in (E) and t -test between EPHA2 and TRA1-81 in (F). **P p < .01, *** P < .001, N.S; no significance between 3 with P > .05.

Article Snippet: For immunofluorescent analyses of human iPSCs, mouse monoclonal anti-human EPHA2 (MAB3035; R&D systems) and goat polyclonal anti-human SOX17 (AF1924, R&D systems) were used.

Techniques: Expressing, Gene Expression, Staining

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 mRNA expression profiles of various tumor samples and paired normal tissues. The height of the bar represents the median expression level (in transcripts per million (TPM)) in each tumor or normal tissue type.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 expression patterns in pancreatic adenocarcinoma (PAAD) patients. (a) EphA2 transcriptional levels in PAAD and normal pancreatic tissues and (b) EphA2 mRNA levels in PAAD primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Associations between pancreatic adenocarcinoma (PAAD) clinical pathological stage and EphA2 expression.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: The prognostic value of EphA2 expression in pancreatic adenocarcinoma (PAAD) patients. Kaplan–Meier plots are shown for (a) overall survival and (b) disease-free survival.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: EphA2 promoter DNA methylation patterns in pancreatic adenocarcinoma (PAAD) primary tumors and normal tissues.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: DNA Methylation Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Correlations of immune cell infiltration and EphA2 expression in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Multivariate Cox proportional risk model of pancreatic adenocarcinoma (PAAD).

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Protein-protein interaction (PPI) analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients. (a) PPI network of EphA2. (b) Potential functions of the proteins of interest and (c) Molecular complex detection components identified in the gene lists.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques:

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Functional enrichment analysis of EphA2 in pancreatic adenocarcinoma (PAAD) patients.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Functional Assay

Journal: The Journal of International Medical Research

Article Title: Identification of therapeutic targets and prognostic biomarkers of the ephrin receptor subfamily in pancreatic adenocarcinoma

doi: 10.1177/03000605231218559

Figure Lengend Snippet: Analysis of EphA2 protein expression using immunohistochemistry (IHC) in tissue samples from pancreatic adenocarcinoma (PAAD) patients. Representative images are shown at 100x magnification. (a) High EphA2 protein expression in pancreatic cancer tissue and (b) Low EphA2 protein expression in paracancerous tissue.

Article Snippet: A mouse anti-EphA2 antibody (MAB3035, 1:50, R&D Systems, Boston, MA, USA) was used.

Techniques: Expressing, Immunohistochemistry

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet:

Article Snippet: Rabbit Anti human/mouse Phospho-EphA2 (Ser897) (D9A1) , Cell Signaling Technology , Cat#6347; RRID: AB_11220420.

Techniques: Recombinant, Modification, Membrane, Transfection, In Vivo, Software

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Treatment failure and evaluation of the underlying mechanism of non-response in TNBC recurrence, post treatment with 5FU (A) Survival curve of DFS in TNBC patients with differential treatment regimens, one SOC containing 5FU and the other cohort received SOC without 5FU. (B) Differential expression of phospho-EphA2 and phospho- c -MET in recurrent TNBC tissue. (C) Schematic representation of ex vivo explants culture. (D and E) Differential expression of Ki67 and Caspase 3c in TNBC patient derived ex vivo explants cultures, stratifying the cohort into responders and non-responder population. (F) Expressional status of phopsho-EphA2 and phospho- c -MET in the non-responder population from explants cultured tissues. Data are representative of triplicate experiments. ∗p < 0.05 represents statistically significant difference and was calculated by the log-rank test. Scale bars: 200 μm and 100 μm (in inset). HGF = hepatocyte growth factor; 5FU = 5-fluorouracil; SOC = standard of care; TNBC = triple negative breast cancer; HE = Hematoxylin & Eosin staining. Also see, Figures S1 and ; Tables S1–S6 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Quantitative Proteomics, Ex Vivo, Derivative Assay, Cell Culture, Staining

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the effect of individual and dual knock down of c-MET and EphA2 genes on MD-MB-231 in the presence of HGF (A) c-MET and EphA2 knockdown/inhibition and its effect on downstream effectors on MDA-MB-231 cells was analyzed by western blot; NTP = non-target pool control. (B) Transwell migration assay post knockdown/inhibition of c-MET or EphA2 or both and HGF as a chemotactic factor on MDA-MB-231 cells. (C) Transwell invasion assay post knockdown/inhibition of EphA2 and c-MET or both and HGF as a chemotactic factor on MDA-MB-231 cells. (D) A schematic representation of the mammosphere formation assay. (E) Mammosphere formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF. (F) Matrigel tube formation assay post knockdown/inhibition of c-MET or EphA2 or both in the presence of HGF. (G) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale. (H) Graph representing the tumor volume vs. the various groups. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. Scale bars: 100 μm. Also see, Figures S3 and .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Knockdown, Inhibition, Western Blot, Control, Transwell Migration Assay, Transwell Invasion Assay, Tube Formation Assay

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the combinatorial effect of Lupeol and 5FU on MDA-MB 231 cells (A) Western blot analysis of MDA-MB-231 cells treated with 5FU, Lupeol, or both in the presence of HGF and probed for phospho-EphA2 (S897), EphA2, phospho- c -MET, total c-MET, pERK1/2, total ERK1/2, MMP2 and Laminin-5Ƴ2 protein expression. β- Actin was used as a loading control. (B) Protein-protein interaction (PPI) network analysis using input proteins from the present study using STRING platform showing interacting molecules and edge confidence levels in the network. (C and D) Transwell migration and invasion of MDA-MB-231 cells upon treatment with Lupeol, 5FU or both, while HGF was used as a chemotactic factor (Scale bars: 100 μm). (E) Matrigel tube formation of MDA-MB-231 cells upon treatment with Lupeol, 5FU, or both in the presence of HGF (Scale bars: 500 μm and 100 μm in inset). (F) Primary and secondary Mammosphere formation of MDA-MB-231 cells in the presence of HGF and treated with 5FU, Lupeol, or both (Scale 200 μm in white and 100 μm in red). (G) Immunofluorescence staining of MDA-MB-231 cells for the detection of E-cadherin (green) and Vimentin (red) post treatment with Lupeol or 5FU or both in the presence of HGF (Scale bars: 100 μm). (H) Western blot analysis of MDA-MB-231 cells treated with Lupeol, 5FU, or both in the presence of HGF and probed for E-cadherin, Vimentin, SNAI1, SLUG, and TWIST. Data are representative of triplicate experiments (mean ± SD). HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Figure S5 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Western Blot, Expressing, Control, Migration, Immunofluorescence, Staining

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: In vivo evaluation of the combinatorial effect of Lupeol and 5FU in a TNBC syngeneic mice model (A) Schematic representation of dosing schedule (all compounds were administered intraperitoneally). (B) In vivo live animal images of the 4T1 luc2 induced tumors in various treatment arms in BALB/c mice at day 5 (initiation of treatment) and at day 15 (experimentation endpoint). (C) Graph representing the log of average radiance vs. Time of the tumor growth. (D) Representative photomicrograph of freshly harvested breast tumors with a ruler (below) for scale. (E) Graph representing the tumor volume vs. the various treatment arms. (F) Representative images of Hematoxylin and Eosin stain followed by IHC staining for Ki67 and Caspase 3c in the sections of harvested tumors developed in BALB/c mice along with their quantitative graphs. (G) Representative images of the IHC staining to evaluate the differential expression of phospho-EphA2 and phospho-cMET in the tumors of the various treatment arms along with their quantitative graphs. ∗p < 0.05 and ∗∗∗p < 0.001 statistically significant difference compared to corresponding control by one-way ANOVA. Data are representative of triplicate experiments (mean ± SD) ns = not significant; HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Scale bars: 200 μm. Also see, Figure S6 and Table S8 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: In Vivo, H&E Stain, Immunohistochemistry, Quantitative Proteomics, Control

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet: Evaluation of the combinatorial effect of 5FU and Lupeol in ex vivo explant culture model (A) Graphical representation of the patient data used in this experiment, pertaining to the nodal status and tumor stage. (B) Representative images of IHC staining of TNBC tissue fragments, post exposure to HGF, 5FU, Lupeol alone or in combination cells for the detection of Ki67 and Caspase 3c protein expression (Scale bars: 200 μm). (C) Graph representing the percentage of cells expressing Ki67 vs. the various treatment arms. (D) Graph representing the IHC score of Caspase 3c vs. various treatment arms. (E) Representative images of IHC staining for the evaluation of phospho-EphA2 and phospho-cMET in various treatment arms (Scale bars: 200 μm and 100 μm in inset). (F) Graph representing the various IHC scores of phospho-EphA2 in various treatment arms. (G) Graph representing the various IHC scores of phospho-cMET in various treatment arms. (H) Graph representing the various IHC scores of Caspase 3c expression in TNBC tumors (N = 7) after treating them with various SOC and 5FU and Lupeol combination. Data are representative of triplicate experiments (mean ± SD). ∗p < 0.05 statistically significant difference compared to corresponding control by one-way ANOVA. HGF = hepatocyte growth factor; HF = HGF+5FU; HL = HGF+Lupeol; HFL = HGF+5FU + Lupeol. Also see, Table S9 .

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Ex Vivo, Immunohistochemistry, Expressing, Control

Journal: iScience

Article Title: Lupeol synergizes with 5-fluorouracil to combat c-MET/EphA2 mediated chemoresistance in triple negative breast cancer

doi: 10.1016/j.isci.2023.108395

Figure Lengend Snippet:

Article Snippet: Rabbit Anti human/mouse EphA2 (D4A2) , Cell Signaling Technology , Cat#6997; RRID: AB_10827743.

Techniques: Recombinant, Modification, Membrane, Transfection, In Vivo, Software