Journal: Cell Reports Medicine

Article Title: iPSC-derived hypoimmunogenic tissue resident memory T cells mediate robust anti-tumor activity against cervical cancer

doi: 10.1016/j.xcrm.2023.101327

Figure Lengend Snippet:

Article Snippet: VEGF , Miltenyi Biotec , Cat# 130-109-386.

Techniques: Recombinant, DNA Extraction, Sequencing, Staining, Flow Cytometry, Cell Stimulation, Software

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 2 ｜Cellular ATP levels and F1F0-ATPase activity in isolated mitochondria in a in SH-SY5Y cell model of Parkinson’s disease. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector + MPP+: LV-Flag-vector- transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag + MPP+: LV-CHCHD2-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Detection of ATP levels in control or MPP+-treated SH-SY5Y cells stably transfected with empty vector or constructs encoding WT or T61I-mutant CHCHD2. (B) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in control or MPP+-treated cells. (C) Effect of WT or T61I-mutant CHCHD2 on ATP synthase specific activity in AVV-transfected mice with or without MPTP treatment. CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); Flag-vector: AVV-Flag (CMV- betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ATP: Adenosine triphosphate; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Activity Assay, Isolation, Plasmid Preparation, Transfection, Cell Culture, Control, Stable Transfection, Construct, Mutagenesis, Saline

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 3 ｜CHCHD2 T61I mutation promotes mitochondrial permeability transition pore (mPTP) opening in SH-SY5Y cells. CHCHD2-Flag: LV-CHCHD2-Flag-transfected SH-SY5Y cells; CHCHD2-T61I: LV-CHCHD2-T61I-Flag-transfected SH-SY5Y cells; Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells. (A) Calcein-AM and CoCl2 were administered to live cells expressing WT or T61I-mutant CHCHD2, and fluorescence intensity was detected by flow cytometry. (B) Effect of WT or the T61I-mutant CHCHD2 on mPTP opening as determined by detecting mitochondrial membrane potential using JC-1 fluorescent probes (white squares) in control or MPP+-treated SH-SY5Y cells. The fluorescence intensity of intracellular JC-1 aggregates (red) in the MPP+ + CHCHD2-Flag group was greater than that in the MPP+ + Flag-vector group. The CHCHD2-T61I group showed an increased amount of JC-1 monomers (green). Scale bars: 10 μm. (C) The mean fluorescence intensity of JC-1 aggregates or monomers in control or MPP+-treated SH-SY5Y cells expressing WT or T61I-mutant CHCHD2. Data are expressed as the mean ± SD (n = 3 independent experiments). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). Calcein-AM: Calcein acetoxymethyl ester; MPP+: 1-methyl-4-phenylpyridinium; mPTP: mitochondrial permeability transition pore; WT: wild type.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Permeability, Transfection, Plasmid Preparation, Expressing, Fluorescence, Flow Cytometry, Membrane, Control

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 5 ｜CHCHD2 T61I mutation aggravates movement deficits and nigral DA neuron function in a mouse model of PD. (A) Experimental design. AAV-CHCHD2, AAV-CHCHD2 T61I, or AAV-Vector was stereotaxically injected into the substantia nigra pars compacta, and 4 weeks later mice were injected intraperitoneally with normal saline or MPTP for 5 weeks. One day after the last MPTP/normal saline injection, behavioral tests were performed, and then the mice were sacrificed. Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector+MPTP: AVV-Flag– transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag+MPTP: AVV-CHCHD2-Flag–infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I+MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (B) The grasping test was used to examine grip strength. (C) The pole-climbing test was used to examine bradykinesia. (D) The rotarod test was used to examine motor coordination. (E) Representative immunohistochemical staining of TH-positive neurons in the substantia nigra (SN). There were fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 800 μm. (F) Quantification of the TH-positive cells shown in E. (G) Representative images of double-immunofluorescent staining for CHCHD2-Flag (green, Alexa Fluor 488) and TH (red, Alexa Fluor 555) (white squares) in the SN. There were significantly fewer TH-positive neurons in the SN in the AAV-CHCHD2-T61I + MPTP group than in the AAV-Flag-vector + MPTP and AAV-CHCHD2-Flag + MPTP groups. Scale bars: 50 μm. (H) Quantification of TH mean fluorescence in the SN as shown in G. (I) Representative immunoblot for TH in the SN. (J) TH expression in the SN. Results are expressed as the mean ± SD (n ≥ 6). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; AAV: adeno-associated virus; DA: dopaminergic; DAPI: 4′,6-diamidino-2-phenylindole; MPTP: 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine; TH: tyrosine hydroxylase; SN: substantia nigra.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Mutagenesis, Plasmid Preparation, Injection, Saline, Transfection, Infection, Immunohistochemical staining, Staining, Fluorescence, Western Blot, Expressing, Virus

Journal: Neural regeneration research

Article Title: CHCHD2 Thr61Ile mutation impairs F1F0-ATPase assembly in in vitro and in vivo models of Parkinson's disease.

doi: 10.4103/1673-5374.378010

Figure Lengend Snippet: Figure 8 ｜CHCHD2 modulates OSCP expression in MPP+-/MPTP-induced PD models. (A, B) Flag-vector: LV-Flag-vector (Ubi-MCS-3FLAG-SV40-puromycin vector)-transfected SH-SY5Y cells cultured in MEM-F12 medium; Flag-vector+MPP+: LV-Flag-vector–transfected SH- SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-Flag+MPP+: LV-CHCHD2-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours); CHCHD2-T61I + MPP+: LV-CHCHD2-T61I-Flag–transfected SH-SY5Y cells treated with MPP+ (500 μM for 24 hours). (A) Representative immunoblot of OSCP, CHCHD2, and ACTB in MPP+-treated SH-SY5Y cells. (B) Quantification of relative OSCP expression shown in A. (C, D) Flag-vector: AVV-Flag(CMV-betaGlobin-MCS-3Flag-SV40 polyA (GV411) vector)-transfected mice treated with normal saline (twice a week, for 5 weeks); Flag-vector + MPTP: AVV-Flag-infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-Flag + MPTP: AVV-CHCHD2-Flag- infected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks); CHCHD2-T61I + MPTP: AVV-CHCHD2-T61I–transfected mice treated with MPTP (25 mg/kg, twice a week, for 5 weeks). (C) Representative immunoblot of OSCP, CHCHD2, and ACTB in control or MPTP-treated mice. (D) Quantification of relative OSCP expression shown in C. Data normalized to SH-SY5Y cells overexpressing Flag-vector or C57BL/6J mice transfected with AAV-Flag are expressed as the mean ± SD (n = 3 independent experiments for cells, n = 6 for animals). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 (one-way analysis of variance followed by Tukey’s post hoc test). ACTB: β-Actin; MPP+: 1-methyl-4-phenylpyridinium; MPTP: 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine; OSCP: oligomycin sensitivity conferral protein.

Article Snippet: Detection of mitochondrial permeability transition pore opening Opening of the mitochondrial permeability transition pore (mPTP) was detected by the calcein-AM/cobalt assay following the manufacturer’s instructions (C2009S, Beyotime Biotechnology).

Techniques: Expressing, Plasmid Preparation, Transfection, Cell Culture, Western Blot, Saline, Infection, Control

Journal: Journal of Animal Science and Biotechnology

Article Title: Endoplasmic reticulum stress-induced apoptosis in intestinal epithelial cells: a feed-back regulation by mechanistic target of rapamycin complex 1 (mTORC1)

doi: 10.1186/s40104-018-0253-1

Figure Lengend Snippet: Tunicamycin induced activation of mTOR signaling. Cells were treated with or without tunicamycin for indicated time periods, protein abundance for mTORC1, p-mTORC1, p70S6K, p-p70S6K, p-4EBP1, and 4EBP1 were determined by Western blot analysis. The histograms represent the statistical analysis of protein abundance from three individual experiments. ( a ) p-mTOR/mTOR. ( b ) p-p70S6K/p70S6K. ( c ) p-4EBP1/4EBP1. Data are presented as mean ± SEM. * P < 0.05

Article Snippet: Rapamycin and antibodies against cleaved-caspase-3, IRE1α, BiP, ATF4, c-myc, phospho-eIF2α (Ser51), eIF2α, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-JNK (Thr183/Tyr185), JNK, phospho-mTORC1 (Ser2448), mTORC1, phospho-p70S6K (Thr389/Thr412), p70S6K, phospho-4EBP1 (Thr70), and 4EBP1 were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Quantitative Proteomics, Western Blot

Journal: Journal of Animal Science and Biotechnology

Article Title: Endoplasmic reticulum stress-induced apoptosis in intestinal epithelial cells: a feed-back regulation by mechanistic target of rapamycin complex 1 (mTORC1)

doi: 10.1186/s40104-018-0253-1

Figure Lengend Snippet: Rapamycin enhances tunicamycin-induced cell apoptosis in IEC-6 cells. a Rapamycin blocked tunicamycin-induced mTORC1 signaling activation. IEC-6 cells were treated with or without tunicamycin (1 μg/mL) in the presence or absence of rapamycin (100 nmol/L) for 12 h. Protein abundances for mTORC1, p-mTORC1, p70S6K, p-p70S6K, p-4EBP1, and 4EBP1 were determined by Western blot analysis. The right panel histograms represents the statistical analysis of protein abundance from three individual experiments. Data are expressed as mean ± SEM. b mTORC1 inhibition by rapamycin sensitized tunicamycin-induced apoptosis. Cells were treated without or with tunicamycin (1 μg/mL) in the presence or absence of rapamycin (10 or 100 nmol/L) for 12 h, cell viability was assessed by using of CCK-8 kit. Data are presented as mean ± SEM in percentage compared to the untreatment group, n = 6. c Flow cytometry analysis of apoptosis in IEC-6 cells. Cells treated as in Fig. were stained by Annexin V-FITC/PI and apoptosis was determined by Facs analysis. d Bar chart for apoptosis in tunicamycin and/or rapamycin treated cells. Data are mean ± SEM, n = 3. TUN, tunicamycin; UT, untreatment; Rap, rapamycin

Article Snippet: Rapamycin and antibodies against cleaved-caspase-3, IRE1α, BiP, ATF4, c-myc, phospho-eIF2α (Ser51), eIF2α, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-JNK (Thr183/Tyr185), JNK, phospho-mTORC1 (Ser2448), mTORC1, phospho-p70S6K (Thr389/Thr412), p70S6K, phospho-4EBP1 (Thr70), and 4EBP1 were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Activation Assay, Western Blot, Quantitative Proteomics, Inhibition, CCK-8 Assay, Flow Cytometry, Staining

Journal: Journal of Animal Science and Biotechnology

Article Title: Endoplasmic reticulum stress-induced apoptosis in intestinal epithelial cells: a feed-back regulation by mechanistic target of rapamycin complex 1 (mTORC1)

doi: 10.1186/s40104-018-0253-1

Figure Lengend Snippet: Effects of mTORC1 inhibition on intestinal barrier function in tunicamycin-challenged cells. Effect of rapamycin on tunicamycin-induced ( a ) transepithelial electric resistance (TEER), and ( b ) permeability in IEC-6 cells. IEC-6 monolayer cells incubated with tunicamycin (1 μg/mL) in the presence or absence of rapamycin (100 nmol/L) for 24 h. TEER and permeability were determined. Data are expressed as mean ± SEM, n = 3. TEER, transepithelial electrical resistance; FITC, fluorescein isothiocyanate; Rap, rapamycin. ( c ) Proposed mechanism for tunicamycin-induced apoptosis in intestinal epithelial cells and its feed-back regulation by mTORC1. TUN, tunicamycin

Article Snippet: Rapamycin and antibodies against cleaved-caspase-3, IRE1α, BiP, ATF4, c-myc, phospho-eIF2α (Ser51), eIF2α, phospho-p38 MAPK (Thr180/Tyr182), p38 MAPK, phospho-ERK1/2 (Thr202/Tyr204), ERK1/2, phospho-JNK (Thr183/Tyr185), JNK, phospho-mTORC1 (Ser2448), mTORC1, phospho-p70S6K (Thr389/Thr412), p70S6K, phospho-4EBP1 (Thr70), and 4EBP1 were obtained from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Inhibition, Permeability, Incubation

Journal: Cell & Bioscience

Article Title: Vascular NRP2 triggers PNET angiogenesis by activating the SSH1-cofilin axis

doi: 10.1186/s13578-020-00472-6

Figure Lengend Snippet: NRP2 modulates angiogenesis by promoting HUVEC migration via a VEGF/VEGFR2-independent pathway. a HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and then transfected with a vector control or NRP2 overexpression plasmid before they were seeded for the capillary tube formation assay. Representative images at 4, 12 and 24 h after plating are shown. b Quantification of the number of complete and broken tubes at 6 h from a representative experiment. Data are shown as the mean ± SD of three independent experiments. * P ≤ 0.05 by Student’s t test. c HUVECs were treated with conditioned medium from BON cells for 24 h and then transfected with a vector or NRP2 overexpression plasmid before they were subjected to a CCK8 assay. d After HUVECs were cultured in the presence or absence of conditioned medium from BON cells (treatment and control, respectively) and transduced with the NRP2-overexpressing plasmid, flow cytometry was performed to assess apoptosis. e Representative images for the wound-healing assay at 0, 24 and 48 h after scratching for the 4 different cell groups (HUVECs with or without NRP2 overexpression cultured in the presence or absence of conditioned medium from BON cells). f Quantification of the healing rate at 48 h after wound-healing assays in HUVECs cultured in the presence or absence of conditioned medium from BON cells followed by transfection with empty vector or NRP2 plasmid. The data are shown as the means ± SD of three independent experiments. *** P ≤ 0.001 by Student’s t test. g HUVECs were transfected with empty vector or an NRP2 overexpression plasmid and then treated with the VEGFR2-specific inhibitor KI8751. Western blotting assays were performed to determine the levels of VEGFR2 phosphorylation at Tyr951 as well as the total protein levels of VEGFR2, CD31, CD34 and GAPDH. h Control and NRP2-overexpressing HUVECs were treated with KI8751 and PBS and evaluated for tube formation. i After HUVECs were cultured in the presence or absence of conditioned medium from BON cells for 24 h, they were transfected with a vector or NRP2 overexpression plasmid. These cells were subsequently treated with PBS (control) or KI8751, and a wound-healing assay was performed. Representative image of three independent experiments is shown. j Qualification of the wound-healing rate at 48 h in HUVEC-vector or HUVEC-NRP2 cells treated with PBS or KI8751. The data are shown as the mean ± SD of three independent experiments. ** P ≤ 0.01 by Student’s t test. k After HUVECs were transfected with empty vector or an NRP2 overexpression plasmid, they were treated with PBS or KI8751 and subjected to the CCK8 assay at 24, 48 and 72 h after treatment. l Flow cytometry assay was performed in the 4 cell groups described in Fig. 2k

Article Snippet: Western blotting was performed with antibodies targeting the following proteins: VEGF receptor 2 (2479S, Cell Signaling), NRP2 (ab185710, Abcam), phospho-VEGF receptor 2 (Tyr951) (4991S, Cell Signaling), CD31 (ab28364, Abcam), CD34 (ab81289, Abcam), GAPDH (5174S, Cell Signaling), cofilin (5175S, Cell Signaling), phosphorylated cofilin (Ser3) (3313S, Cell Signaling), and SSH1 (ab76943, Abcam).

Techniques: Migration, Cell Culture, Control, Transfection, Plasmid Preparation, Over Expression, Capillary Tube Formation Assay, CCK-8 Assay, Transduction, Flow Cytometry, Wound Healing Assay, Western Blot, Phospho-proteomics

Journal: The FASEB Journal

Article Title: PARP‐1 involves in UVB‐induced inflammatory response in keratinocytes and skin injury via regulation of ROS‐dependent EGFR transactivation and p38 signaling

doi: 10.1096/fj.202002285rr

Figure Lengend Snippet: FIGURE 1 PARP-1 deficiency exacerbated the UVB-induced reaction in mouse back skin phenotypically. WT and Parp-1−/− mice’s back were irradiated with UVB 200 mJ/cm2 for 4 days. A, The gross appearance of mouse back was photographed on each day. Representative pictures from at least three independent experiments were shown. B, Mice were sacrificed on day 4 and the back skin tissues were excised and subjected to H&E staining. C, Mice back skin thickness was measured on day 4. #P < .05 (mean ± S.E.M. n = 6) compared with control group. *P < .05 (mean ± S.E.M. n = 6) compared with WT. D, E, Physiological parameters for barrier permeability function TEWL (D) and erythema (E) were measured before the treatment on each day from day 0 to day 4. *P < .05, **< .01 (mean ± S.E.M. n ≥ 5) compared with WT. F, Mice were sacrificed on day 4 and the back skin tissues were excised and subjected to immunohistochemical staining with COX-2 and phosphorylated p38

Article Snippet: PARP- 1 (#9532), γH2AX (#9718), p- EGFR (#2234), p- p38 (#9211), and p- p38 for IHC (#4511) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Irradiation, Staining, Control, Permeability, Immunohistochemical staining

Journal: The FASEB Journal

Article Title: PARP‐1 involves in UVB‐induced inflammatory response in keratinocytes and skin injury via regulation of ROS‐dependent EGFR transactivation and p38 signaling

doi: 10.1096/fj.202002285rr

Figure Lengend Snippet: FIGURE 6 PARP-1-dependent EGFR/p38 signaling axis contributes to UVB-induced inflammation. NHEK was treated with DMSO, olaparib (10 μM) (A), gefitinib (D), or SB203580 (E) for 30 minutes, or transfected with siCTL or siPARP-1 for 48 hours (B, C). After irradiation with UVB 30 mJ/cm2 (A, B, D, E) or treatment with EGF (100 ng/mL) (C) for the indicated times, phosphorylated EGFR, and p38 as well as PAR, PARP-1, and COX-2 were determined by western blot (A-E), and the gene expression of COX-2, TNF-α, and IL-8 were determined by QPCR (E). *P < .05 indicates UVB-induced gene expression. #P < .05 indicates the inhibition of UVB response by SB203580

Article Snippet: PARP- 1 (#9532), γH2AX (#9718), p- EGFR (#2234), p- p38 (#9211), and p- p38 for IHC (#4511) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Transfection, Irradiation, Western Blot, Gene Expression, Inhibition

Journal: The FASEB Journal

Article Title: PARP‐1 involves in UVB‐induced inflammatory response in keratinocytes and skin injury via regulation of ROS‐dependent EGFR transactivation and p38 signaling

doi: 10.1096/fj.202002285rr

Figure Lengend Snippet: FIGURE 7 PARP-1 positively regulates UVB-induced ROS regulation and subsequent EGFR activation. A, NHEK was silenced with siCTL, siPARP-1or siEGFR for 48 hours, or treated with DMSO or olaparib (10 μM) for 30 minutes, then irradiated with UVB 30 mJ/cm2. H2DCFDA staining and flow cytometry analysis were performed after different times. B, NHEK was treated with NAC (1 mM) for 30 minutes then applied UVB 30 mJ/cm2. EGFR, p38, and PAR were then measured. C, NHEK was treated with NAC (1 mM) for 30 minutes and then, EGF (100 ng/mL) for the indicated times. EGFR, p38, and PAR were measured with western blot. *P < .05 indicates UVB-induced ROS response. #P < .05 indicates the inhibition of UVB response by siPARP-1 and siEGFR. D, UVB-induced DNA damage and ROS production lead to PARP-1 activation and EGFR transactivation, which in turn causes p38 signaling pathway for upregulation of COX-2, MMP-1, TNF-α, and IL-8 expression. However, PARP-1 also can negatively regulate TNF-α and IL-8 expression via a mechanism independent of EGFR upon UVB stimulation

Article Snippet: PARP- 1 (#9532), γH2AX (#9718), p- EGFR (#2234), p- p38 (#9211), and p- p38 for IHC (#4511) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA).

Techniques: Activation Assay, Irradiation, Staining, Flow Cytometry, Western Blot, Inhibition, Expressing

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN alleviates ferroptosis of A/R-induced H9c2 cardiomyocytes via downregulation of VDAC1. (A) Cell Counting Kit-8 detection of viability in A/R-induced cells following TSN or Fer-1 pretreatment. (B) LDH, (C) MDA, (D) total iron, (E) GSH, GSSG, GSH/GSSG and (F) ROS were determined by quantitative kits in A/R-induced cells following TSN or Fer-1 treatment (magnification, ×200; scale bar, 50 μ m). (G) Expression of (H) ferroptosis-related proteins and VDAC1 were detected by western blot analysis in A/R-induced cells following TSN or Fer-1 pretreatment. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, Tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; Fer-1, ferrostatin-1; LDH, lactate dehydrogenase; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; ROS, reactive oxygen species; PTGS2, Prostaglandin endoperoxide synthase 2; GPX, Glutathione peroxidase 4.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Cell Counting, Expressing, Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN binds to VDAC1. (A) Chemical structure of TSN. (B) Molecular structure of VDAC1. (C) 3D and (D) 2D diagram of the interaction between TSN and VDAC1. TSN, Tanshinone IIA; VDAC1, Voltage-dependent anion channel 1.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques:

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN inhibits ferroptosis of A/R-induced H9c2 cardiomyocytes by downregulating VDAC1. (A) Cell Counting Kit-8 detection of viability in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC pretreatment. (B) LDH, (C) MDA, (D) total iron, (E) GSH, GSSG, GSH/GSSG and (F) ROS were determined by quantitative kits in A/R-induced cells following TSN, pAd/VDAC1 and pAd/NC treatment (magnification, x200; scale bar, 50 μ m). (G) Expression of (H) ferroptosis-associated proteins and VDAC1 were detected by western blot analysis in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC pretreatment. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, tanshinone IIA; A/R, anoxia/reoxygenation; VDAC1, voltage-dependent anion channel 1; NC, negative control; LDH, lactate dehydrogenase; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; ROS, reactive oxygen species; PTGS2, Prostaglandin endoperoxide synthase 2; GPX4, Glutathione peroxidase 4.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Cell Counting, Expressing, Western Blot, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN improves mitochondrial function and integrity in H9c2 cardiomyocytes exposed to A/R by downregulating VDAC1. (A) Fluorescent probe BBcellProbe M61 indicating mPTP opening was detected by flow cytometry with the FL1-A: B525-FITC channel. (B) mPTP flow cytometry. (C) Flameng score and (D) transmission electron microscopy of H9c2 cells (magnification, ×8,000; scale bar, 2 μ m). Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; mPTP, Mitochondrial permeability transition pore; NC, negative control.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Flow Cytometry, Transmission Assay, Electron Microscopy, Permeability, Negative Control

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: TSN inhibits apoptosis of A/R-induced H9c2 cardiomyocytes by downregulating VDAC1. (A) Expression of (B) apoptosis-associated proteins was detected by western blot analysis in A/R-induced cells following TSN, pAd/VDAC1 and pAd/NC pretreatment. (C) Caspase-3 activity was measured using a Caspase-3 kit in A/R-induced cells after TSN, pAd/VDAC1 and pAd/NC treatment. (D) MMP and (E) apoptosis were detected by flow cytometry. (F) MMP levels detected by JC-1 in H9c2 cells indicated by the red/green fluorescence ratio. (G) Apoptotic rate measured by Annexin V-FITC/PI flow cytometry. Data are expressed as the mean ± SD (n=3). *** P<0.05. TSN, Tanshinone IIA; A/R, Anoxia/reoxygenation; VDAC1, Voltage-dependent anion channel 1; NC, negative control; MMP, mitochondrial membrane potential.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Expressing, Western Blot, Activity Assay, Flow Cytometry, Fluorescence, Negative Control, Membrane

Journal: International Journal of Molecular Medicine

Article Title: Tanshinone IIA confers protection against myocardial ischemia/reperfusion injury by inhibiting ferroptosis and apoptosis via VDAC1

doi: 10.3892/ijmm.2023.5312

Figure Lengend Snippet: Potential mechanism of TSN in myocardial ischemia/reperfusion injury. TSN pretreatment upregulates the expression of VDAC1, thereby decreasing the accumulation of ROS and iron and abnormal lipid metabolism, maintaining mitochondrial function and protecting the myocardium against anoxia/reoxygenation-induced ferroptosis and apoptosis. TSN, tanshinone IIA; VDAC1, Voltage-dependent anion channel 1; ROS, reactive oxygen species; MDA, malondialdehyde; GSH, Glutathione; GSSG, Glutathione disulfide; LDH, lactate dehydrogenase; MMP, Mitochondrial membrane potential.

Article Snippet: An equal amount of total protein (40 μ g/lane) in each sample was separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to polyvinylidene fluoride membranes and blocked with 5% non-fat dry milk in Tris-buffered saline with 0.1% Tween-20 buffer at room temperature for 2 h. Subsequently, membranes were incubated with primary antibodies against PTGS2 (ProteinTech Group, Inc.; cat. no. #12375-1-AP; 1:1,000), VDAC1 (ProteinTech Group, Inc.; cat. no. #55259-1-AP; 1:1,000), GPX4 (ZENBIO; cat. no. #381958; 1:1,000), Bcl-2 (ZENBIO; cat. no. #250412; 1:1,000), Bax (ZENBIO; cat. no. #380709; 1:1,000) and β-actin (ProteinTech Group, Inc.; cat. no. #20536-1-AP; 1:1,000) at 4°C overnight in a shaker.

Techniques: Expressing, Membrane

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 1. The role of CD34− accessory cells in CD34+ stem cell–derived endothelial progenitor cell (EPC) commitment. A, A schematic diagram of the human EPC colony-forming assay (CFA) used to evaluate the effect of functional CD34− cells on EPC colony-forming units (CFUs). B, Differentiation of 2 types of EPC colony clusters, small EPC-CFUs and large EPC-CFUs, from 3 kinds of cell populations. Small EPC-CFUs were round-shaped, and large EPC-CFUs were spindle-shaped (magnification, ×40). C, Standard quantification of EPC-CFUs was performed by counting the number of small, large, and total EPC-CFUs. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). D, Schematic diagram of the insert culture assay used to assess the effect of functional CD34− cells on CD34+ cell- mediated endothelial cell (EC)-lineage commitment. E, Standard quantification of EPC-CFUs was performed by calculating the number of expanded EC progenitor colonies using CD34+ cells (lower chamber) cocultured with CD34− cells, CD11b+/CD34− cells (macrophage), or CD11b−/CD34− cells (upper chamber). The results were shown as the mean±SEM (*P<0.05 and **P<0.01 vs CD34+ cells). F, Expression of angiogenic cytokines in tumor necrosis factor (TNF)α-treated or untreated CD34− cell–derived macrophages. G, Effect of stromal cell– derived factor (SDF)-1α and vascular endothelial growth factor (VEGF) on human EPC-CFUs. In response to stimulation with SDF-1α and VEGF, the frequency of large EPC-CFUs was significantly increased (*P<0.05 and **P<0.01 vs basal control cytokines: stem cell factor, VEGF, interleukin-3, basic fibroblast growth factor, epidermal growth factor, insulin-like growth factor-1). H, Expression of endothelial lin- eage markers for kinase insert domain receptor, C-X-C chemokine receptor 4, and Tie2 on transwell-cultured cells with or without CD34− cells. HUCB indicates human umbilical cord blood; and MNC, mononuclear cell.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Derivative Assay, Functional Assay, Expressing, Control, Cell Culture

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 2. Characterization and functional capacity of CD34−/CD34+ cell–derived endothelial colony-forming cells (hybrid-dECFCs) and CD34+ cell–derived ECFCs (stem-dECFCs). A, Hybrid-dECFCs and stem-dECFCs are spindle-shaped cells (similar to outgrowth endothelial cells [OECs]; magnification, ×40). B, Human ECFCs were characterized by the endothelial cell (EC) markers CD31, vascular endothelial growth factor (VEGFR)2 (KDR), and von Willebrand factor (vWF), as well as pivotal markers of functional ECFCs, including phosphor-protein kinase B, endothelial nitric oxide synthase (eNOS), and p-eNOS. C, Expression of surface markers for CD34, CD133, kinase insert domain receptor (KDR), C-X-C chemokine receptor 4 (CXCR4), and c-Kit on 2 types of ECFCs measured by flow cytometry. Fluorescence-activated cell sorter (FACS) analysis indicated the ratios of CD34/CD133, KDR, CXCR4, or c-Kit positive cells. D, Hybrid- dEPC (passages 6–20) and stem-dEPC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blot- ting using anti-cyclin-dependent kinase (Cdk)-2, anticyclin E, anti-Cdk4, and anticyclin D1 antibodies. E, Both types of ECFCs (passages 6–16) were treated with VEGF (100 ng/mL), and cell proliferation was examined via the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetra- zolium bromide assay after 24 hours. F, Senescence-associated β-galactosidase (SA-β-gal) activity of the 2 types of ECFCs (passages 6–16). The green cells were counted as positive for senescence (magnification, ×40). G, Hybrid-dECFC (passages 6–20) and stem-dECFC (passages 6–16) lysates containing equal amounts of total protein were analyzed by Western blotting using senescence markers with anti-senescence marker protein (SMP)-30 and antip21 antibodies. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs hybrid- dECFCs). DAPI indicates 4',6-diamidino-2-phenylindole; EBM, endothelial basal medium; and SMP, senescence marker protein.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Functional Assay, Derivative Assay, Expressing, Flow Cytometry, Fluorescence, Western Blot, Activity Assay, Marker

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: CD34 Hybrid Cells Promote Endothelial Colony-Forming Cell Bioactivity and Therapeutic Potential for Ischemic Diseases

doi: 10.1161/atvbaha.112.301052

Figure Lengend Snippet: Figure 5. Proliferation, survival, and secretion of angiogenic growth factors of endothelial colony-forming cells (ECFCs) in hind-limb isch- emia. At day 3 after surgery, samples harvested from hind-limb ischemic tissues were stained to determine the proliferation, survival, and secretion of angiogenic growth factors of the transplanted ECFCs (passage 6). A, Proliferative cells in ischemic injury sites visualized by immunofluorescent staining for proliferating cell nuclear antigen (PCNA; red). B, Standard quantification of proliferating cells represented as the number of PCNA/4',6-diamidino-2-phenylindole (DAPI) double-positive cells per high-power field. Results are shown as the mean±SEM (*P<0.05 and **P<0.01 vs sham, ##P<0.05 vs CD34+ cell–derived ECFCs [stem-dECFCs]). C, Proliferative transplanted cells at the ischemic injury sites indicated by human nuclear antigen (HNA; red), Ki-67 (green), and DAPI (blue) triple-positive cells. White color indicates triple-positive cells in the merged images. D, Apoptotic transplanted cells in ischemic injury shown as HNA (red), caspase-3 (green), and DAPI (blue) triple-positive cells. E, Standard quantification of proliferating transplanted cells represented as the number of HNA/Ki-67/DAPI triple-positive cells (white) per high-power field. F, Standard quantification of apoptotic cells represented as the num- ber of HNA/caspase-3/DAPI triple-positive cells (white) per high-power field. Results are shown as the mean±SEM (**P<0.01 vs CD34−/ CD34+ cell–derived ECFCs [hybrid-dEPC]). G–K, Secretion of angiogenic growth factor from transplanted ECFCs in injury sites visualized by fibroblast growth factor (FGF-2; G), hepatocyte growth factor (HGF; H), stromal cell–derived factor (SDF)-1α (I), vascular endothelial growth factor (VEGF; J), and interleukin (IL)-8 (K) staining (green) and HNA staining (red). L At day 3 after surgery, samples harvested from hind-limb ischemic tissues were analyzed to confirm the secretion of angiogenic growth factor at the injury sites by Western blot- ting. Western blots of ischemic tissue homogenates indicated secretion of FGF-2, HGF, SDF-1α, VEGF, and IL-8. Hvf indicates high visual field.

Article Snippet: SDF-1α and VEGF levels were determined by Quantikine ELISA Human SDF-1α Immunoassay and Quantikine ELISA Human VEGF Immunoassay (R&D Systems, Minneapolis, MN, USA) following the manufacturer’s instructions.

Techniques: Staining, Derivative Assay, Western Blot

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Binding activities of BsAb: ( A ) Surface plasmon resonance sensor grams showing the binding kinetics of anti-EGFR/VEGFR2 BsAb to antigens EGFR and VEGFR2 as detected by a Biacore T200 optical biosensor instrument. Black line represents the constant concentration of anti-EGFR/VEGFR2 BsAb (8 µg/mL) and colored lines represent the nM concentrations of antigens (EGFR or VEGFR2). ( B ) ELISA binding assay showing that anti-EGFR/VEGFR2 BsAb binds to both EGFR and VEGFR2 comparable with cetuximab and ramucirumab, respectively. ( C ) Flow cytometry experiment was performed to assess the binding of anti-EGFR/VEGFR2 BsAb, cetuximab, ramucirumab with cell surface EGFR and VEGFR2 expressed on MDA-MB-231, BT-20, MDA-MB-468 and HUVEC cells. Human IgG was used as isotype control. ( D ) Whole cell lysate of MDA-MB-231 cells was subjected to co-immunoprecipitation assay to assess the binding of anti-EGFR/VEGFR2 BsAb with EGFR and VEGFR2 comparable to parental antibodies. Unprocessed western blot images are available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Binding Assay, SPR Assay, Concentration Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Control, Co-Immunoprecipitation Assay, Western Blot

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Anti-EGFR/VEGFR2 BsAb inhibited ligand-induced activation of EGFR and VEGFR2. ( A ) MDA-MB-231 cells were serum starved overnight and then pre-treated with different antibodies at concentration of 10 µg/mL for 1 h followed by a co-treatment of EGF and VEGF-A (EGF + VEGF) at a concentration of 50 ng/mL each for 30 min. After treatments, WCL was prepared and subjected to Western blot analysis to determine the phosphorylation of EGFR. ( B ) MDA-MB-231 cells were treated as described in A and then phosphorylation levels of Akt was determined by western blot analysis. ( C ) BT-20 cells were exposed to the treatment conditions as described in the A and analyzed by western blotting to assess the phosphorylated and total levels of EGFR, ERK and AKT. ( D ) HUVEC cells were treated with antibodies and EGF + VEGF as described in A. After treatments, WCL was analyzed by western blot to determine the phosphorylated and total levels of VEGFR2, ERK, Akt using respective antibodies. ( E ) MDA-MB-231 cells were serum starved overnight in media containing 1% FBS and then treated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 or left untreated. Western blotting was carried out to evaluate the expression of total VEGFR2. Unprocessed western blot images and available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Activation Assay, Concentration Assay, Western Blot, Phospho-proteomics, Expressing

Journal: Cancers

Article Title: A Novel Bispecific Antibody Targeting EGFR and VEGFR2 Is Effective against Triple Negative Breast Cancer via Multiple Mechanisms of Action

doi: 10.3390/cancers13051027

Figure Lengend Snippet: Anti-EGFR/VEGFR2 BsAb downregulates cancer cell-induced VEGFR2 signaling in HUVEC cells ( A ) The levels of VEGF-A were determined by ELISA assay in supernatants of MDA-MB-231, MDA-MB-468 and BT-20 cells after culturing the cells in a 6-well-plate for 2 days at 37 °C. ( B ) MDA-MB-231 cells were cultured for 2 days in serum free media and then conditioned media was harvested. HUVEC cells were incubated for 30 min and 2 h with the conditioned media obtained from MDA-MB-231. After incubation, Western blot analysis was performed to assess the phosphorylated and total levels of VEGFR2 and ERK. ( C ) HUVEC cells were pretreated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 BsAb or left untreated and then incubated with conditioned media obtained from MDA-MB-231 cells. Post-incubation, western blotting was performed to assess the phosphorylated and total levels of VEGFR2 and ERK. ( D ) HUVEC cells were cultured in the top chamber of trans-well co-culture plates and MDAMB231 cells were cultured in a separate 6-well plate in serum free media overnight. Both HUVEC and MDAMB231 cells were then treated with cetuximab, ramucirumab, anti-EGFR/VEGFR2 BsAb for 1 h or left untreated. After treatments, the trans-well upper chamber inserts containing HUVEC were placed on top of 6-well plate containing MDA-MB-231 cells for 30 min. WCL of HUVEC cells was collected and subjected to Western blot analysis to evaluate the phosphorylated levels of ERK and VEGFR2. ( E ) HUVEC cells and MDA-MB-231 cells were cultured in trans-well co-culture plate as described in D and treated with 1, 5 and 20 µg/mL of anti-EGFR/VEGFR2 BsAb. WCL of HUVEC cells was then subjected to Western blot analysis to assess the levels of ERK and VEGFR2. Unprocessed western blot images are available in .

Article Snippet: Then the supernatants were collected and tested for human VEGF expression with a Human VEGF PicoKin ELISA Kit (Boster Bio, Pleasanton, CA, USA) as per manufacturer’s instructions.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture, Incubation, Western Blot, Co-Culture Assay

Journal: EBioMedicine

Article Title: Blood glutamate EAAT 2 -cell grabbing therapy in cerebral ischemia

doi: 10.1016/j.ebiom.2018.11.024

Figure Lengend Snippet: Examination of MSC characteristics. Phase-contrast image of matrigel angiogenic assay in MSCs − (A) and MSCs + (B), (scale bar, 100 μm). (C) Analysis of ring formation of MSCs − and MSCs + . Data are shown as mean ± SD ( n = 3). (D) VEGF release determined at different time-points in MSCs − and MSCs + . Data are shown as mean ± SD (n = 3). (E) Analysis of mesenchymal phenotype markers (CD90 + , CD73 + , and CD45 − ) in MSCs − (upper panel) and MSCs + (lower panel) by flow cytometry (n = 3). Non-transfected MSCs (MSC − ), transfected MSCs (MSC + ), fluorescein isothiocyanate (FITC), allophycocyanin (APC), peridinin chlorophyll protein c complex (PerCP), phycoerythrin (PE), yellow fluorescent protein (YFP).

Article Snippet: Vascular endothelial growth factor (VEGF) analysis in cell culture supernatants: The levels of VEGF in MSCs − and MSCs + were measured with a Quantikine VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) following the manufacturer's instructions.

Techniques: Flow Cytometry, Transfection

Journal: Signal Transduction and Targeted Therapy

Article Title: Hypoxia induced VEGF secretion promotes resistance to bispecific T-cell engagers

doi: 10.1038/s41392-025-02505-3

Figure Lengend Snippet: Immunophenotyping of patient samples. a M02 top row: Recurrent high grade serous carcinoma (hematoxylin and eosin stain, 400x), and peritoneal cytologic specimen. M02 bottom row: nuclear immunohistochemical expression for PAX-8, and peritoneal tumor cells stained for WT1. M10 top row: Liver core biopsy of recurrent high grade ovarian adenocarcinoma (hematoxylin and eosin stain, 100x), and nuclear immunohistochemical expression for PAX-8. b Western blot analysis performed on lysates derived from OC cell lines and cells from OC patient ascites for M02 (M02-E represents M02 cells passed through a fibroblast exclusion column and M02-R represents the cells retained in the column). OVCAR3 cells included as positive control for CA125, PAX8, Mesothelin and E-Cadherin. A2780 cell line included as positive control for N-Cadherin and Vimentin expression. c Western blot analysis performed on lysates derived from for M10. Jurkat cells included as positive controls for Twist 1/2 and Vimentin. IMR-90 and H226 cells were used as negative controls for Twist 1/2 and snail. Equal amounts of proteins were separated on 4–12% SDS-PAGE gels and probed for Tumor-associated and EMT-related protein expression. d CA125 expression on the M02, M10, and control OVCAR3 cell lines determined by flow cytometry. *** p < 0.001. e Serum CA125 trend over time for patient M02 (top), patient M10 (bottom). f In vitro CA125 secretion of cultured OVCAR3, M02-E cells, M02-R cells and M10 cells as measured by ELISA. Equal numbers of cells were plated, and the supernatant was collected after 72 h in culture

Article Snippet: Human CA125/MUC16 ELISA kit and Human VEGF ELISA Kit (R&D Systems, MN, USA) were used according to the manufacturer’s protocol.

Techniques: H&E Stain, Immunohistochemical staining, Expressing, Staining, Western Blot, Derivative Assay, Positive Control, SDS Page, Control, Flow Cytometry, In Vitro, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Signal Transduction and Targeted Therapy

Article Title: Hypoxia induced VEGF secretion promotes resistance to bispecific T-cell engagers

doi: 10.1038/s41392-025-02505-3

Figure Lengend Snippet: Evaluation of VEGF secretion under HX conditions. a Evaluation of VEGF secretion by ELISA in OVCAR3, OVCAR4, M02 and M04 cells cultured for the indicated timepoints. b Evaluation of MUC16-BITED cytotoxicity in OVCAR4 cells in the presence of OVCAR3 HX conditioned media (top row) and OVCAR3 cells in the presence of OVCAR4 HX conditioned media (bottom row). c Evaluation of MUC16-BITED cytotoxicity in OVCAR3 and OVCAR4 cells in the presence of M02-conditioned media (M02 NXS). Conditioned media from OVCAR3 HX (NXS) and OVCAR4 (NXS) were used as controls. d Evaluation of MUC16-BITED cytotoxicity in OVCAR3 NX and HX cells with or without bevacizumab. Data are expressed as the means ± SEM from two independent measurements for ( a ), ( b ), and ( c ), and two independent measurements for ( d ). Differences between the groups were analyzed using the Two-way ANOVA. ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Human CA125/MUC16 ELISA kit and Human VEGF ELISA Kit (R&D Systems, MN, USA) were used according to the manufacturer’s protocol.

Techniques: Enzyme-linked Immunosorbent Assay, Cell Culture

Journal: Signal Transduction and Targeted Therapy

Article Title: Hypoxia induced VEGF secretion promotes resistance to bispecific T-cell engagers

doi: 10.1038/s41392-025-02505-3

Figure Lengend Snippet: T-cell suppression by VEGF and hypoxia. a T-cell proliferation evaluated by CFSE labeling of T-cells stimulated in the context of NX or HX conditioned media. b BH3 profiling assay evaluating activated T-cells cultured under normoxic conditions (NX) with normoxic supernatant (NXS) or hypoxic supernatant (HXS) and activated T-cells cultured under hypoxic conditions (HX) with normoxic supernatant (NXS) or hypoxic supernatant (HXS) and treated with indicated peptides for 60 min prior to fixation and cytochrome c detection. Data are expressed as the means ± SEM from two independent measurements. Differences between the groups were analyzed using the Two-way ANOVA. ** p < 0.01; *** p < 0.001; **** p < 0.0001

Article Snippet: Human CA125/MUC16 ELISA kit and Human VEGF ELISA Kit (R&D Systems, MN, USA) were used according to the manufacturer’s protocol.

Techniques: Labeling, Cell Culture