Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Distribution of EGF and EGFR and phosphorylation status of EGFR in EGF treated cells. ( A ) ICC for EGF and EGFR in untreated cells and cells treated with EGF for 30 min Confocal imaging was done on laser scan confocal Nikon Ti microscope at 100x magnification; staining was done for nuclei (Hoechst 33342), EGF (ab9595 Abcam, Cambridge, United Kingdom) and EGFR (antibody recognizing extracellular domain of human EGFR: epitope 6-273 aa, antibody clone sc-101; Santa Cruz Biotechnology, TX, United States). The scale bars are 50 μm. ( B ) Phosphorylation of EGFR upon treatment with EGF was determined by Western blot. Whole cell lysates were obtained from cells without treatment or 30 min after treatment with recombinant EGF. Three independent experiments were done and imaged using Li-Cor system; representative Western blots are shown. Normalization was done using α-Tubulin as a loading control (Catalog number 926-42213 Li-Cor, NE, United States). Antibodies for phosphorylated EGFR included Y1068 (Catalog number #3777, Cell Signaling, MA, United States), Y1086 (Catalog number #2220S, Cell Signaling, MA, United States) and Y1173 (Catalog number ab32578 Abcam, Cambridge, United Kingdom). ( C ) Western blot quantification was done using software provided by Li-Cor and plotted using Graph Pad Prizm. This bar graph represents relative quantification of phosphorylation of EGFR (for tyrosine positions Y1068, Y1086 and Y1173) for wild type ME180 cells and mutant clone VII11 at 30 min after EGF treatment. To show relative phosphorylation values, the highest phosphorylation signal (that for Tyrosine 1068 in wild type cells) was set as 1. Results are presented as the means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Phospho-proteomics, Imaging, Microscopy, Staining, Western Blot, Recombinant, Control, Software, Quantitative Proteomics, Mutagenesis

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of EGFR downstream signaling in wild type cells and clones upon treatment with EGF. ( A ) Western blots were performed on whole cell lysates isolated from cells serum starved for 1 h and then incubated with recombinant EGF for 30 min in serum free media with 1% BSA. Control cells were incubated in serum free media for 30 min. Experiments were performed independently from at least 3 different passages of cells and representative blots are shown. Antibodies used in this work came from antibody panel ab283852 (Abcam, Cambridge, United Kingdom). ( B ) Quantification of Western blot results was performed using Li-cor quantification software. The results were normalized to protein expression in wild type cells not treated with EGF set at 100%. Data are presented as the mean ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Clone Assay, Western Blot, Isolation, Incubation, Recombinant, Control, Software, Expressing

Journal: Scientific Reports

Article Title: Changes in EGFR activity following CRISPR/Cas9-editing of the EGF binding domain

doi: 10.1038/s41598-026-37579-8

Figure Lengend Snippet: Evaluation of cell cycle distribution and expression of cell cycle regulator proteins. ( A ) Representative cell cycle profiles of wild type ME180 cells and mutant clones based on DAPI staining and incorporation of EdU. The usual horseshoe pattern cell distribution allowed separation of cell populations into G1, S and G2/M stage of the cell cycle. ( B ) Histogram showing cell cycle distribution in wild type cells and mutant clones based on flow cytometry data done on 5 independent experiments using cells from 5 different cell passages. The data are shown as means ± SD from five independent experiments including cells from three different cell cycle passages. ( C ) Western blots performed on whole cell lysates from wild type cells and mutant clones growing in cell culture or additionally treated with recombinant EGF for 30 min. Experiments were performed independently using at least three different cell passages and representative blots are shown. ( D ) Quantification of Western blot results was performed using Li-Cor quantification software and the results are presented values relative to protein expression in untreated wild type cells set as 100%. Data are presented as means ± SD of at least three independent experiments.

Article Snippet: For the EGF and TGF-α binding test, cells were grown on glass cover slips and incubated with 1% BSA in McCoy’s serum free medium for 1 h, and then placed on ice for 10 min. After removal of the medium, either EGF AlexaFluor ® 555 (Invitrogen, MA, United States200 fold dilution of 1 μg/μl stock solution) or recombinant EGF protein (Catalog number 236-EG, R&D systems, MN, United States, stock 0.5 mg/ml, diluted 1:200), were diluted in McCoy’s Medium with 1% BSA and incubated with cells.

Techniques: Expressing, Mutagenesis, Clone Assay, Staining, Flow Cytometry, Western Blot, Cell Culture, Recombinant, Software

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Connexin43 promotes angiogenesis through activating the HIF-1α/VEGF signaling pathway under chronic cerebral hypoperfusion

doi: 10.1177/0271678X211010354

Figure Lengend Snippet: Cx43 KO mice displayed defective reparative angiogenesis after BCAS. (a) Immunohistochemical analysis of CD31 in the cortex and striatum of the brain slices removed from the four mouse groups 30 days after surgery. Brown strips indicated CD31-positive capillaries. Scale bar =100μm. (b) Quantification of density and length of CD31-positive capillaries in the cortex and striatum. (c) Ki-67 and CD31 immunohistochemical double-staining. Ki-67 was stained in green and CD31 in red. Scale bar =100μm. (d) Immunoblot analysis of VEGF expression of brain tissues with GAPDH used as loading control. (e) Quantitative analysis of VEGF relative to GAPDH. (f) Cortical microvascular perfusion was evaluated by in vivo multiphoton microscopy analysis of 2,000,000 Da FITC-conjugated dextran. Scale bar =100μm. (g) Quantitative analysis of perfused capillary length from angiograms in Figure 2(f) . ****P < 0.0001 ***P < 0.001 **P < 0.01 *P < 0. 05 versus sham-operated mice, #### P < 0.0001 ### P < 0.001 ## P < 0.01 # P < 0.05 versus BCAS-operated Cx43 +/+ mice, n ≥ 6 mice in each group.

Article Snippet: The concentration of VEGF in the culture medium of each group was assessed using an VEGF ELISA kit (RayBiotech, Atlanta, United States, ELM-VEGF).

Techniques: Immunohistochemical staining, Double Staining, Staining, Western Blot, Expressing, Control, In Vivo, Microscopy

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Connexin43 promotes angiogenesis through activating the HIF-1α/VEGF signaling pathway under chronic cerebral hypoperfusion

doi: 10.1177/0271678X211010354

Figure Lengend Snippet: Cx43 regulated VEGF-induced angiogenesis through the HIF-1α-PKA signaling pathway. The brain tissues removed from the four mouse groups 30 days after surgery were used for western blots and RT-PCR. (a) Representative images of western blots for HIF-1α, AKT, and p-AKT using GAPDH as an internal control. (b) Differences in the band intensities of HIF-1α, AKT, and p-AKT were compared among the four mouse groups. (c) Representative images of separate western blot analyses for the HIF-1α/P-AKT pathway in white and gray matter using β-actin as an internal control. (d) Quantitative analysis of the band intensities relative to β-actin. (e) HIF-1α mRNA expression in the four mouse groups was evaluated using RT-PCR. (f) RT-PCR measures of VEGF mRNA in each group of mice. ****P < 0.0001 ***P < 0.001 **P < 0.01 versus sham-operated mice, #### P < 0.0001 ### P < 0.001 ## P < 0.01 # P < 0.05 versus BCAS-operated Cx43 +/+ mice, n ≥ 6 mice for each group.

Article Snippet: The concentration of VEGF in the culture medium of each group was assessed using an VEGF ELISA kit (RayBiotech, Atlanta, United States, ELM-VEGF).

Techniques: Western Blot, Reverse Transcription Polymerase Chain Reaction, Control, Expressing

Journal: Journal of Cerebral Blood Flow & Metabolism

Article Title: Connexin43 promotes angiogenesis through activating the HIF-1α/VEGF signaling pathway under chronic cerebral hypoperfusion

doi: 10.1177/0271678X211010354

Figure Lengend Snippet: Cx43 mediated VEGF-induced angiogenesis by activating the HIF-1α-PKA signaling pathway in vitro . (a) VEGF contents in the culture medium of each group of cells were analyzed using a VEGF ELISA kit. (b) HIF-1α mRNA expression in four cell groups were studied by RT-PCR. (c) RT-PCR results indicating VEGF mRNA levels in each cell group. (d) Western blot images of HIF-1α, AKT, and p-AKT from the cultured bEnd.3 samples in four difference groups. β-actin was an internal control. (e) Quantitative analysis of protein levels relative to β-actin. (f) Western blot analysis of HIF-1α expression in lentivirus-transfected cells. (g) Quantitative analysis of HIF-1α protein level relative to β-actin. (h) Representative images of western blots for VEGF, AKT, and p-AKT expression in the three group bEnd.3 cell groups treated with 375 μmol/L CoCl 2 for 24 h. (i) Quantitative analysis of the band intensities relative to β-actin. (j) VEGF concentrations in the culture medium of the three group bEnd.3 cell groups were analyzed using a VEGF ELISA kit. ****P < 0.0001 ***P < 0.001 **P < 0.01 *P < 0.05 versus control cells, #### P < 0.0001 ### P < 0.001 ## P < 0.01 # P < 0.05 versus CoCl 2 -treated control cells. All these studies were conducted independently at least three times.

Article Snippet: The concentration of VEGF in the culture medium of each group was assessed using an VEGF ELISA kit (RayBiotech, Atlanta, United States, ELM-VEGF).

Techniques: In Vitro, Enzyme-linked Immunosorbent Assay, Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Cell Culture, Control, Transfection

Journal: Materials Today Bio

Article Title: Injectable hyaluronate-L- cysteine gel potentiates photothermal therapy in osteosarcoma via vorinostat-copper cell death

doi: 10.1016/j.mtbio.2024.101368

Figure Lengend Snippet: An Illustrative Depiction of the Fabrication of CHASA and Its Promising Anti-Tumor Mechanism, Featuring Cuproptosis Potentiated by Copper-Death Enhancer SAHA and Photothermal Effect Triggered by 808 nm near-infrared laser. Abbreviations: poly-Cys (hyaluronate-L-cysteine hydrogel precursors, poly-HA-Cys), SAHA (Vorinostat), CHASA (SAHAm@{[Cu(HA-Cys) 2 ]Cl 2 }n hydrogel), SLC31A1(Solute Carrier Family 31 Member 1), ATP7A/B(Copper-transporting P-type ATPase)，TCA cycle (Tricarboxylic Acid Cycle), DLAT (Dihydrolipoamide S-Acetyltransferase)，LIAS(Lipoic Acid Synthase)，LA (Lipoic Acid).

Article Snippet: Vorinostat (SAHA, Cat no.8638-EG-050) was purchased from R&D Systems Inc., Minneapolis, MN, USA.

Techniques:

Journal: bioRxiv

Article Title: Targeting Neuropilin-1 to Enhance Immunotherapy in Melanoma: Reducing Tumour Progression and Peripheral Treg-Mediated Immunosuppression

doi: 10.1101/2024.09.27.615359

Figure Lengend Snippet: (A) Oncoprint depicting the frequency and types of alterations of the NRP1 gene in 212 melanoma cancer and 2683 pan-cancer patient samples from the cBioPortal database. (B) Bar graph representation of alteration frequency of NRP1 gene in 2683 cancer patient samples from cBioPortal database. (C) Fold change in mRNA expression of NRP1 gene when compared between tumorigenic B16-F10 and non-tumorigenic NIH-3T3 cells. (D) Bar graphs representing apoptosis in tumorigenic B16-F10 and non-tumorigenic NIH-3T3 cells upon treatment with different concentrations of NRP1 inhibitor EG00229, which is determined by Annexin & 7-AAD dual staining. Representative bar diagrams are of three independent experiments. ns, non-significant; * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001.

Article Snippet: EG00229 trifluoroacetate (an inhibitor for NRP1, cat no.- 6986) was from Tocris (United Kingdom).

Techniques: Expressing, Staining

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: M2 macrophage-derived factors regulated by CYP4A drive lung pre-metastatic niche formation. ( a ) TGF-β, IGF-1, VEGF, SDF-1, PDGF-B, MMP-2 and MMP-9 in M2 macrophages (M2) and CYP4A10 high M2 were determined by qPCR. ( b ) VEGF and SDF-1 from the culture supernatants were determined by ELISA. ( c ) The conditioned medium (CM) from M2 or CYP4A10 high M2 incubated with or without neutralizing anti-VEGF (20 μg/ml) or anti-SDF-1 (10 μg/ml) antibodies for 48 h was added to the lower chamber. VEGFR1 + myeloid cells were added to the upper chamber, and then incubated over night. The cells migrated to the lower chamber were counted. ( d ) L929 fibroblast (L929) and lung-derived fibroblast (LF) were stimulated with the CM from CYP4A10 high M2 with or without neutralizing anti-TGF-β (10 μg/ml), anti-VEGF (20 μg/ml), anti-SDF-1 (10 μg/ml), anti-PDGF-B (10 μg/ml) or anti-MMP-9 (20 μg/ml) antibodies for 48 h. α-SMA was determined by qPCR. ( e ) L929 and LF were pre-treated with or without TGF-β receptor inhibitor (A83-01, 10 μ M ) for 1 h, and then incubated with the CM from CYP4A10 high M2 for another 48 h. α-SMA and fibronectin were measured by western blot. ( f ) L929 and LF were stimulated with the CM from M2 or CYP4A10 high M2 with or without a combination of neutralizing anti-TGF-β (10 μg/ml), anti-VEGF (20 μg/ml) and anti-SDF-1 (10 μg/ml) antibodies for 48 h. α-SMA was determined by qPCR. ( g ) M2 CM, CYP4A10 high M2 CM, M2 CM plus VEGF (800 pg/ml) or SDF-1 (60 pg/ml) or TGF-β (200 pg/ml), and M2 CM plus combination cytokines (VEGF, SDF-1 and TGF-β) were intraperitoneally injected into BALB/c mice daily for 2 weeks. VEGFR1 + myeloid cells in the lungs were analyzed by flow cytometry ( n =5). The value is presented as the mean±s.e.m. # P <0.05, ## P <0.01 vs M2, ^ P <0.05, ^^ P <0.01 vs CYP4A10.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Derivative Assay, Enzyme-linked Immunosorbent Assay, Incubation, Western Blot, Injection, Flow Cytometry

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: CYP4A/20-HETE–STAT3 signaling is crucial for M2 polarization of macrophages and migration of VEGFR1 + myeloid cells in vitro . ( a , b ) Mouse peritoneal macrophages (PNMS) or human monocyte-derived macrophages (MDMs) were stimulated with IL-4/IL-13 (20 ng/ml) for 12 h or the tumor-conditioned medium (CM) for 72 h to generate M2 macrophages, and then treated with HET0016 (5 μ M ), DDMS (10 μ M ) or vehicle for 24 h. STAT3 and p-STAT3 was determined by western blot. ( c ) Immunostaining analysis of CD206 (red) and p-STAT3 (green) in PNMS from the above treated groups was shown. Scale bars, 20 μm. ( d ) The effect of siRNA against STAT3 on the protein levels of STAT3 was assayed by western blot. ( e ) The effect of Stattic on p-STAT3 was assayed by western blot. ( f ) MDMs and PNMS were pre-treated with or without STAT3 inhibitor (Stattic, 20 μ M ) for 1 h, and then incubated with IL-4/IL-13 for 12 h or the tumor CM for 72 h, followed by treatment with or without 20-HETE (1 μ M ) for another 24 h. In another experiment, MDMs and PNMS were pre-treated with or without STAT3 siRNA (STAT3-siRNA) and scramble siRNA for 24 h, and then incubated with IL-4/IL-13 for 12 h or the tumor CM for 72 h, followed by treatment with 20-HETE (1 μ M ) for another 24 h. Th2 cytokines (TGF-β and IL-10) were determined by ELISA. ( g ) VEGF and SDF-1 from the culture supernatants in the above groups were determined by ELISA. ( h ) The CM in the above treated groups was added to the lower chamber. VEGFR1 + myeloid cells were added to the upper chamber, and then incubated over night. The cells migrated to the lower chamber were counted. Each value presents the mean±s.e.m. of three independent triplicate experiments. * P <0.05, ** P <0.01 vs control, # P <0.05, ## P <0.01 vs CYP4A, ^ P <0.05, ^^ P <0.01 vs 20-HETE.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Migration, In Vitro, Derivative Assay, Western Blot, Immunostaining, Incubation, Enzyme-linked Immunosorbent Assay, Control

Journal: Oncogene

Article Title: CYP4A in tumor-associated macrophages promotes pre-metastatic niche formation and metastasis

doi: 10.1038/onc.2017.118

Figure Lengend Snippet: A proposed mechanism to explain the role of CYP4A/20-HETE in TAMs in pre-metastatic niche formation and metastasis. 20-HETE, 20-hydroxyeicosatetraenoic acid; CYP4A, cytochrome P450 4A; MMP-9. matrix metalloproteinase-9; SDF-1, stromal cell-derived factor-1; STAT3, signal transducer and activator of transcription 3; TAMs, tumor-associated macrophages; TGF-β, transforming growth factor-β VEGF, vascular endothelial growth factor.

Article Snippet: Mouse recombinant VEGF, SDF-1 and TGF-β were purchased from R&D Systems (Oxford, UK).

Techniques: Derivative Assay