Journal: Journal of cell science

Article Title: STAT1 and STAT3 do not participate in FGF-mediated growth arrest in chondrocytes.

doi: 10.1242/jcs.017160

Figure Lengend Snippet: Fig. 1. FGFR3 interacts with STAT1 and STAT3. FLAG-tagged wt, FGFR3-K650E or FGFR3-K508M were expressed in HeLa cells (A) or RCS chondrocytes (B) and the whole cell lysates were subjected to WB for indicated molecules (left panel). Actin serves as a loading control. The same lysates were subjected to STAT1 (middle panel) and STAT3 (right panel) immunoprecipitation (IP) followed by WB. Please note that the transfected FGFR3- FLAG was detected by FLAG antibody in A and by FGFR3 antibody in B. (C) FLAG- tagged wt or FGFR3-K650E, immunoprecipitated from CHO cells, or recombinant tyrosine kinase (TK) domain of FGFR3 were subjected to an in vitro kinase assay with recombinant STAT1 as a substrate. The level of STAT1 phosphorylation was determined by WB with antibody recognizing STAT1 only when phosphorylated at Y701. Samples including the FGFR inhibitor SU5402 (20 M) or those with ATP omitted serve as negative controls for the kinase reaction. The levels of total STAT1 and FGFR3 serve as controls for the substrate and kinase quantity, respectively. Note that the FGFR3 antibody was raised against the extracellular domain of FGFR3 and thus cannot recognize FGFR3-TK. (D) The kinase assay was carried out as described above, using FGFR3-TK as a kinase, and recombinant STAT1 and/or recombinant FRS2 as substrates. The level of STAT1 phosphorylation was determined as described above, whereas FRS2 tyrosine phosphorylation was determined by WB with the 4G10 phosphotyrosine antibody.

Article Snippet: Antibodies against the following proteins were used: actin, FGFR3 and FRS2 (Santa Cruz Biotechnology, Santa Cruz, CA); ERK1/2, P-ERK1/2T202/Y204, STAT1, STAT3, STAT5, P-STAT1Y701, PSTAT3Y705, P-STAT5Y694, P-STAT1S727, P-STAT3S727 (Cell Signaling, Beverly, MA); FLAG (Sigma, St Louis, MO); SRC (Biosource, Camarillo, CA); and 4G10 (Upstate Biotechnology, Lake Placid, NY).

Techniques: Control, Immunoprecipitation, Transfection, Recombinant, In Vitro, Kinase Assay, Phospho-proteomics

Journal: Molecular cancer therapeutics

Article Title: Cigarette Smoke Containing Acrolein Contributes to Cisplatin Resistance in Human Bladder Cancers through the Regulation of HER2 Pathway or FGFR3 Pathway.

doi: 10.1158/1535-7163.MCT-21-0725

Figure Lengend Snippet: Figure 3. Analysis of HER2 and FGFR3 sig- naling pathways in acrolein- induced cisplatin-resistant RT4 and T24 clones. Western blot anal- ysis of HER2 and FGFR3 down- stream signaling pathways was performed in RT4 Acr-clone#3 (A) and T24 Acr-clone#1 (B) com- pared with parental cells. mRNA expression of HER2 and FGFR3 was analyzed in RT4 Acr-clone#3 (upper panel of C) and T24 Acr-clone#1 (lower panel of C) compared with parental cells using qRT-PCR assay. Values were pre- sented as the mean SD. Stu- dent’s t tests were used to deter- mine statistical significance, and two-tailed P values are shown. , P < 0.01 compared with paren- tal cells. Dose and time effects of acrolein on HER2 and FGFR3 expression, p38 activation, and ERK activation in RT4 Acr-clone#3 (D) and T24 Acr-clone#1 (E) cells were compared with that of paren- tal cells using Western blot analy- sis. For the dose and time effects, the cells were treated with differ- ent concentrations of acrolein (0–10 mmol/L) for 24 hours or acrolein (7.5 mmol/L) for 3 to 24 hours, respectively. h, hour.

Article Snippet: Briefly, blots were blocked with 5% nonfat milk and hybridized with primary antibodies overnight at 4 C. The antibodies against phosphoHER2/ErbB2 (Tyr1221/1222; 1:1000, Cell Signaling Technology #2249), HER2 (1:1000, Cell Signaling Technology #9212), phosphoFGFR3 (Tyr724; 1:1000, Abcam #ab155960), FGFR3 (1:1000, Novus Biologicals #JM81–10), phospho-p38 (Thr180/Tyr182; 1:1000, Cell Signaling Technology #9211), p38 (1:1000, Cell Signaling Technology #9212), phospho-p44/42MAPK (Erk1/2; Thr202/Tyr204; 1:1000, Cell Signaling Technology #9101), p44/42 MAPK (Erk1/2; 1:1000, Cell Signaling Technology #9102), PARP-1 (1:1000, Cell Signaling Technology #9542), caspase-9 (1:1000, Cell Signaling Technology #9502), caspase-3 (1:1000, Cell Signaling Technology #9662), E-cadherin (1:1000, Cell Signaling Technology #3195), EpCAM (gifted from Prof. Ying Chih Chang from Genomic Research Center at Academia Sinica, Taipei, Taiwan), vimentin (1:1000, Taiclone # TCEA19744), Snail AACRJournals.org Mol Cancer Ther; 21(6) June 2022 1011 D ow nloaded from http://aacrjournals.org/m ct/article-pdf/21/6/1010/3153401/1010.pdf by guest on 28 O ctober 2024 (1:1000, Cell Signaling Technology #C15D3), and GAPDH (1:5000, Cell Signaling Technology #5174).

Techniques: Clone Assay, Western Blot, Protein-Protein interactions, Expressing, Quantitative RT-PCR, Two Tailed Test, Activation Assay

Journal: Molecular cancer therapeutics

Article Title: Cigarette Smoke Containing Acrolein Contributes to Cisplatin Resistance in Human Bladder Cancers through the Regulation of HER2 Pathway or FGFR3 Pathway.

doi: 10.1158/1535-7163.MCT-21-0725

Figure Lengend Snippet: Figure 4. Effect of trastuzumab or PD173074 combined with cisplatin in acrolein-induced cisplatin-resistant RT4 and T24 clones. RT4 Acr-clone#3 was treated with cisplatin (2.5 mg/mL), or cisplatin (2.5 mg/mL) with trastuzumab (1 mg/mL), or trastuzumab (1 mg/mL), or cisplatin (2.5 mg/mL) with PD173074 (5 mmol/L), or PD173074 (5 mmol/L) for 24 hours. T24 Acr-clone#1 was treated with cisplatin (1 mg/mL, 24 hours), or cisplatin (1 mg/mL) with PD173074 (5 mmol/L), or PD173074 (5 mmol/L), or cisplatin (1 mg/mL) with trastuzumab (1 mg/mL), or trastuzumab (1 mg/mL) for 24 hours. Cytotoxicity of trastuzumab combined with cisplatin in RT4 Acr-clone#3 (A) and in T24 Acr-clone#1 (D) and cytotoxicity of PD173074 combined with cisplatin in T24 Acr-clone#1 (G) and RT4 Acr-clone#3 (J) compared with that of the parental cells were analyzed using MTT assays. Values are presented as the mean SD. Student t tests were used to determine statistical significance, and two-tailed P values are shown. , P < 0.05, compared with vehicle treatment, ##, P < 0.01; ###, P < 0.005 compared with cisplatin treatment group. Western blot analysis of apoptotic proteins, including cleavage of PARP, caspase-9, and caspase-3, in RT4 Acr-clone#3 (B, K) and T24 Acr-clone#1 (E, H) cells compared with that of the parental cells was performed. Western blot analysis of HER2 and FGFR3, p38 activation, and ERK activation in RT4 Acr-clone#3 (C, L) and T24 Acr-clone#1 (F, I) cells compared with that of the parental cells was performed. c-Cas.: cleavage form of caspase; pro-Cas.: proform of caspase.

Article Snippet: Briefly, blots were blocked with 5% nonfat milk and hybridized with primary antibodies overnight at 4 C. The antibodies against phosphoHER2/ErbB2 (Tyr1221/1222; 1:1000, Cell Signaling Technology #2249), HER2 (1:1000, Cell Signaling Technology #9212), phosphoFGFR3 (Tyr724; 1:1000, Abcam #ab155960), FGFR3 (1:1000, Novus Biologicals #JM81–10), phospho-p38 (Thr180/Tyr182; 1:1000, Cell Signaling Technology #9211), p38 (1:1000, Cell Signaling Technology #9212), phospho-p44/42MAPK (Erk1/2; Thr202/Tyr204; 1:1000, Cell Signaling Technology #9101), p44/42 MAPK (Erk1/2; 1:1000, Cell Signaling Technology #9102), PARP-1 (1:1000, Cell Signaling Technology #9542), caspase-9 (1:1000, Cell Signaling Technology #9502), caspase-3 (1:1000, Cell Signaling Technology #9662), E-cadherin (1:1000, Cell Signaling Technology #3195), EpCAM (gifted from Prof. Ying Chih Chang from Genomic Research Center at Academia Sinica, Taipei, Taiwan), vimentin (1:1000, Taiclone # TCEA19744), Snail AACRJournals.org Mol Cancer Ther; 21(6) June 2022 1011 D ow nloaded from http://aacrjournals.org/m ct/article-pdf/21/6/1010/3153401/1010.pdf by guest on 28 O ctober 2024 (1:1000, Cell Signaling Technology #C15D3), and GAPDH (1:5000, Cell Signaling Technology #5174).

Techniques: Clone Assay, Two Tailed Test, Western Blot, Activation Assay

Journal: Molecular cancer therapeutics

Article Title: Cigarette Smoke Containing Acrolein Contributes to Cisplatin Resistance in Human Bladder Cancers through the Regulation of HER2 Pathway or FGFR3 Pathway.

doi: 10.1158/1535-7163.MCT-21-0725

Figure Lengend Snippet: Figure 5. Effect of PD173074 combined with cisplatin in acrolein-induced cisplatin-resistant T24 clones using an xenograft mouse model. The animal experimental protocol is described in the Materials and Methods. A, Overall view of tumors formed by T24 Acr-clone#1 cells treated with the vehicle (PBS, 50 mL, i.p. once weekly; and corn oil, 50 mL, i.p. once weekly), cisplatin (5 mg/kg in PBS, 50 mL, i.p. once weekly), cisplatin (5 mg/kg in PBS, 50 mL, i.p. once weekly) combined with PD173074 (10 mg/kg in corn oil, 50 mL, i.p. once weekly), or PD173074 (10 mg/kg in corn oil, 50 mL, i.p. once weekly). Tumor growth curves (B) and body weight (C) of nude mice in different experimental groups (n ¼ 5). D, Representative H&E (top), TUNEL staining (middle), and IHC staining of FGFR3 (bottom) in tumor sections from the vehicle, cisplatin, and/or PD173074 treatment groups. E, Quantification of the TUNEL-positive area shown in the middle panel of (D). The apoptotic tumor cells in the middle panel are stained brown. F, Quantification of the FGFR3-positive area shown in the lower panel of (D). Student t tests were used to determine statistical significance, and two-tailed P values are shown. , P < 0.01; , P < 0.005 compared with vehicle group; #, P < 0.05; ##, P < 0.01 compared with cisplatin alone; &&, P < 0.01; &&&, P < 0.005 compared with cisplatin þ PD173074.

Article Snippet: Briefly, blots were blocked with 5% nonfat milk and hybridized with primary antibodies overnight at 4 C. The antibodies against phosphoHER2/ErbB2 (Tyr1221/1222; 1:1000, Cell Signaling Technology #2249), HER2 (1:1000, Cell Signaling Technology #9212), phosphoFGFR3 (Tyr724; 1:1000, Abcam #ab155960), FGFR3 (1:1000, Novus Biologicals #JM81–10), phospho-p38 (Thr180/Tyr182; 1:1000, Cell Signaling Technology #9211), p38 (1:1000, Cell Signaling Technology #9212), phospho-p44/42MAPK (Erk1/2; Thr202/Tyr204; 1:1000, Cell Signaling Technology #9101), p44/42 MAPK (Erk1/2; 1:1000, Cell Signaling Technology #9102), PARP-1 (1:1000, Cell Signaling Technology #9542), caspase-9 (1:1000, Cell Signaling Technology #9502), caspase-3 (1:1000, Cell Signaling Technology #9662), E-cadherin (1:1000, Cell Signaling Technology #3195), EpCAM (gifted from Prof. Ying Chih Chang from Genomic Research Center at Academia Sinica, Taipei, Taiwan), vimentin (1:1000, Taiclone # TCEA19744), Snail AACRJournals.org Mol Cancer Ther; 21(6) June 2022 1011 D ow nloaded from http://aacrjournals.org/m ct/article-pdf/21/6/1010/3153401/1010.pdf by guest on 28 O ctober 2024 (1:1000, Cell Signaling Technology #C15D3), and GAPDH (1:5000, Cell Signaling Technology #5174).

Techniques: Clone Assay, TUNEL Assay, Staining, Immunohistochemistry, Two Tailed Test

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: RT-PCR Analysis and Immunohistochemical Analysis of FGFR3IIIc Expression in Esophageal Squamous Cell Carcinoma.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Immunohistochemical staining, Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Enhanced expression of FGFR3IIIc was associated with proliferating esophageal carcinoma (EC) cells. (A) Immunofluorescence staining of FGFR3IIIc (a, d) and SCC-112 (b, e) in non-cancerous mucosa (NCM; N, non-tumor) and esophageal squamous cell carcinoma (ESCC) (T, tumor), which was diagnosed as stage 0. In confocal microscopic images, strong staining of FGFR3IIIc (red) was observed in EC cells (d) but not in normal esophageal epithelium cells (a). FGFR3IIIc-positive cells in ESCC were consistent with SCC-112-positive cells (green) (d, e, f: white arrows). Nuclei were stained with DAPI (blue). (B) Immunofluorescence staining of Ki-67 (red; a, d) and SCC-112 (green; b, e) in NCM (N) and ESCC (T). The strong staining of Ki-67 was observed in EC cells, and Ki67-positive cells were consistent with SCC-112-positive cells in ESCC (d, e, f: white arrows). On the other hand, staining of Ki-67 and SCC-112 were not observed in NCM (a, b, c). (C) Immunofluorescence staining of DAPI (a, d), FGFR3IIIc (b), Ki-67 (e) and merged images (c, f) in ESCC samples. The expression of FGFR3IIIc was detected in the same cells, which also expressed Ki-67 in consecutive sections (c, f; white arrows). Scale, 20 μm.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Expressing, Immunofluorescence, Staining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Expression of FGFR3IIIc was enhanced in esophageal carcinoma cells diagnosed as stage IA (A, B, and C), stage IB (D and E), stage IIIA (F, G and H), but not in normal esophageal epithelium cells. FGFR3IIIc was not stained by anti-FGFR3IIIc antibody after pre-adsorption with the recombinant human FGFR3IIIc Fc chimera (H). Hematoxylin and eosin staining (H&E stain, A, D, and F), FGFR3IIIc immunostaining (B, E, and G), and SCC-112 immunostaining (C). Strong staining for FGFR3IIIc was observed in esophageal carcinoma cells (B, E, and G: white arrows), and FGFR3IIIc expression was clearly restricted in the carcinoma area, with a clear border. Normal esophageal epithelium cells did not express FGFR3IIIc (B, E, and G: black arrows). FGFR3IIIc-positive cells were consistent with SCC-112-positive cells in the nuclei on consecutive sections (C: white arrows). Scale, 20 μm.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Expressing, Staining, Adsorption, Recombinant, Immunostaining

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: PCR Primer Sequences.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Sequencing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Enhanced expression of FGFR3IIIc accelerated cell proliferation. (A) Cell proliferation was significantly lower (by 15%) in EC-GI-10 cells treated with FGFR3 siRNA (siFGFR3) than in EC-GI-10 cells treated with sicontrol (sicontrol) after 5 days in culture. Cell proliferation in FGFR3IIIc-overexpressed siFGFR3-EC-GI-10 cells (FGFR3IIIc) was significantly higher than that in siFGFR3-EC-GI-10 cells (by 1.3-fold), whereas the overexpression of FGFR3IIIb (FGFR3IIIb) did not significantly affect cell proliferation rates. Cell proliferation was significantly lower (~25%) in EGFP-infected siFGFR3-EC-GI-10 cells (EGFP) than in siFGFR3-EC-GI-10 cells. The parental EC-GI-10 cells were not treated with siRNA (-) and not infected (-). Cell numbers are the mean ± S.E.M. The experiment was performed with n=6 samples, and repeated thrice. (#, p<0.05 versus the sicontrol cells and *, p<0.05 versus the siFGFR3 cells; t-test, N.S., not significant). (B) Western blotting shows FGFR3 expression levels of parental cells and cells treated with sicontrol, EGFP, siFGFR3, FGFR3IIIb, or FGFR3IIIc. β-actin was expressed equally among the cells. (C) RT-PCR shows that FGF2 was endogenously expressed in parental EC-GI-10 cells.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Expressing, Over Expression, Infection, Western Blot, Reverse Transcription Polymerase Chain Reaction

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: FGFR3IIIb and FGFR3IIIc Expression in Esophageal Carcinoma (EC) and Non-Cancerous Mucosa (NCM).

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Expressing

Journal: Journal of Histochemistry and Cytochemistry

Article Title: Enhanced Expression of Fibroblast Growth Factor Receptor 3 IIIc Promotes Human Esophageal Carcinoma Cell Proliferation

doi: 10.1369/0022155415616161

Figure Lengend Snippet: Gene expression of FGFR3 isoforms in esophageal carcinoma (EC) and non-cancerous mucosa (NCM). Total RNA was extracted from the specimens of EC patients. The gene expression of FGFR3 in 16 patients (E1 to E16) was analyzed by RT-PCR. FGFR3IIIc expression was clearly detected in the EC (T; tumor) of 12 patients (E2, E3, E4, E5, E6, E7, E8, E10, E13, E14, E15, and E16) and in the NCM (N; non-tumor) of 6 patients (E1, E3, E10, E13, E14, and E15). FGFR3IIIb expression was clearly detected in the EC (T) of 12 patients and in the NCM (N) of 11 patients. GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Article Snippet: The cell lysates were electrophoresed through 7.5% polyacrylamide gels and transferred to PVDF membranes (Immobilon-P; Millipore), and were analyzed by immunoblotting with the monoclonal anti-β-actin antibody (Sigma-Aldrich; 1:2000 dilution), anti-FGFR3 antibody (C-15) (Santa Cruz Biotechnology, Dallas, TX; 1:2000 dilution) and monoclonal anti-FGFR3IIIc antibody (cat#MAB7662, lot.GHK411041, R&D Systems; 1:500 dilution).

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: Calcified Tissue International

Article Title: Fibroblast Growth Factor 23 (FGF23) and Alpha-Klotho Stimulate Osteoblastic MC3T3.E1 Cell Proliferation and Inhibit Mineralization

doi: 10.1007/s00223-011-9501-5

Figure Lengend Snippet: FGFRs were expressed and modulated by FGF23 + Klotho, but not Klotho or FGF23 alone (B-DNA analysis). Cells were cultured in 12-well plates in differentiation medium (alpha-MEM/10% FBS/ascorbic acid [50 μg/mL]/BGP [10 mM]) and supplemented with huFGF23R176Q (F) (1 and 1,000 ng/mL), murine Klotho (KL) (50 ng/mL), their combination, or FGF2 (bFGF) for 14 days. a FGFR “IIIc” forms are preferentially expressed in MC3T3.E1 cells exposed to differentiation medium alone. b FGFR1(IIIc). c FGFR2(IIIc). d FGFR3(IIIc). Gene panel FGFR1(IIIb), FGFR1(IIIc), FGFR2(IIIb), FGFR2(IIIc), FGFR3(IIIc) and FGFR4. Bars represent mean ± SEM. * P < 0.05, ** P < 0.01 compared with differentiation medium ( Diff. Medium )

Article Snippet: The p38 inhibitor SB203580 (PHZ1253, lot 72547179A, stock 50 mg/mL, 132 mM) was from Invitrogen; the phosphoinositide 3-kinase (P13 K) inhibitor LY294002 (9901, lot 9, stock 15.3 mg/mL, 50 mM) was from Cell Signaling (Hayward, CA); FGFR2(IIIc) neutralizing antibody (MAB716, lot FSQ02, stock 5 mg/mL, 33 μM (ND50 = 0.67–2.7 nM) and FGFR3 neutralizing antibody (MAB710, lot FTD02, stock 5 mg/mL, 33 μM (ND50 = 20–80 nM) were from R&D Systems.

Techniques: Cell Culture

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: ( A ) Following biotin-X-DHPE labeling on FGFR3-overexpressing cells (PDC #1), FGFR3 expression levels on the cell surface were analyzed by FACS analysis. Biotin-X-DHPE was detected using Streptavidin-FITC, and FGFR3 was detected using PE-direct conjugated antibody. ( B ) The optimal reaction temperature and time for labeling the living cells with biotin-X-DHPE were analyzed by FACS analysis. ( C ) Optimal reaction buffer analysis for attaching magnetic beads on cells. ( D ) The capture yield of live cells attached with magnetic beads was compared using both direct and indirect method through cell counting and microscopic observation. *** p < 0.001 paired T test. ( E ) Microscopic observation of live cells attached with magnetic beads (magnification 400×).

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Labeling, Expressing, Magnetic Beads, Cell Counting

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: ( A ) Phage pool binding to FGFR3-overexpressing cells in a flow cytometer; comparison of cell surface binding between amplified phage pools obtained using conventional bio-panning (blue histogram) only or with the introduction of cell panning (red histogram). Comparison of CDR-H3 sequence similarity of isolated antibodies through cell panning introduction. ( B ) Phylogenetic tree of similar sequences built on the basis of CDR-H3 (Kabat numbering) using CLUSTAL W multiple sequence alignment programs. Each phylogenetic tree was analyzed for 13 clones selected through conventional bio-panning only and 6 clones selected through introduction of cell panning. The A1D06, S2D05, S3A06, and S3B09 clones were grouped into “Clade A”. ( C ) Comparison of CDR-H3 loop structure alignment using POSA analysis (interactive multiple protein structure alignment). The CDR-H3 of the A1D06 (yellow), S2D05 (green), S3A06 (cyan), and S3B09 (magenta) clones of Clade A were aligned based on the VH region in the Fv modeling annotation. CDR-H3 loop structure and length are different for each clone (gray, framework).

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Flow Cytometry, Comparison, Amplification, Sequencing, Isolation, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Physicochemical property analysis of anti-FGFR3 antibodies derived from cell panning. ( A ) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis of purified antibodies in non-reducing and reducing conditions. In the non-reducing condition, 150 kDa band indicates the whole IgG, and in the reducing condition, 25 kDa and 50 kDa indicate the light chain and heavy chain of the antibody, respectively. ( B ) The purity of the anti-FGFR3 antibodies were analyzed using size exclusion-high performance liquid chromatography (SEC-HPLC). All anti-FGFR3 antibodies showed more than 95% purity of monomer.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Derivative Assay, Polyacrylamide Gel Electrophoresis, SDS Page, Purification, High Performance Liquid Chromatography

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Binding characterization analysis of anti-FGFR3 antibody derived following the introduction of semi-automated bio-panning. ( A ) The binding specificity of selected FGFR3 antibodies to FGFR3 isoforms and interspecies was evaluated using ELISA. ( B ) The binding ability of FGFR3 antibodies to FGFR3-overexpressing cells (PDC#1) and FGFR3 negative cells (PDC#2) was determined using flow cytometry.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Derivative Assay, Enzyme-linked Immunosorbent Assay, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Binding affinities (KD) of selected clones to human FGFR3 isotypes and interspecies FGFR3.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Binding Assay, Clone Assay

Journal: International Journal of Molecular Sciences

Article Title: Semi-Automated Cell Panning for Efficient Isolation of FGFR3-Targeting Antibody

doi: 10.3390/ijms22126240

Figure Lengend Snippet: Biological functional assay of anti-FGFR3 antibodies. Inhibitory effect of clone S3B09 (Clade A) and S3C04 selected from conventional bio-panning on proliferation of FGFR3-overexpressing cells (PDC #1) was evaluated. Cells were cultured in the presence of 10 ng/mL FGF1 plus 10 μg/mL heparin sulfate or with S3B09 or S3C04 antibodies. Relative cell growth was evaluated through ( A ) cell viability after 96 h incubation with antibodies. Data represent mean ± SD; ***, p < 0.001 using one-way ANOVA. ( B ) Cell surface FGFR3 degradation assay. Individual antibodies were treated on FGFR3-overexpressing cells (PDC #1) and incubated for 1 h at 37 °C, and FGFR3 expression on the cell surface was detected through FACS analysis. ( C ) Total FGFR3 degradation assay. After the FGFR3-overexpressing cells (PDC #1) were treated with the antibody, total FGFR3 contained in the cell lysate was detected through sandwich ELISA. Data represent mean ± SD; *, p < 0.03 using one-way ANOVA.

Article Snippet: After washing with 1% FBS (pH 7.4, PBS), the cells were stained with human FGFR3 PE-conjugated antibody (R&D systems, FAB766P, Minneapolis, MN, USA) and the FGFR3 expression level on the cell surface was analyzed by FACS analysis.

Techniques: Functional Assay, Cell Culture, Incubation, Degradation Assay, Expressing, Sandwich ELISA