Stock cultures of RPTEC/TERT1 cells were obtained from American Type Culture Collection and were grown using serum‐free conditions as previously described by this laboratory.15 , 16 The composition of the growth formulation was also as described previously.12 , 13 , 15 Confluent cultures of the immortalized RPTEC/TERT1 cell line were sorted into two different cell populations, namely HRTPT (CD133+/CD24+) cells and HREC24T (CD133‐CD24+) cells, using BD FACSAria (BD Biosciences) as detailed previously.12 Both cell lines were subcultured at 1:3 ratio, allowed to reach confluence and then used in the described experimental protocols. For treatment with FGFR inhibitor, SU5402 (Selleck Chemicals), HRTPT cells were grown to confluency and subcultured into growth media containing 5uM SU5402 inhibitor or DMSO for control, grown to confluency and harvested to obtain pellets for RNA and protein analyses.
Su5402
Manufactured by Selleck Chemicals
SU5402 is a laboratory reagent that inhibits the activity of the fibroblast growth factor receptor (FGFR) protein. It is commonly used in cell-based assays and in vitro experiments to study the role of FGFR signaling in various biological processes.
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Lab products found in correlation
Erdafitinib, by Selleck Chemicals (1 mentions)
Cycloheximide (chx), by Cell Signaling Technology (1 mentions)
Ly2874455, by Selleck Chemicals (1 mentions)
Azd4547, by Selleck Chemicals (1 mentions)
Facsaria, by BD (1 mentions)
Doxycycline, by Merck Group (1 mentions)
Sirtinol, by Merck Group (1 mentions)
Fk866, by Cayman Chemical (1 mentions)
Su5402, by Merck Group (1 mentions)
5 bromo 2 deoxyuridine brdu, by Merck Group (1 mentions)
5 protocols using su5402
1
RPTEC/TERT1 cell culture and sorting
2
SU5402 Treatment for Zebrafish Surgery
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SU5402 (Selleckchem, Houston, TX) was dissolved in DMSO as a 17 mM stock and added to fish water at a final concentration of 17 μM as described [27 (link)], tanks were kept in the dark. Up to 5 fish were treated in 250 mL of water, tanks were maintained at 28.5°C, and drug solutions were replaced every 24 h. Drug treatments were performed 24 h before surgery and no significant mortality was noted.
3
Inhibitor-based Xenopus Embryo Assay
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FGFR inhibitors SU5402 (catalog no. S7667), AZD4547 (catalog no. S2801), Erdafitinib (catalog no. S8401), and Ly2874455 (catalog no. S7057) were purchased from Selleckchem. CHX was purchased from Cell Signaling Technology (catalog no. 2112S). These chemical inhibitors were used to treat cells and Xenopus embryos at concentrations indicated in the figure legends.
4
Tail Regeneration in Zebrafish Larvae
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Wild-type and sirt1 mutant larvae at 3 dpf were anesthetized in 0.2 mg/ml tricaine for 20 min before amputation with a resection line past the rostral tip of the notochord to remove tail fin fold tissue. Amputated larvae were recovered in E3 for 40 min to 1 hour before incubation in Sirtinol (20μM, Sigma), FK-866 (100μM, Cayman) or SU5402 (5μM, Selleckchem) for 4 hours for the in situ hybridization and the qRT-PCR experiments. For the regeneration growth assay, the treated larvae were then transferred to NAM (10 mM, Sigma) and incubated for 3 days before evaluated for regeneration. 3 dpf amputated sirt1 mutant larvae were recovered in E3 for 2 hours before incubation in 10 mM NAM or 60 μg/mL doxycycline (Sigma) for 3 days. Regeneration length was measured as the distance from the tip of the notochord to the point where the body A-P axis intersects with the edge of the newly formed larval fin fold. All data were derived from 3 biologically independent experiments. For the sirt1 mutant fin development analyses, 1 dpf mutant embryos were incubated in 10 mM NAM for 48 hours until larval fin fold is formed.
5
Fin Regeneration and Proliferation Assay
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Fgf inhibition was performed with 17 μM SU5402 (Selleckchem) or the vehicle control dimethylsulfoxide (DMSO, final percentage 0.1 %) in fish water at the times indicated. Solutions were changed daily. 50 mg/ml 5-Bromo-2-deoxyuridine (BrdU) (Sigma-Aldrich #B5002) stock solutions were prepared in DMSO and used at 5 mM (Figures 5 A–5F and 7 A–7F) or 2.5 mM (Figures 6 C–6H) in selected experiments, either in fish water, in fish water supplemented with NFP (Sigma-Aldrich) or DMSO (3 % DMSO maximum), or in fish water supplemented with SU5402 or DMSO. BrdU treatment was performed during the last 6 hours of day 5 post amputation by incubation. NTR mediated ablation was performed with 1 μM NFP in fish water. Fin regenerates were either fixed and/or photographed at 5 dpa or zebrafish were allowed to recover from treatment for 2 days before fin regenerates were fixed and/or photographed.
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