To determine cell viability, cells were seeded in 96 well plates at a density of 4 × 103 cells per well. After a recovery time of 24 h, the cells were exposed to diverse compounds at different drug concentrations in triplicates. The small molecule inhibitors ponatinib, nintedanib, AZD-4547, dovitinib, erdafitinib, avapritinib, and dasatinib were purchased from Selleck Chemicals (Houston, TX, USA). Upon 72 h incubation, cell survival was determined with the commercially available CellTiter-Glo® Luminescent Cell Viability Assay (Promega, Madison, WI, USA) according to manufacturer’s instructions and luminescence signals were measured with the Tecan infinite 200Pro (Zurich, Switzerland). Dose–response curves were generated and anti-cancer activity was expressed as IC50 values calculated by GraphPad Prism 8.0.1 (GraphPad Software, La Jolla, CA, USA) using point-to-point function.
Nintedanib
Manufactured by Selleck Chemicals
Sourced in United States, Germany
Nintedanib is a chemical compound used in laboratory research. It is a tyrosine kinase inhibitor that targets multiple pathways involved in cellular processes. The core function of Nintedanib is to inhibit the activity of various tyrosine kinases, which play crucial roles in cellular signaling and regulation.
Automatically generated - may contain errors
Lab products found in correlation
Azd4547, by Selleck Chemicals (5 mentions)
Pirfenidone, by Selleck Chemicals (5 mentions)
Trametinib, by Selleck Chemicals (5 mentions)
Ponatinib, by Selleck Chemicals (4 mentions)
Dasatinib, by Selleck Chemicals (3 mentions)
Imatinib, by Selleck Chemicals (3 mentions)
Axitinib, by Selleck Chemicals (3 mentions)
Lapatinib, by Selleck Chemicals (3 mentions)
Sch772984, by Selleck Chemicals (2 mentions)
Cabozantinib, by Selleck Chemicals (2 mentions)
Gefitinib, by Selleck Chemicals (2 mentions)
Pf 3758309, by Selleck Chemicals (2 mentions)
Murine anti pd l1 mab anti pd l1 migg1e3 invivofit, by InvivoGen (2 mentions)
4 oht, by Merck Group (2 mentions)
Tamoxifen, by Merck Group (2 mentions)
Rpmi 1640, by Thermo Fisher Scientific (2 mentions)
Pirfenidone, by Merck Group (2 mentions)
Nintedanib, by Merck Group (2 mentions)
6 thioguanine, by Merck Group (2 mentions)
Dimethyl sulfoxide (dmso), by Thermo Fisher Scientific (2 mentions)
34 protocols using nintedanib
1
Cell Viability Quantification by Luminescence
2
Astrocyte-Neuron Co-Culture Conditioned Media Modulation of Microglial Phenotype
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To prepare conditioned medium (human) astrocyte and (mouse) neuron co-cultures were generated as previously described, except that at DiV 4, cells were washed once and media replaced with Neurobasal-A medium in the absence of serum. At DiV 7, media was harvested and co-cultures discarded. Flasks of (rat) microglia, prepared as described above, were detached, centrifuged and pellets were resuspended in either naive Neurobasal-A medium, or conditioned medium, with cells diluted to 2 X 105 cells/ml, and plated in 24 well plates. The following drug treatments were added at plate-down: Nintedanib (500 nM, Selleckchem), AZD8797 (10 μM, Selleckchem), Vactosertib (100 μM, Selleckchem), LY2109761 (3 μM, Selleckchem) mouse TGF-β2 (20 ng/ml, R&D Systems). Microglia were incubated for 72 h, after which they were processed for either immunohistochemistry or RNA isolation and qPCR.
3
Isolation and Analysis of Human Lung Cells
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Human lung single-cell isolation and flow cytometry analysis were performed as described previously (10 (link), 13 (link)). In brief, human lung tissues were minced and then digested with 2 mg/mL Dispase II, followed by digestion with 10 U/mL elastase and 100 U/mL DNase I. Finally, cells were filtered through a 100 μm cell strainer and lysed with red blood cell lysis to get single-cell suspensions. Antibody staining was similar in human and mouse cells. Flow cytometry was performed with a Fortessa and FACSAria III flow cytometer and analyzed with FlowJo 10.6.1 software (BD Biosciences). Anti–human CD31 (clone WM59, catalog 303118, RRID AB_2247932), CD45 (clone WI30, catalog 304016, RRID AB_314404), and EpCAM (clone 9C4, catalog 324212, RRID AB_756086) were from BioLegend. SLC39A8 (ZIP8) polyclonal antibody (catalog PA5-26368, RRID AB_2543868) and goat anti–mouse IgG/IgM (catalog A-10680, RRID AB_2534062) were from Thermo Fisher Scientific. HTII-280 (52 (link)) was a gift from the laboratory of L. Dobbs (UCSF). Human SIRT1 monoclonal mouse IgG1 (clone 834918, catalog IC7714S) from R&D Systems was used for intracellular staining. Pirfenidone was from MilliporeSigma (catalog P2116), and nintedanib was from Selleck Chemicals (catalog S1010).
4
3D Lung Fibroblast Culture and Alveospheres
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Thick matrices were produced from primary Human control and IPF lung fibroblast using an adapted protocol from Castelló-Cros and Cukierman, 2009 (link). Control primary lung fibroblasts were next seeded on these matrices and cultured for 48 hr as described in Castelló-Cros and Cukierman, 2009 (link). Control primary lung fibroblasts were next seeded on this 3D matrices and treated with Imatinib (10 µg/ml), Nintedanib (10 nM) or PDGFR V inhibitor (10 nM) (all from Selleckchem, Boston USA) in DMEM medium supplemented with 2% of FCS. Forty-eight hr after treatment, cells were harvested for mRNA analysis (as described below).
Lung alveosphere culture in matrigel was performed following an adapted protocol from Konishi et al., 2022 (link) using wild-type and Prrx1-/- MEFs as stromal cells with wild-type primary mouse lung derived epithelial cells. Spheres were counted at day 15.
Lung alveosphere culture in matrigel was performed following an adapted protocol from Konishi et al., 2022 (link) using wild-type and Prrx1-/- MEFs as stromal cells with wild-type primary mouse lung derived epithelial cells. Spheres were counted at day 15.
5
Drug Resistance Induction in Prostate Cancer Cell Lines
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The Pca cell lines 22RV1 (CRL-2505), LNCaP (CRL-1740), DU145 (HTB-81) and PC3 (CRL-1435) and 293 cells were purchased from the American Type Culture Collection (Manassas, VA, USA) and were maintained in RPMI-1640 medium (Invitrogen; Thermo Fisher Scientific, Inc., Waltham, MA, USA) or Dulbecco's modified Eagle's medium (Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) that were supplemented with 10% fetal bovine serum (Invitrogen; Thermo Fisher Scientific, Inc.) at 37°C in a humidified atmosphere with 5% CO2. All drugs were purchased from Selleck Chemicals (Houston, TX, USA), and Pca cells were treated at the following concentrations: Nintedanib (3 µM for 4 weeks to develop resistance), PI3K inhibitor buparlisib (1.5 µM for 4 weeks) (17 (link)), protein kinase B (Akt) inhibitor MK2206 (10 µM for 4 weeks) (18 (link)), extracellular-signal-regulated kinase (ERK)1/2 inhibitor SCH772984 (3 µM for 4 weeks) (19 (link)), CDC42 inhibitor ML141 (2 µM for 4 weeks) (20 (link)), or ROCK1/2 inhibitor Y-27632 (10 µM for 1 week) (21 (link)). For drug treatment experiments, all the cells were seeded at a density of 1×105 cells/well in 6-well culture plates in the corresponding medium. Following 24 h of incubation at 37°C for attachment, the cells were treated with drugs.
6
Optimized Compound Screening Protocol
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Drugs were purchased from the following: NVP-BHG712 (S2202), AZD (S7106), CCT129202 (S1519), Nintedanib (S1010), R406 (S2194), Axitinib (S1005), Bosutinib (S1014), selumetinib (S1008), Navitoclax (ABT263, S1001), PF561772 (S2890, FAK inhibitor), and SB202190 (S1077, P38 inhibitor) from Selleckchem (Houston, TX, USA). Staurosporine (S9300), etoposide (E1383), and H2O2 (216763) from Sigma-Aldrich (St. Louis, MO, USA). They were prepared 10 mM stock solution in DMSO. They were added in culture medium to obtain a suitable working solution. As a vehicle, DMSO as the same volume was added in culture medium. The stock solution was stored at -20 °C.
7
Isolating and Treating pmATII Cells
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The pmATII cells were isolated from mice as previously described [8 (link)]. The pmATII cells were seeded in 12 well-tissue culture plates and cultured in DMEM-F12 supplemented with 10% FCS, 2 mM 1-glutamine, 1% penicillin/streptomycin, 3.6 mg/ml glucose and 10 mM HEPES for 24 h to allow attachment. Cells were then cultured for 12 h in fresh 0.1% FCS containing medium. Subsequently, cells were pre-treated with Nintedanib (1 μM) (Selleck, Houston, TX) or Pirfenidone (500 μM) (Selleck, Houston, TX) or respective DMSO control for 48 h.
8
High-throughput Screening of Kinase Inhibitors
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High-throughput inhibitor screening was performed using the SCADS Inhibitor kit-1, 3 or 4, provided by the Screening Committee of Anticancer Drugs supported by a Grant-in-Aid for Scientific Research on Innovative Areas, Scientific Support Programs for Cancer Research, from the Ministry of Education, Culture, Sports, Science and Technology of Japan. ASP3026 (ChemieTek), TAE684, AEW541 (ActiveBiochem), AP26113-analog (Biochempartner), cabozantinib, foretinib, ponatinib (Selleck), afatinib (BIBW2992, ChemieTek), E7080 (Selleck), motesanib (ChemieTek), CEP701 (Calbiochem) and nintedanib (Selleck) were appended to the library (for a total of 282 compounds). Wild-type CD74-ROS1, F2004V- and F2075C-mutated cells were seeded in triplicate in 96-well plates, and each inhibitor was added at 10, 100 nM, 1, or 3 μM. A DMSO-treated well used in each plate as a control. After 72 h of exposure to the inhibitors, cell viability was measured by the CellTiter-Glo assay.
9
Nintedanib Inhibits NSCLC Cell Growth
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Nintedanib and gefitinib were purchased from Selleck Chemicals (Houston, TX, USA). Growth inhibition was assessed by the MTS assay to examine the effect of Nintedanib on the 16 NSCLC cell lines. Cell suspensions (5,000 cells/well) were seeded into 96-well plates and increasing concentrations of Nintedanib (0, 0.01. 0.1, 1.0 and 10 μM) were added. After incubation at 37ºC for 72 h, MTS was added to each well and incubated at 37ºC for 2 h, after which absorbance was measured using a microplate reader with a test wavelength of 450 nm. The IC50 value was defined as the concentration needed for 50% reduction of the growth by treatment with Nintedanib by SigmaPlot12 (Hulinks, Inc., Tokyo, Japan). A549 and PC-1R cells (5,000 cells/well) were seeded into 96-well plates for 24 h, and then incubated in the various concentrations of Nintedanib at 37ºC for 72 h after exposure to miR-200 mimic, miR-141 mimic or miR-mimic-control (Cont-mimic) at a final concentration of 40 nM for 24 h.
10
Long-Term Clonogenic Proliferation Assay
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Primary mPDAC cell cultures were isolated from autochthonous PDAC and cultured as described previously59 (link)
. All cells were cultivated for less than 30 passages, authenticated by genotyping and tested for mycoplasma contamination by PCR. Conventional human PDAC cell lines and primary patient derived low passaged PDAC cell cultures were established and cultured as previously reported58 (link)
.
For long-term clonogenic cell proliferation assays, cells were seeded into 24-well plates (density of 1-2×103 cells/well, depending on growth rate. The following day, plates were treated with different concentrations of drugs as indicated. Every 7 days, media and drug were refreshed. Cells were fixed and stained with 0.2% crystal violet in an ethanol/water solution 7 to 13 days after the start of treatment, according to the confluence reached by the untreated control. Crystal violet was solubilized with 10% acetic acid and absorbance was quantified at 595 nm. The resulting values were used to calculate the Bliss synergy score with the online software Synergy Finder (v1.0)60 (link)
. All assays were performed independently at least three times. Trametinib, nintedanib, AZD-4547, imatinib and PF-3758309 were obtained from Selleckchem, 4-OHT from Sigma, murine anti PD-L1 mAb (Anti PD-L1-mIgG1e3 InvivoFit™) was purchased from InvivoGen, and tamoxifen for in vivo treatment from Sigma.
. All cells were cultivated for less than 30 passages, authenticated by genotyping and tested for mycoplasma contamination by PCR. Conventional human PDAC cell lines and primary patient derived low passaged PDAC cell cultures were established and cultured as previously reported58 (link)
.
For long-term clonogenic cell proliferation assays, cells were seeded into 24-well plates (density of 1-2×103 cells/well, depending on growth rate. The following day, plates were treated with different concentrations of drugs as indicated. Every 7 days, media and drug were refreshed. Cells were fixed and stained with 0.2% crystal violet in an ethanol/water solution 7 to 13 days after the start of treatment, according to the confluence reached by the untreated control. Crystal violet was solubilized with 10% acetic acid and absorbance was quantified at 595 nm. The resulting values were used to calculate the Bliss synergy score with the online software Synergy Finder (v1.0)60 (link)
. All assays were performed independently at least three times. Trametinib, nintedanib, AZD-4547, imatinib and PF-3758309 were obtained from Selleckchem, 4-OHT from Sigma, murine anti PD-L1 mAb (Anti PD-L1-mIgG1e3 InvivoFit™) was purchased from InvivoGen, and tamoxifen for in vivo treatment from Sigma.
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