Journal: Molecular pharmacology

Article Title: Dynamic Expression of Transient Receptor Potential Vanilloid-3 and Integrated Signaling with Growth Factor Pathways during Lung Epithelial Wound Repair following Wood Smoke Particle and Other Forms of Lung Cell Injury.

doi: 10.1124/molpharm.121.000280

Figure Lengend Snippet: Fig. 7. Inhibition of EGFR and growth factor pathway components prevented TRPV3 mRNA upregulation after injury. (A) HBEC3-KT cells were treated with 5 mM solutions of inhibitors of multiple ErbB receptor tyrosine kinase isoforms: ErbB1 (EGFR)-specific inhibitor AG-1478, ErbB pan-inhibitors AZD8931 and Afatinib, and a selective ErbB2 (HER2) inhibitor CP-724 for 2 hours in the cell passaging injury model, fol- lowed by analysis of TRPV3 mRNA. (B) Downstream targets of EGFR activation were also inhibited, and TRPV3 expression was subsequently measured 2 hours after cell passaging injury. Inhibitors included 5 mM TWS119 (GSK3b), 20 mM CCT036477 (b-catenin), 10 mM PD169316 (p38 MAPK), and 10 mM SP600125 (JNK). (C) Inhibition of NF-jB (20 mM BMS-345541) and effects on TRPV3 mRNA 2 hours after cell passaging injury. (D) Inhibitors of Wnt7a and Fzd signaling were also used: 10 ng/ml secreted Frizzled-related protein (sFRP) and IWP 2 (Porcupine inhibitor) reduced TRPV3 upregulation after injury. Furthermore, SB 431542 (TGFbRI inhibitor) and 150 mM ITD-1 (TGFbRII inhibitor) were also tested, and SB 431542 treatment prevented TRPV3 expression as well. Values were normalized to vehicle controls and are presented as mean ± S.D. from n $ 3 replicates. Statistical significance was determined using one-way ANOVA with the Dunnett post-test. ***P < 0.001, ****P < 0.0001. For Fig. 4C, an unpaired t test was used. ****P < 0.0001.

Article Snippet: Afatinib dimaleate (EGFR, ErbB2, HER4/receptor tyrosine kinase 4), IWP 2 (inhibitor of Wnt production 2, targeting porcupine), SB 431542 (TGFbRI), and ITD 1 (1,4-dihydropyridine inducer of TGFbRII degradation-1) were purchased from Tocris (Minneapolis, MN).

Techniques: Inhibition, Passaging, Activation Assay, Expressing

Journal: Translational Lung Cancer Research

Article Title: PKC-iota drives EGFR-TKI resistance in EGFR-mutated NSCLC by phosphorylating FASN to reprogram lipid metabolism

doi: 10.21037/tlcr-2025-aw-1260

Figure Lengend Snippet: PKC-iota/FASN complex drives tumor growth and osimertinib resistance in vitro and in vivo. (A-C) PC9 cells were cotransfected with siRNA-FASN or siRNA-NC and Flag-PKC-iota or empty vector (worked as a control) for 16–18 hours, and then plated onto 96-well plates (5,000 cells per well), followed by 72 hours gefitinib (A, 0–100,000 nmol/L), afatinib (B, 0–100 nmol/L), or osimertinib (C, 0–100,000 nmol/L), CCK-8 (10 µL) was then added for 2 hours to quantitate by a colorimetric method. (D) H1975 cells were cotransfected with siRNA-FASN or siRNA-NC and Flag-PKC-iota or empty vector for 16-18 hours, and then plated onto 96-well plates (5,000 cells per well), followed by osimertinib (1 µmol/L) or DMSO treatment, and cell numbers were counted at 96 hours. The osimertinib-induced cell decrease rate = (cell counts in the control group – cell counts in the osimertinib-treated group)/cell counts in the control group × 100%. Data were also shown in Figure S4A . ns, not significant; *, P<0.05. (E) After 24 hours of transfection (siRNA-FASN or siRNA-NC and Flag-PKC-iota or empty vector), osimertinib (10 nmol/L) or DMSO was added into H1975 cells for 7 days, followed by colony formation assay. The osimertinib-induced colony number decrease rate = (colony number in control group – colony number in osimertinib-treated group)/colony number in control group × 100%. Representative images of the colony formation were also shown in Figure S4B . ns, nonspecific; *, P<0.05. (F) After 24 hours of transfection (siRNA-FASN or siRNA-NC and Flag-PKC-iota or empty vector), H1975 cells were treated with osimertinib (1 µmol/L) or DMSO for 72 hours, followed by detection of cell apoptosis by flow cytometry. The osimertinib-induced cell apoptotic rate = apoptotic rate in osimertinib-treated group – apoptotic rate in control group. Representative images of percentage of apoptosis cells were also shown Figure S4C . ns, nonspecific; *, P<0.05; ***, P<0.001. (G-L) Nude mice were injected with 5×10 6 H1975 cells stable overexpressing PKC-iota or empty vector and shRNA-FASN or shRNA-NC: (G) the volume of tumors was measured at regular intervals, followed by osimertinib treatment at the 10th day. Days means days after injection; (H) image of tumors isolated from nude mice; (I) statistical analysis of tumor weight was performed separately for control groups and osimertinib-treated groups. ns, nonspecific; *, P<0.05. (J) Osimertinib-induced tumor volume decrease rate (tumor volume in control group – tumor volume in osimertinib-treated group)/tumor volume in control group × 100% was analyzed using the volume of 31 day. ns, nonspecific; *, P<0.05. (K) Osimertinib-induced tumor weight decrease rate (tumor weight in control group – tumor weight in osimertinib-treated group)/tumor weight in control group × 100% was analyzed using isolated tumors. ns, nonspecific; *, P<0.05. (L) Representative images of H&E staining, Oil Red O staining and Ki67 IHC staining of tumor samples (scale bar, 100 µm). CCK-8, Cell-Counting Kit-8; DMSO, dimethyl sulfoxide; H&E, hematoxylin and eosin; IHC, immunohistochemistry; FASN, fatty acid synthase; NC, negative control; PKC-iota, protein kinase C-iota.

Article Snippet: The following additional reagents were in the present study: MG132 (HY-13259, MCE, NJ, USA); cycloheximide (HY-12320, MCE); EGFT-TKIs gefitinib (HY-50895, MCE), afatinib (HY-10261, MCE), and osimertinib (HY-15772, MCE); Dynasore (HY-15304, MCE); Pitstop2 (HY-115604, MCE); TMA-DPH (HY-D0986, MCE); EGF (HY-P7109, MCE).

Techniques: In Vitro, In Vivo, Plasmid Preparation, Control, CCK-8 Assay, Transfection, Colony Assay, Flow Cytometry, Injection, shRNA, Isolation, Staining, Immunohistochemistry, Cell Counting, Negative Control