Journal: Frontiers in Pharmacology

Article Title: Oxyresveratrol as a novel ferroptosis inducer exhibits anticancer activity against breast cancer via the EGFR/PI3K/AKT/GPX4 signalling axis

doi: 10.3389/fphar.2024.1527286

Figure Lengend Snippet: ORes induces ferroptosis in breast cancer cells via the EGFR/PI3K/AKT/GPX4 signalling axis. (A) The table indicates the top 10 connectivity scores between the input gene signature and the gene signatures of compounds in the LINCS L1000 touchstone dataset. (B) The table displays the top 10 scores between the input gene signature and the gene signatures of compounds in the DGDB dataset. (C) GSEA of HTS 2 results of Osimertinib dimesylate–treated MDA-MB-231 cells. (D) GSEA of HTS 2 results of AZ-5104–treated MDA-MB-231 cells. (E) Volcano plot of the DEGs in EGFR knockdown MDA-MB-231 cells, with red and blue dots indicating upregulated and downregulated genes, respectively. Differential gene screening criteria: |FoldChange|>1.3 and p- value < 0.05. (F) Representative Western blot results. (G) Quantification of western blots. Experiments were performed in triplicate, and data are presented as mean ± SD. *** p < 0.001; ns, no significance.

Article Snippet: The membranes were incubated overnight at 4°C with primary antibodies against GPX4 (Cell Signaling Technology, 59735, 1:1,000), phosphorylated EGFR (ZenBio, R26283, 1:1,000), EGFR (Selleck, A5858, 1:1,000), phosphorylated PI3K (ZenBio, 310164, 1:1,000), PI3K (ZenBio, 200900, 1:1,000), phosphorylated AKT (Cell Signaling Technology, 13038, 1:1,000), and AKT (Cell Signaling Technology, 4691, 1:1,000).

Techniques: Knockdown, Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 is upregulated in gefitinib-resistant cells. a-b Human NSCLC HCC827P (a) and PC9P (b) cells treated with 10nM gefitinib at indicated days were immunoblotted with antibodies against phosphorylated EGFR (p-EGFR), total EGFR (T-EGFR), phosphorylated ERBB2 (p-ERBB2), total ERBB2 (T-ERBB2), phosphorylated ERBB3 (p-ERBB3), total ERBB3 (T-ERBB3), DARPP-32, and α-tubulin (loading control). c-d HCC827P, HCC827GR (c), PC9P, PC9GR2, and PC9GR3 (d) cells treated with 100nM gefitinib were lysed and antibody-reactive protein bands were detected using anti-p-EGFR, EGFR, p-ERBB2, ERBB2, p-ERBB3, ERBB3, DARPP-32, and α-tubulin antibodies. Immunoblotting experiments were repeated independently at least three times, and a representative experimental result is shown.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Gefitinib blocks EGFR phosphorylation in EGFR-mutated human NSCLC cells. a-b HCC827P (a) and PC9P (b) cells were lysed in RIPA buffer and phosphorylated EGFR (p-EGFR), total EGFR (T-EGFR), phosphorylated ERBB2 (p-ERBB2), total ERBB2 (T-ERBB2), phosphorylated ERBB3 (p-ERBB3), total ERBB3 (T-ERBB3), DARPP-32, and α-tubulin (loading control) proteins were detected by immunoblotting of cell lysates.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Overexpression of DARPP-32 isoforms increases p-ERBB3 expression. a Human lung cancer cell line PC9P treated with vehicle (UT) or 100nM gefitinib (T) were transduced with retrovirus containing control (LacZ)-, DARPP-32- or t-DARPP-overexpressing clones and immunofluorescence experiments were performed using primary antibodies against p-ERBB3 (green) and p-EGFR (red). Nuclei were stained with DAPI (blue). b-c Average red (b) and green (c) fluorescence intensity of 6-10 random microscopic fields for each sample was reported. Experiments were repeated at least three times. Scale bar, 20 μm. Bar graphs indicate mean ±SEM (n=3). *P<0.05 and **P<0.01, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Over Expression, Expressing, Transduction, Clone Assay, Immunofluorescence, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Expression of p-ERBB3 is controlled by DARPP-32. a PC9GR3 cells were transduced with lentivirus containing control (LacZ) or DARPP-32 shRNAs. Cells treated with vehicle (UT) or 100nM gefitinib (T) were fixed, permeabilized, and incubated with primary antibodies that detect p-ERBB3 (green) and p-EGFR (red) proteins. DAPI-stained nuclei were represented in blue color. b-c Expression of p-EGFR (b) and p-ERBB3 (c) was reported by calculating average fluorescence intensity of 6-10 random microscopic fields for each sample. Each circle on a graph represents an independent experiment. Scale bar, 20 μm. Results represent mean ±SEM (n=3). *P<0.05 and **P<0.01, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Expressing, Transduction, Incubation, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 physically associates with EGFR and ERBB3. a-b EGFR-mutated human NSCLC HCC827P (a) and PC9P (b) cells transduced with retrovirus encoding control (LacZ)-, DARPP-32- or t-DARPP-overexpressing clones were immunoprecipitated using antibodies against FLAG (detects both DARPP-32 isoforms), and ERBB3. Immunoprecipitated protein complexes and total cell lysates (input) were immunoblotted using anti-EGFR, ERBB3, FLAG, and α-tubulin antibodies. c-d Human lung adenocarcinoma HCC827P, HCC827GR (c), PC9P, PC9GR2, and PC9GR3 (d) cells were lysed and immunoprecipitated with anti-DARPP-32 (recognizes endogenous DARPP-32 and t-DARPP) and anti-ERBB3 antibodies. Immunoprecipitated lysates along with total cell lysates were separated on SDS-PAGE followed by immunoblot analysis using antibodies against EGFR, ERBB3, DARPP-32, and α-tubulin. Immunoprecipitation experiments were repeated at least three times.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Transduction, Clone Assay, Immunoprecipitation, SDS Page, Western Blot

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Depletion of DARPP-32 reduces p-EGFR to p-ERBB3 heterodimer formation. a Proximity ligation assays (PLA) were performed in PC9GR3 cells stably transduced with control (shLacZ) or DARPP-32 shRNAs (shDP32) using antibodies against phosphorylated ERBB3 and EGFR following 24h incubation with either vehicle (UT) or 100 nM gefitinib (T). The images show a maximum intensity projection of the raw image based on 10 z-planes. PLA signals are shown in red and the DAPI-stained nuclei in blue. Scale bar, 20 μm. b Total number of PLA signals per cell were reported after calculating red fluorescence signals of 6-10 random microscopic fields for each group. Each circle on a graph represents an independent experiment. Bar graphs represent mean ±SEM of three independent experiments. *P<0.05, 2-way unpaired t-test.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Ligation, Stable Transfection, Transduction, Incubation, Staining, Fluorescence

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 activates AKT and ERK signaling in the presence of gefitinib. a Immunoblotting was performed in gefitinib-treated HCC827P and PC9P cells stably overexpressing control (LacZ), DARPP-32 or t-DARPP using antibodies against phosphorylated AKT (p-Akt), total AKT (Akt), phosphorylated ERK (p-Erk1/2), total ERK (Erk1/2), DARPP-32, and α-tubulin (loading control). b Gefitinib-resistant human lung cancer cell lines, HCC827GR, PC9GR2, and PC9GR3, were transduced with lentivirus containing LacZ or DARPP-32 shRNAs and treated with 100nM gefitinib for 24h. Cell lysates were separated and antibody-reactive protein bands were detected using anti-p-AKT, AKT, p-ERK, ERK, DARPP-32, and α-tubulin antibodies. Three independent immunoblotting experiments have been performed and representative results from one experiment have been shown.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Western Blot, Stable Transfection, Transduction

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: DARPP-32 silencing inhibits EGFR TKI refractory tumor growth in vivo a Luciferase-labeled human HCC827GR cells transduced with control (LacZ) or DARPP-32 shRNA were injected into the left thorax of SCID mice (n=7 mice per group), imaged for luminescence, administered 25mg/Kg gefitinib on indicated days. b Quantification of tumor growth was reported by determining the difference in relative luciferase units (RLU) before and after drug treatment. c Immunohistochemistry was performed using monoclonal Ki-67 antibody on formalin-fixed, paraffin-embedded lung tissue (n=3 mice per group) obtained from human lung tumor xenograft model. For evaluation of the morphology, slides were stained with hematoxylin and eosin (H&E) dye. Scale bar, 20 μm.

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: In Vivo, Luciferase, Labeling, Transduction, shRNA, Injection, Immunohistochemistry, Formalin-fixed Paraffin-Embedded, Staining

Journal: bioRxiv

Article Title: DARPP-32 promotes ERBB3-mediated resistance to molecular targeted therapy in EGFR-mutated lung adenocarcinoma

doi: 10.1101/2021.02.12.430856

Figure Lengend Snippet: Expression of DARPP-32, p-EGFR, and p-ERBB3 proteins is elevated in EGFR TKI resistant lung adenocarcinoma. a Tumor tissue was biopsied before EGFR TKI treatment (i.e. baseline) and following EGFR TKI resistance (i.e. progressive disease after first-line gefitinib or erlotinib therapy) from lung adenocarcinoma patients with EGFR activating mutations (n=30 patients in each group). Paired baseline (top) and EGFR TKI resistance (bottom) lung tumor specimens were immunostained for DARPP-32, phosphorylated EGFR (p-EGFR), total EGFR, p-ERBB3, and total ERBB3. b IHC score was calculated by multiplying the staining intensity score (0-3) by the percent of positive tumor cells. Each circle on the plots represents single patient. *P<0.05, **P<0.01, ****P<0.0001,2-way unpaired t-test

Article Snippet: Permeabilized cells were then used for immunofluorescence staining using primary antibodies against phosphorylated-EGFR (Y845; BD Biosciences; Cat No.: 558381; Dilution 1:200) and phosphorylated-ERBB3 (Y1289; Cell Signaling Technology; Cat No.: 2842; Dilution 1:200).

Techniques: Expressing, Staining