Journal: bioRxiv

Article Title: Ellagic acid ameliorates atherosclerosis by targeting the epidermal growth factor receptor

doi: 10.1101/2023.09.14.557846

Figure Lengend Snippet: (A) Effect of EA (treatment for 24 h) on LDLR mRNA levels in HepG2 cells. (B) The effect of EA on the stability of LDLR mRNA, observed under the mRNA synthesis of HepG2 cells was inhibited by actinomycin D. (C) Effects of different concentrations of EA (10, 20, and 40 μM) on p-ERK and p-EGFR levels in HepG2 cells. (D) Effects of EA on p-ERK, p-EGFR, and LDLR levels after treatment with cetuximab to block the EGFR pathway in HepG2 cells. Data represent the mean ± SEM (n = 3). *P < 0.05, **P < 0.01, and ***P < 0.001. EA, ellagic acid; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; LDLR, low-density lipoprotein receptor; p-EGFR, phosphorylated-EGFR; p-ERK, phosphorylated-ERK; SEM, standard error of the mean.

Article Snippet: Other antibodies included anti-LDLR (ET1606, Huabio, Hangzhou, China), anti-EGFR (2085S, Cell Signaling Technology, MA, USA), anti-phospho-EGFR (48576SF, Cell Signaling Technology, MA, USA), anti-ERK1/2 (4696S, Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2 (4370S, Cell Signaling Technology, MA, USA), and anti-β-actin (66009-1-IG, Proteintech, Wuhan, Hubei, China).

Techniques: Blocking Assay

Journal: bioRxiv

Article Title: Ellagic acid ameliorates atherosclerosis by targeting the epidermal growth factor receptor

doi: 10.1101/2023.09.14.557846

Figure Lengend Snippet: (A) Diagram of the EA-naps composite and appearance of the EA-naps powder. (B) Protein levels of LDLR, p-ERK, and p-EGFR in HepG2 cells treated with EA or EA-naps. (C) mRNA levels of LDLR in HepG2 cells treated with EA or EA-naps. Data represent the mean ± SEM (n = 3). *P < 0.05 and **P < 0.01. EA, ellagic acid; EA-naps, ellagic acid-loaded human serum albumin nanoparticles; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HSA, human serum albumin; LDLR, low-density lipoprotein receptor; p-EGFR, phosphorylated-EGFR; p-ERK, phosphorylated-ERK; RT-PCR, real-time polymerase chain reaction; SEM, standard error of the mean.

Article Snippet: Other antibodies included anti-LDLR (ET1606, Huabio, Hangzhou, China), anti-EGFR (2085S, Cell Signaling Technology, MA, USA), anti-phospho-EGFR (48576SF, Cell Signaling Technology, MA, USA), anti-ERK1/2 (4696S, Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2 (4370S, Cell Signaling Technology, MA, USA), and anti-β-actin (66009-1-IG, Proteintech, Wuhan, Hubei, China).

Techniques: Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction

Journal: bioRxiv

Article Title: Ellagic acid ameliorates atherosclerosis by targeting the epidermal growth factor receptor

doi: 10.1101/2023.09.14.557846

Figure Lengend Snippet: (A) mRNA levels of LDLR. (B) Protein levels of LDLR, p-ERK, and p-EGFR in the liver of ApoE -/- mice. *P < 0.05, **P < 0.01, and ***P < 0.001. Data represent the mean ± SEM of six biological replicates. HFD group: high-fat and high-cholesterol diet, and treatment with uncoated ellagic acid nanoparticles; HFD+EA-naps group: high-fat and high-cholesterol diet, and treatment with coated ellagic acid nanoparticles. ApoE, apolipoprotein E; EA-naps, ellagic acid-loaded human serum albumin nanoparticles; EGFR, epidermal growth factor receptor; ERK, extracellular signal-regulated kinase; HFD, high-fat diet; IHC, immunohistochemistry; LDLR, low-density lipoprotein receptor; p-EGFR, phosphorylated-EGFR; p-ERK, phosphorylated-ERK; SEM, standard error of the mean.

Article Snippet: Other antibodies included anti-LDLR (ET1606, Huabio, Hangzhou, China), anti-EGFR (2085S, Cell Signaling Technology, MA, USA), anti-phospho-EGFR (48576SF, Cell Signaling Technology, MA, USA), anti-ERK1/2 (4696S, Cell Signaling Technology, MA, USA), anti-phospho-ERK1/2 (4370S, Cell Signaling Technology, MA, USA), and anti-β-actin (66009-1-IG, Proteintech, Wuhan, Hubei, China).

Techniques: Immunohistochemistry

Journal: bioRxiv

Article Title: EGFR-to-Src family tyrosine kinase switching in proliferating-DTP TNBC cells creates a hyperphosphorylation-dependent vulnerability to EGFR TKI

doi: 10.1101/2023.07.17.549374

Figure Lengend Snippet: (A) Schematic of Proliferating drug-tolerant persister (PDTP) model. Different subtypes of Triple-negative Breast cancer (TNBC) parental cell (PC) lines MDAMB4-68, HCC70, HS478T, and MDA-MB-453 were treated short-term with chemotherapy cytotoxic doses (IC 80-95 ) in vitro. After a few days, quiescent drug-tolerant cancer cells were observed. Over time, these quiescent cancer cells resumed growth, establishing “recurrent” colonies. The PDTPs were resistant to the last doses of drugs given for selection. (B) The volcano plot below depicts the protein expression levels of 207 proteins as observed by mass spectrometry. The p-values are FDR-adjusted prior to further analysis and the level of significance is set at 0.05. Following these 15 significantly upregulated genes have been plotted in red and 52 significantly downregulated genes have been plotted in blue. EGFR is found to be the most down-regulated. (C) Levels of EGFR and p-EGFR (Y 1068 ) were determined in different subtypes of parental PC of TNBC and their Paclitaxel-PDTP cells. GAPDH served as their loading control. The expression levels of the indicated proteins were assessed by WB. Band intensities were quantified by ImageJ software and presented as fold of PC in the heat map at the right-side panel (red indicates increase and green indicate lower than PC) (D) Representative confocal images of the indicated TNBC PC and their Paclitaxel (PDTP-P) and Doxorubicin (PDTP-D) PDTP stained with EGFR and DAPI as described in Materials and Methods. Scale bar, 10 µm. (E) Mean fluorescence intensity of total cellular EGFR and cell-membrane localized EGFR were quantitated using Image J software in PC, PDTP-P and PDTP-D of MDA-MB468. Similarly, membrane and cytoplasmic EGFR were quantitated in PC of MDA-MB468 and HS578T (F) Effects of short-term treatment of Paclitaxel (10 nM) on the levels of EGFR and p-EGFR protein in the indicated cell lines as estimated by WB analysis at different time points of treatment. Heatmaps presented in the right panel show the fold change in the protein expression quantified by densitometric analysis using ImageJ and expressed as folds of untreated (UT) control cells (G) The indicated TNBC cell lines were treated with different concentrations of paclitaxel (see Materials and Methods) for 24 hours and levels EGFR and pEGFR proteins were assessed by WB. Beta-tubulin served as their loading control. Heatmaps at the right panel show the fold change in the protein expression quantified by densitometric analysis using ImageJ and expressed as folds of untreated (UT) control of respective cells.

Article Snippet: The following antibodies were purchased from Cell Signaling Technology (USA): rabbit anti-EGFR (4267), rabbit anti-phospho EGFR Y-1068 (3777), mouse anti–STAT3 (9139), and rabbit anti-phospho STAT3 Y-705 (9145), rabbit anti-Src (2123), rabbit anti-phospho Src Y-416 (6943), rabbit anti-Lyn (2796), rabbit anti-Fyn (4023), rabbit anti-AKT (9272), rabbit anti-phospho AKT S-473 (4058), rabbit anti-ERK1/2 (9102), rabbit anti-phospho ERK1/2 T-202 Y-204 (9101), rabbit anti-GAPDH (5174), rabbit anti-P38 (9212) rabbit anti-pP38 (4511), rabbit anti-NDRG1 (9485).

Techniques: In Vitro, Selection, Expressing, Mass Spectrometry, Software, Staining, Fluorescence

Journal: Nature Communications

Article Title: HCK induces macrophage activation to promote renal inflammation and fibrosis via suppression of autophagy

doi: 10.1038/s41467-023-40086-3

Figure Lengend Snippet: A Endogenous HCK interacted with autophagy proteins ATG2A and CBL in macrophage cell line Raw264.7 by IP/WB. B Overexpression of HCK inhibited autophagy flux as more LC3 HiBiT reporter remained. HCK knockdown promoted autophagy flux as less LC3 HiBiT reporter remained. C HCK KO could promote autophagy by increasing LC3II/LC3I and decrease P62 levels with and without 3-MA treatment in mice BMDM cells. Western blots ( D ) and quantification ( E ) showed phospho-PI3K and phospho-AKT decreased in HCK KO BMDMs. However, there were no significant differences for phospho-Erk1/2 and phospho-EGFR with HCK KO. F Effect of HCK KO was abrogated in autophagy inducing by PP242 treatment. Baf1: bafilomycin A1. 3-MA: 3-methyladenine. Source data are provided as a Source Data file.

Article Snippet: Lysates were subjected to immunoblot analysis using the following antibodies: HCK (CST #14643), V5 tag (GenScript Inc #A01724), phosphor-Y410 HCK (Abcam #ab61055), LC3A/B (CST #4108), Fyn (CST #4023 S), p62/SQSTM1 (NOVUS BIOLOGICALS NBP1-48320SS), Atg2A (CST #15011), c-Cbl (CST #2747), phospho-c-Cbl (Tyr700) (D16D7) (CST #8869), MMR/CD206 (R&D Systems #AF2535), iNOS (BioLegend #690902), Arginase 1 (BioLegend #678802), Phospho-PI3K p85 /p55 (CST #17366), Phospho-Akt (CST #4060), Akt (CST #4691), Phospho-p44/42 MAPK (Erk1/2) (CST #9101), p44/42 MAPK (Erk1/2) (CST #9102), EGFR (CST #4267), Phospho-EGFR (CST #3777), SYK (CST #13198), pY20 (Invitrogen # 14-5001-82), a-SMA (Sigma-Aldrich #A5228) and GAPDH mAb (CST #2118).

Techniques: Over Expression, Western Blot