Journal: Journal of Advanced Research

Article Title: Squalene epoxidase promotes paraquat-induced pulmonary toxicity through endoplasmic reticulum-mediated ferroptosis

doi: 10.1016/j.jare.2025.05.064

Figure Lengend Snippet: SQLE inhibitors attenuate PQ-induced lung epithelial cell death. (A-G) Cells were treated with PQ for 24 h in the presence or absence of LNT postreatment for 30 mins. CCK-8 assay (A), ATP release (B) in A549 cells. CCK-8 assay (C), ATP release (D) in BEAS-2B cells. CCK-8 assay (E), ATP release (F) in RLE-6TN cells. CCK-8 assay (G) in AECⅡ. (H) Cells were treated with PQ for 24 h in the presence or absence of NB598 postreatment for 30 mins in A549 cells. Followed by CCK-8 assay. Morphological changes. Bar = 400 µm. (I), and PI staining (J). Data are presented as Mean ± S.D. of at least three independent biological replicates (n = 3). *P < 0.05 , ** P < 0.01 , *** P < 0.001 show significant differences from each group.

Article Snippet: A549 human alveolar epithelial cells, RLE-6TN rat alveolar epithelial cells, and HEK-293 T cells were sourced from the American Type Culture Collection (ATCC, Manassas, VA, USA), while BEAS-2B human bronchial epithelial cells were obtained from Beyotime.

Techniques: CCK-8 Assay, Staining

Journal: Future science OA

Article Title: Hesperidin alleviates pulmonary fibrosis by regulating EI24-mediated autophagy.

doi: 10.1080/20565623.2025.2483147

Figure Lengend Snippet: Figure 1. Hes activated autophagy in TGF-β1-induced RLE-6TN cells. RLE-6TN cells were treated with 10 ng/mL TGF-β1 for 48 h, as well as 25, 50 and 100 µM Hes for 24 h. (A) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 were examined by western blot. Data were expressed with the normalization with β-ACTIN. (B) The expression level of LC3B was detected by IF. Scale bar = 25 µm. **P < 0.01 vs. control (con); #P < 0.05 and ##P < 0.01 vs. TGF-β1. Note: Hes: hesperidin; TGF-β1: transforming growth factor β1; EI24: etoposide-induced protein 2.4; IF: immunofluorescence.

Article Snippet: Rle-6tN cells were stimulated with 10 ng/ml tGF-β1 (#GF346, sigma-aldrich) for 48 h to construct the pulmonary fibrosis model in vitro according to the earlier research [25]. small interfering RNa against ei24 (si-ei24), and the negative control (si-Nc) were purchased from GenePharma (shanghai, china) to downregulate the expression of ei24 in Rle-6tN cells. additionally, 3-Ma (1 mM, #19312, Medchem express), an inhibitor of autophagy, was used to incubate with Rle-6tN cells for 24 h, to verify the role of autophagy. the concentration of 3-Ma used in this study was based on the previous research [25].

Techniques: Western Blot, Expressing, Control, Immunofluorescence

Journal: Future science OA

Article Title: Hesperidin alleviates pulmonary fibrosis by regulating EI24-mediated autophagy.

doi: 10.1080/20565623.2025.2483147

Figure Lengend Snippet: Figure 2. Hes suppressed TGF-β1-induced fibrosis in RLE-6TN cells. (A) The relative mRNA expressions of α-SMA, collagen I and fibronectin were measured by RT-qPCR in RLE-6TN cells. Data were expressed with the normalization with GAPDH. (B) The relative protein expressions of α-SMA, COLLAGEN I and FIBRONECTIN were determined by western blot in RLE-6TN cells. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con); #P < 0.05 and ##P < 0.01 vs. TGF-β1. Note: Hes: hesperidin; TGF-β1: transforming growth factor β1; mRNA: messenger ribonucleoprotein; RT-qPCR: reverse transcription quantitative polymerase chain reaction; GAPDH: reduced glyceraldehyde-phosphate dehydrogenase; α-SMA: alpha smooth muscle Actin.

Article Snippet: Rle-6tN cells were stimulated with 10 ng/ml tGF-β1 (#GF346, sigma-aldrich) for 48 h to construct the pulmonary fibrosis model in vitro according to the earlier research [25]. small interfering RNa against ei24 (si-ei24), and the negative control (si-Nc) were purchased from GenePharma (shanghai, china) to downregulate the expression of ei24 in Rle-6tN cells. additionally, 3-Ma (1 mM, #19312, Medchem express), an inhibitor of autophagy, was used to incubate with Rle-6tN cells for 24 h, to verify the role of autophagy. the concentration of 3-Ma used in this study was based on the previous research [25].

Techniques: Quantitative RT-PCR, Western Blot, Control, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Future science OA

Article Title: Hesperidin alleviates pulmonary fibrosis by regulating EI24-mediated autophagy.

doi: 10.1080/20565623.2025.2483147

Figure Lengend Snippet: Figure 3. Hes inhibited fibrosis by regulating EI24-mediated autophagy in TGF-β1-induced RLE-6TN cells. RLE-6TN cells were transfected with si-EI24. After 48 h of transfection, cells were collected to treat with 10 ng/mL TGF-β1 for 48 h, as well as 100 µM Hes and 1 mM 3-MA for 24 h. (A) The relative protein expression of EI24 was examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con). (B) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 were examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. (C) The expression level of LC3B was detected by IF. Scale bar = 25 µm. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. (D) The relative protein expressions of α-SMA, COLLAGEN I and FIBRONECTIN were determined by western blot in RLE-6TN cells. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. control (con); ##P < 0.01 vs. TGF-β1; &P < 0.05 vs. TGF-β1 + 100 µM Hes. Note: Hes: hesperidin; TGF-β1: transforming growth factor β1; EI24: etoposide-induced protein 2.4; IF: immunofluorescence; α-SMA: alpha smooth muscle Actin.

Article Snippet: Rle-6tN cells were stimulated with 10 ng/ml tGF-β1 (#GF346, sigma-aldrich) for 48 h to construct the pulmonary fibrosis model in vitro according to the earlier research [25]. small interfering RNa against ei24 (si-ei24), and the negative control (si-Nc) were purchased from GenePharma (shanghai, china) to downregulate the expression of ei24 in Rle-6tN cells. additionally, 3-Ma (1 mM, #19312, Medchem express), an inhibitor of autophagy, was used to incubate with Rle-6tN cells for 24 h, to verify the role of autophagy. the concentration of 3-Ma used in this study was based on the previous research [25].

Techniques: Transfection, Expressing, Western Blot, Control, Immunofluorescence