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: Protein samples (20 μg) were electrophoresed with 10% sDs-PaGe and transferred onto PVDF membranes (#iPVh00010, eMD Millipore, Billerica, Ma, usa). the membranes were blocked with 5% Bsa Blocking Buffer (#sW3015, solarbio) at room temperature for 1 h, and then treated with primary antibodies at 4 °c overnight. the membranes were incubated with the secondary antibodies of Goat anti-Rabbit igG h&l (hRP) (1:10000, #ab6721, abcam) or Donkey anti-Goat igG h&l (hRP) (1:10000, #ab6728, abcam) (1:5000, #ab6885, abcam) for 1 h at room temperature. the bands were developed using a Beyoecl Plus kit (#P0018s, Beyotime, shanghai, china), and the gray values were measured with image-ProPlus software (Media cybernetics, inc., Rockville, MD, usa). β-actiN acted as the internal reference. the primary antibodies included rabbit monoclonal to ei24 (#42328, cell signaling technology, inc., Danvers, Ma, usa), rabbit polyclonal to BecliN1 (1:1000, #3738, cell signaling technology), rabbit polyclonal to lc3ii/lc3i (1:1000, #4108, cell signaling technology), rabbit polyclonal to P62 (1:1000, #5114, cell signaling technology), goat polyclonal to alpha smooth muscle actin (α-sMa) (1:1000, #ab21027, abcam, cambridge, uK), rabbit polyclonal to cOllaGeN i (1:1000, #ab254113, abcam), rabbit polyclonal to FiBRONectiN (1:1000, #63779, cell signaling technology) and rabbit polyclonal to β-actiN (1:1000, #4967, cell signaling technology).

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 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: Protein samples (20 μg) were electrophoresed with 10% sDs-PaGe and transferred onto PVDF membranes (#iPVh00010, eMD Millipore, Billerica, Ma, usa). the membranes were blocked with 5% Bsa Blocking Buffer (#sW3015, solarbio) at room temperature for 1 h, and then treated with primary antibodies at 4 °c overnight. the membranes were incubated with the secondary antibodies of Goat anti-Rabbit igG h&l (hRP) (1:10000, #ab6721, abcam) or Donkey anti-Goat igG h&l (hRP) (1:10000, #ab6728, abcam) (1:5000, #ab6885, abcam) for 1 h at room temperature. the bands were developed using a Beyoecl Plus kit (#P0018s, Beyotime, shanghai, china), and the gray values were measured with image-ProPlus software (Media cybernetics, inc., Rockville, MD, usa). β-actiN acted as the internal reference. the primary antibodies included rabbit monoclonal to ei24 (#42328, cell signaling technology, inc., Danvers, Ma, usa), rabbit polyclonal to BecliN1 (1:1000, #3738, cell signaling technology), rabbit polyclonal to lc3ii/lc3i (1:1000, #4108, cell signaling technology), rabbit polyclonal to P62 (1:1000, #5114, cell signaling technology), goat polyclonal to alpha smooth muscle actin (α-sMa) (1:1000, #ab21027, abcam, cambridge, uK), rabbit polyclonal to cOllaGeN i (1:1000, #ab254113, abcam), rabbit polyclonal to FiBRONectiN (1:1000, #63779, cell signaling technology) and rabbit polyclonal to β-actiN (1:1000, #4967, cell signaling technology).

Techniques: Transfection, Expressing, Western Blot, 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 5. Hes activated autophagy in BLM-induced fibrosis via EI24. (A) The relative protein expressions of α-SMA, COLLAGEN I and FIBRONECTIN in lung tissues were determined by western blot. Data were expressed with the normalization with β-ACTIN. (B) The relative protein expressions of EI24, BECLIN1, LC3II/LC3I and P62 in lung tissues were examined by western blot. Data were expressed with the normalization with β-ACTIN. **P < 0.01 vs. sham; ##P < 0.01 vs. BLM; &P < 0.05 vs. BLM + 100 mg/kg Hes. Note: Hes: hesperidin; EI24: etoposide-induced protein 2.4; BLM: bleomycin; α-SMA: alpha smooth muscle Actin.

Article Snippet: Protein samples (20 μg) were electrophoresed with 10% sDs-PaGe and transferred onto PVDF membranes (#iPVh00010, eMD Millipore, Billerica, Ma, usa). the membranes were blocked with 5% Bsa Blocking Buffer (#sW3015, solarbio) at room temperature for 1 h, and then treated with primary antibodies at 4 °c overnight. the membranes were incubated with the secondary antibodies of Goat anti-Rabbit igG h&l (hRP) (1:10000, #ab6721, abcam) or Donkey anti-Goat igG h&l (hRP) (1:10000, #ab6728, abcam) (1:5000, #ab6885, abcam) for 1 h at room temperature. the bands were developed using a Beyoecl Plus kit (#P0018s, Beyotime, shanghai, china), and the gray values were measured with image-ProPlus software (Media cybernetics, inc., Rockville, MD, usa). β-actiN acted as the internal reference. the primary antibodies included rabbit monoclonal to ei24 (#42328, cell signaling technology, inc., Danvers, Ma, usa), rabbit polyclonal to BecliN1 (1:1000, #3738, cell signaling technology), rabbit polyclonal to lc3ii/lc3i (1:1000, #4108, cell signaling technology), rabbit polyclonal to P62 (1:1000, #5114, cell signaling technology), goat polyclonal to alpha smooth muscle actin (α-sMa) (1:1000, #ab21027, abcam, cambridge, uK), rabbit polyclonal to cOllaGeN i (1:1000, #ab254113, abcam), rabbit polyclonal to FiBRONectiN (1:1000, #63779, cell signaling technology) and rabbit polyclonal to β-actiN (1:1000, #4967, cell signaling technology).

Techniques: Western Blot