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 4. Hes regulated EI24 to mitigate BLM-induced fibrosis of rat lung tissue. Rats were intratracheally treated with 6 IU/ kg BLM on day 7, intragastrically administered with 100 mg/kg Hes from day 1 to day 28, and treated with 30 μL of ad-sh-EI24 (5 × 10 transducing unit) from day 1 to day 28. (A) Rats body weight on day 28. (B) Measurement of the lung tissues wet/dry ration. (C) Detection of lung resistance. (D) Examination of the hydroxyproline concentration. (E) Pathological examination by H&E staining. (F) The fibrosis was determined by masson trichome staining. **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 H&E: hematoxylin and eosin.

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: Concentration 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 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

Journal: Cellular & molecular biology letters

Article Title: Etoposide-induced protein 2.4 homolog promotes argininosuccinate synthase 1 and cancer cell survival upon arginine deprivation.

doi: 10.1186/s11658-025-00726-6

Figure Lengend Snippet: Fig. 1 EI24 increased ASS1 upon arginine deprivation in ASS1-deficient breast cancer cells. A Urea cycle-related enzymes (CPS1, carbamoyl phosphate synthetase 1). B Expression of arginine metabolism enzymes in a subset of breast cancer cell lines. C Viability of breast cancer cells upon arginine deprivation with EI24 overexpression (OE) by doxycycline (MDA-MB-231) or adenovirus (Hs 578T). D Expression of ASS1 upon arginine deprivation (left) or lysine supplementation for 24 h (right) in MDA-MB-231 with EI24 OE by doxycycline. E Enzymatic activity of ASS1 upon time-dependent deprivation of arginine with EI24 OE by doxycycline in MDA-MB-231. F Expression of ASS1 upon arginine deprivation in 24 h in Hs 578T cells with EI24 OE by adenovirus (left) and knockdown by siRNA (right). G Arginine level in human breast cancer samples classified by EI24 gene expression. H Correlation of EI24 expression and Kegg_arginine_and_ proline_metabolism pathway determined by GSEA in two different breast cancer datasets

Article Snippet: Primary antibodies were purchased from Cell Signaling Technology for EI24 (#42328), ASS1 (#70720), ARG2 (#55003), eNOS (#32027), pAKT (Ser473) (#9271), AKT (#9272), MDM2 (#86934), pP70S6K (#9204), P70S6K (#9202), pS6 (#2211), LC3 (#4108), and p21 (#2947); from Santa Cruz for ODC1 (sc-390366), ASL (sc-374353), and β-actin (sc-69879); from Novus for HIF1α (NB100-449); and from Sigma-Aldrich for puromycin (MABE343).

Techniques: Expressing, Over Expression, Activity Assay, Knockdown, Gene Expression

Journal: Cellular & molecular biology letters

Article Title: Etoposide-induced protein 2.4 homolog promotes argininosuccinate synthase 1 and cancer cell survival upon arginine deprivation.

doi: 10.1186/s11658-025-00726-6

Figure Lengend Snippet: Fig. 2 EI24 regulates protein synthesis of ASS1. A Expression of ASS1 upon arginine deprivation and/or cycloheximide (CHX, 10 µg/ml, 24 h), MG132 (10 µM, 6 h), and bafilomycin A1 (Baf, 10 nM, 2 h) treatment in MDA-MB-231 cells; murine double minute 2 (MDM2), hypoxia-inducible factor 1 subunit alpha (Hif1α), and microtubule-associated protein 1 A/1B-light chain 3 (LC3) were included as markers for treatment effects. B Global protein synthesis level with EI24 OE by adenovirus in MDA-MB-231 cells. C Integration of pS6 to ribosome upon EI24 OE by adenovirus in MDA-MB-231 cells examined by Co-IP. D ASS1 mRNA distribution analyzed by qPCR following polysome fractionation in MDA-MB-231 with EI24 OE by doxycycline (upper) and Hs 578T with EI24 OE by adenovirus (lower). E Phosphorylation of AKT upon time-dependent deprivation of arginine in MDA-MB-231 with EI24 OE by doxycycline. F Phosphorylation of AKT upon time-dependent deprivation of arginine in Hs 578T with knockdown of EI24 by siRNA. G ASS1 expression and phosphorylation of AKT upon arginine deprivation and/or PI3K inhibitors LY294002 (LY, 10 µM) and wortmannin (Wort, 50 µM) in 24 h in MDA-MB-231 with EI24 OE by doxycycline

Article Snippet: Primary antibodies were purchased from Cell Signaling Technology for EI24 (#42328), ASS1 (#70720), ARG2 (#55003), eNOS (#32027), pAKT (Ser473) (#9271), AKT (#9272), MDM2 (#86934), pP70S6K (#9204), P70S6K (#9202), pS6 (#2211), LC3 (#4108), and p21 (#2947); from Santa Cruz for ODC1 (sc-390366), ASL (sc-374353), and β-actin (sc-69879); from Novus for HIF1α (NB100-449); and from Sigma-Aldrich for puromycin (MABE343).

Techniques: Expressing, Co-Immunoprecipitation Assay, Fractionation, Phospho-proteomics, Knockdown

Journal: Cellular & molecular biology letters

Article Title: Etoposide-induced protein 2.4 homolog promotes argininosuccinate synthase 1 and cancer cell survival upon arginine deprivation.

doi: 10.1186/s11658-025-00726-6

Figure Lengend Snippet: Fig. 3 Activation of PI3K/AKT-regulating ASS1 translation is through nitric oxide (NO)-regulated nitrosylation of PTEN. A Relative nitrite amount in MDA-MB-231 cell lysis upon arginine deprivation with EI24 OE by adenovirus. B Expression of ASS1 and pAKT upon arginine deprivation and/or H2O2 and NO donor (deta-NO) treatment in MDA-MB-231 with EI24 OE by adenovirus (NAC, N-acetylcysteine). C ASS1 mRNA distribution in polysome fractions upon NO donor (deta-NO) treatment in MDA-MB-231. D. Effect of L-NAME on ASS1 and pAKT in MDA-MB-231 with EI24 OE by adenovirus. E Cell viability upon arginine deprivation and L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. F ASS1 mRNA distribution in polysome fractions upon L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. G S-nitrosylation of PTEN upon arginine deprivation and L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. H Model of EI24-regulated translation of ASS1 via nitrosylation of PTEN

Article Snippet: Primary antibodies were purchased from Cell Signaling Technology for EI24 (#42328), ASS1 (#70720), ARG2 (#55003), eNOS (#32027), pAKT (Ser473) (#9271), AKT (#9272), MDM2 (#86934), pP70S6K (#9204), P70S6K (#9202), pS6 (#2211), LC3 (#4108), and p21 (#2947); from Santa Cruz for ODC1 (sc-390366), ASL (sc-374353), and β-actin (sc-69879); from Novus for HIF1α (NB100-449); and from Sigma-Aldrich for puromycin (MABE343).

Techniques: Activation Assay, Lysis, Expressing

Journal: Cellular & molecular biology letters

Article Title: Etoposide-induced protein 2.4 homolog promotes argininosuccinate synthase 1 and cancer cell survival upon arginine deprivation.

doi: 10.1186/s11658-025-00726-6

Figure Lengend Snippet: Fig. 4 EI24 enhanced ASS1 expression in various ASS1-deficient cancers, independent of p53 as a transcription factor for ASS1. A Expression of ASS1 in p53-null H1299 upon transfection of wildtype or point-mutated p53 plasmids; p21 and MDM2 were included as positive control for p53 transfection. B Expression of ASS1 upon arginine deprivation in p53-mutated MDA-MB-231 with wildtype p53 plasmid transfection and EI24 OE by adenovirus. C mRNA expression of urea cycle-related enzymes upon arginine deprivation and/or 10074-G5 (G5) treatment in 24 h in MDA-MB-231. D Expression of ASS1 upon arginine deprivation and/or 10074-G5 (G5) treatment in 24 h in MDA-MB-231. E ASS1 expression in a subset of renal cell carcinoma (RCC) and lung adenocarcinoma (LUAD) cells. F Cell viability upon time-dependent arginine deprivation in Caki-1 and A549 with EI24 OE by adenovirus. G Expression of ASS1 and pAKT upon arginine deprivation for 24 h in Caki-1 and A549 with EI24 OE by adenovirus. H Prognostic value of EI24, and ASS1 in breast cancer determined by survival analysis on GENT2

Article Snippet: Primary antibodies were purchased from Cell Signaling Technology for EI24 (#42328), ASS1 (#70720), ARG2 (#55003), eNOS (#32027), pAKT (Ser473) (#9271), AKT (#9272), MDM2 (#86934), pP70S6K (#9204), P70S6K (#9202), pS6 (#2211), LC3 (#4108), and p21 (#2947); from Santa Cruz for ODC1 (sc-390366), ASL (sc-374353), and β-actin (sc-69879); from Novus for HIF1α (NB100-449); and from Sigma-Aldrich for puromycin (MABE343).

Techniques: Expressing, Transfection, Positive Control, Plasmid Preparation