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: PQ induces ferroptosis in lung epithelial cells. (A) A549 cells were treated with PQ (0–2000 μM) and cell viability was assessed. (B-C) A549 cells treated with PQ (1000 μM, 0–24 h) (B) or PQ (0–1000 μM,24 h) (C) and the lipid ROS were measured using C11-BODIPY staining. (D-E) The labile iron pool was evaluated via Calcein-AM staining in A549 cells treated with PQ (1000 μM) (D) for 24 h (E). Images were captured with InCucyte. Bar = 400 µm. (F) MDA content in A549 cells. (G) Iron content in A549 cells. (H) Mito-tracker staining was evaluated for mitochondrial morphological change in A549 cells following 24-hour PQ (1000 μM) treatment. Bar = 10 µm. (I) Fe 2+ levels were measured by FerroOrange staining in A549 cells after 24-hour PQ (1000 μM) treatment. Bar = 10 µm. (J, K, L) Cells were treated with PQ (1000 μM) for 24 h in the presence or absence of DFO (50 μM), DFP (150 μM), RSL3 (1 μM), FeCl 3 (10 μM), FeSO 4 (10 μM) or FAC (10 μM) pretreatment for 1 h, and the cell viability was determined by CCK-8 assay. (M) Effect of PQ on GPX4 and SLC7A11 expression. 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, **** P < 0.0001 show significant differences from each group .

Article Snippet: Antibodies for SQLE (#67206–1-Ig), GPX4 (#67763–1-Ig), solute carrier family 7 member 11 (SLC7A11, #26864–1-AP), and PERK (#20582–1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Staining, CCK-8 Assay, Expressing

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: ER stress mediates PQ-induced ferroptosis. (A-B) A549 cells were transfected with siRNA of PERK or NC (negative control) and then treated with PQ for 24 h. The protein expression (A) and the cell viability were determined (B). (C) Western blotting analysis of p-PERK, PERK, p-eIF2α, eIF2α, and CHOP in A549 cells pre-treated with 1 μM GSK157 for 1 h followed by PQ (1000 μM) treatment for 24 h. (D-E). A549 cells pre-treated with GSK157 (0.25, 0.5, 1 μM) for 1 h followed by PQ (1000 μM) treatment for 24 h, then ATP release (D) and CCK-8 assay (E) were determined. (F) Effects of GSK157 on PQ-induced lipid peroxidation. A549 Cells were pretreated with GSK157 for 1 h and then exposed to PQ for 24 h, followed by C11-BODIPY staining and determined by flow cytometry analysis. (G) Effects of GSK157 on PQ-induced iron content elevation. Cells were pretreated with GSK157 for 1 h and then exposed to PQ for 24 h, followed by calcein-AM staining and determined by flow cytometry analysis. (H) The staining of ER-Tracker (green) and FerroOrange (red) with Hoechst (blue) was photographed by the confocal microscope (Bar = 10 μm). (I) Iron content in A549 cells. (J) Effect of RSL3 on PQ-induced ER-stress. Cells were treated with PQ (1000 μM) for 24 h in the presence or absence of RSL3 (1 μM) and GSK157 (1 μM) pretreatment for 1 h. CCK-8 assay. (K) The expression levels of GPX4 and SLC7A11 were determined by Western blotting. 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. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies for SQLE (#67206–1-Ig), GPX4 (#67763–1-Ig), solute carrier family 7 member 11 (SLC7A11, #26864–1-AP), and PERK (#20582–1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Transfection, Negative Control, Expressing, Western Blot, CCK-8 Assay, Staining, Flow Cytometry, Microscopy

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 inhibition attenuates PQ-induced lung epithelial cell death by inhibiting ferroptosis. (A-C) A549 sgSQLE cells were treated with PQ (1000 μM). Confocal microscopy images show FerroOrange (red) and Hoechst (blue) staining (Bar = 10 μm) (A), GSH content was assessed (B), and Western blotting analysis evaluated GPX4 expression (C). (D) Cells were transfected with siRNA of SQLE or NC and then treated with PQ for 24 h. Western blotting analysis evaluates the expression of GPX4. (E-F) A549 sgSQLE cells were treated with PQ (1000 μM) for 24 h with or without pretreatment of RSL3 (1 μM) and FeCL3 (10 μM) for 1 h, measured by CCK-8. (G-I) A549-SQLE-WT and A549-SQLE-Y195F cells were treated with PQ (1000 μM) for 24 h with or without pretreatment of inhibitors (RSL3 1 μM, FeCL 3 10 μM, FeSO 4 10 μM) for 1 h, assessed by CCK-8. (J) HEK-293 T cells were transfected with the plasmid of SQLE-WT or SQLE-Y195F and then treated with PQ (1000 μM) for 24 h. Western blotting analysis evaluates the expression of SQLE and GPX4. 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. ns , no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies for SQLE (#67206–1-Ig), GPX4 (#67763–1-Ig), solute carrier family 7 member 11 (SLC7A11, #26864–1-AP), and PERK (#20582–1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Inhibition, Confocal Microscopy, Staining, Western Blot, Expressing, Transfection, CCK-8 Assay, Plasmid Preparation

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: LNT attenuates PQ-induced lung epithelial cell death via inhibiting ferroptosis. Cells were treated with PQ (1000 μM) for 30 mins and then post-treated with LNT for 24 h. followed by measurement C11-BODIPY in flow cytometry (A). Calcein-AM staining by captured with InCucyte. Bar = 400 µm (B) and measured by flow cytometry (C). (D) MDA content. (E) Mito tracker (green) with Hoechst (blue) was photographed by the confocal microscope. Bar = 10 μM. (F) FerroOrange (red) with Hoechst (blue) was photographed by the confocal microscope. Bar = 10 μM. (G) Iron content. (H-I) Cells were treated with PQ (1000 μM) for 30 mins in the presence or absence of inhibitors (RSL3, FeCL 3 , FeSO 4 , and FAC) for 1 h pretreatment, and then LNT (20 μM) posttreatment for 24 h in A549 cells. Cell viability was determined by CCK-8 assay. (J) Western blotting analysis of GPX4 and SLC7A11. 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. ns , no significance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Antibodies for SQLE (#67206–1-Ig), GPX4 (#67763–1-Ig), solute carrier family 7 member 11 (SLC7A11, #26864–1-AP), and PERK (#20582–1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Flow Cytometry, Staining, Microscopy, CCK-8 Assay, Western Blot

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: LNT alleviates PQ-induced ALI via inhibiting ferroptosis. (A) MDA in lung tissue. (B) MDA in BALF. (C) Iron content in lung tissue. (D) Prussian Blue staining of lung tissue and IHC sections of GPX4, SQLE, and SLC7A11 staining. Bar = 100 μM. (E) Western blotting analysis for SLC7A11 and GPX4. 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. ns , no significance.

Article Snippet: Antibodies for SQLE (#67206–1-Ig), GPX4 (#67763–1-Ig), solute carrier family 7 member 11 (SLC7A11, #26864–1-AP), and PERK (#20582–1-AP) were purchased from Proteintech Group, Inc. (Wuhan, China).

Techniques: Staining, Western Blot

Journal: European journal of histochemistry : EJH

Article Title: Autofluorescence in freshly isolated adult human liver sinusoidal cells.

doi: 10.4081/ejh.2021.3337

Figure Lengend Snippet: Figure 1. Autofluorescence in primary human LSECs. A) Conventional brightfield image of freshly isolated CD31+ LSECs in culture 17 h after plating; scale bar: 50 μm. B) Same area of cells imaged in a Nikon fluorescence microscope using brightfield (top), green filter (middle) and red filter (bottom); scale bar: 100 μm. C) A large proportion of non-treated CD31+ LSECs labelled only with DAPI shows autofluorescence in all four channels (blue, green, red, and far red) at normal laser intensity; scale bar: 10 μm; to watch a video of the autofluorescence through the entire spectrum of a Zeiss confocal microscope equipped with diode, Argon, DPSS and HeNe lasers, please visit Google drive at https://tinyurl.com/CD31autofluorescence. D) A large proportion of freshly isolated, non-treated, and light- ly fixed CD31+ cells show autofluorescence in the 12-channel layout using AMNIS® imaging flow cytometry; the image is a represen- tative view of 200 cells imaged.

Article Snippet: CD31+ liver cells, enriched in LSEC, were resuspended in AIM-VTM medium (cat.no: 12055091; Gibco, Amarillo, TX, USA) and plated in 8 well Ibidi μ-slides coated with quick coating solution (cat. no: cAP-01, Angio-Proteomie; Boston, MA, USA) at a density of 150,000-250,000 cells/cm2.

Techniques: Isolation, Fluorescence, Microscopy, Imaging, Flow Cytometry

Journal: European journal of histochemistry : EJH

Article Title: Autofluorescence in freshly isolated adult human liver sinusoidal cells.

doi: 10.4081/ejh.2021.3337

Figure Lengend Snippet: Figure 3. Autofluorescence and exogenous fluorophore detection in immunolabelled LSEC-marker expressing cells. A) CD31+ LSECs were cultured for 24 h, fixed, treated with TrueBlack™ for 2 min and immunolabelled using (left) mouse anti-human FcɣRII (green) and (right) goat anti-hMR (green) before being imaged using moderate laser intensity (0.5% laser gain, 700 mV amplitude); the inten- sity of the red laser was increased to 1% laser gain, 780 mV amplitude for the autofluorescence to reappear after TrueBlack-quenching (red); nuclei are stained with DAPI; scale bar: 20 μm. B) Lysosomal labelling with mouse anti-human LAMP-1 antibody (green) shows that the autofluorescent material (red) is located within LAMP-1 positive vesicles (arrow heads); cells were treated with TrueBlack™ for 1 min prior to immunolabelling to quench autofluorescence in the green channel while keeping some autofluorescence in the red channel; scale bar: 10 μm. C) TEM micrograph of a human LSEC displaying large perinuclear vacuoles (arrows) filled with material, displacing an indented (arrowheads) nucleus (N); scale bar: 2 μm.

Article Snippet: CD31+ liver cells, enriched in LSEC, were resuspended in AIM-VTM medium (cat.no: 12055091; Gibco, Amarillo, TX, USA) and plated in 8 well Ibidi μ-slides coated with quick coating solution (cat. no: cAP-01, Angio-Proteomie; Boston, MA, USA) at a density of 150,000-250,000 cells/cm2.

Techniques: Marker, Expressing, Cell Culture, Staining