Journal: Cell Reports Medicine

Article Title: Multi-omics profiling unveils biological and clinical insights into pulmonary sarcomatoid carcinoma

doi: 10.1016/j.xcrm.2026.102887

Figure Lengend Snippet: Derepression of ferroptosis decreases cell proliferation in PSC (A) Western blot showing the knockdown efficiency of SLC3A2 and FTL in PLA-801D cells. (B) The cell proliferation of PLA-801D cells measured using CCK-8 assays following the knockdown of SLC3A2 (left) and FTL (right), respectively. (C) The cell proliferation of PLA-801D cells measured using colony formation assays following the knockdown of SLC3A2 (upper) and FTL (bottom), respectively. Following the knockdown of SLC3A2 or FTL , PLA-801D cells were plated and cultured for 14 days. All colonies were subsequently stained with crystal violet and quantified using ImageJ software. (D) The extracellular GSH concentration of PLA-801D cells was tested following the knockdown of SLC3A2 . (E) Following the knockdown of FTL , PLA-801D cells were stained by FerroOrange and quantified using the microplate reader, respectively. (F) The lysates of PLA-801D cells were immunoblotted with the indicated antibodies following the knockdown of FTL . (G) Following the knockdown of SLC3A2 , PLA-801D cells were treated with 10 μM ferrostatin-1 for 24 h. Afterward, the extracellular GSH concentration was tested. (H) Following the knockdown of FTL , PLA-801D cells were treated with 10 μM ferrostatin-1 for 24 h. Then the lysates of PLA-801D cells were immunoblotted with the indicated antibodies. (I) Following the knockdown of SLC3A2 (left) or FTL (right), PLA-801D cells were treated with DMSO or 10 μM ferrostatin-1 for 24 h. Then the cell proliferation of PLA-801D cells was detected by CCK-8 assays, respectively. (J and K) Following the knockdown of SLC3A2 (left) or FTL (right), PLA-801D cells were treated with DMSO or 10 μM ferrostatin-1 for 24 h. Then the fluorescent probes were added into the culture medium to test the relative level of ROS, respectively (J); the cell lysates were collected for bicinchoninic acid assay and the detection of lipid peroxidation MDA (K). (L) The graphic model of the repressed ferroptosis in PSC. NC, negative control; ns, not significant; ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001.

Article Snippet: Cells were lysed with 1% Triton X-100 buffer (Beyotime, ST1723) and then boiled in 5× loading buffer for 10 min. Each sample was electrophoresed on 10% PAGE gels, then transferred to PVDF membranes and immunoblotted with the antibodies targeting SLC3A2 (Abclonal, A24375), FTL (Abclonal, A11241), GPX4 (Abclonal, A1933) and β-Actin (Abclonal, AC004).

Techniques: Western Blot, Knockdown, CCK-8 Assay, Cell Culture, Staining, Software, Concentration Assay, Acid Assay, Negative Control

Journal: Journal of Animal Science and Biotechnology

Article Title: Rotavirus infection induces ferroptosis through NCOA4-mediated ferritinophagy in IPEC-J2 cells

doi: 10.1186/s40104-026-01457-0

Figure Lengend Snippet: NCOA4 knockdown attenuates rotavirus (RV)-induced ferritinophagy and viral replication in IPEC-J2 cells. IPEC-J2 cells were pretreated with control siRNA or NCOA4 siRNA before being infected with RV at 10 MOI. Cells were co-incubated with RV at 37 °C for 1 h, washed with PBS to remove unbound virus, and then incubated for another 24 h in fresh medium. A and B NCOA4 protein expression and quantification at different time points after RV infection. C and D Representative Western blot images and quantification of FTH1, FTL, and the ratio of LC3-II to LC3-I at different time points after RV infection. E qRT-PCR analysis of NCOA4 mRNA levels after transfection with three different NCOA4 siRNAs. F and G Western blot validation and quantification of NCOA4 knockdown efficiency. H Cell viability of IPEC-J2 cells after transfection with different NCOA4 siRNAs. I Effect of NCOA4 knockdown on cell viability in RV-infected IPEC-J2 cells. J Relative mRNA expression of VP6 detected by qRT-PCR. K and L Representative immunofluorescence images and quantification of VP6 protein. M and N Representative FerroOrange fluorescence images and quantification of intracellular Fe 2+ levels. O and P Representative DCFH-DA fluorescence images and quantification of intracellular ROS levels. Q and R Representative immunofluorescence images and quantification of NCOA4 and LC3 colocalization. S and T Representative Western blot images and quantification of NCOA4, FTH1, FTL, the ratio of LC3-II to LC3-I, and VP6 protein levels. Data are presented as the mean ± SEM from at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Scale bar = 50 μm

Article Snippet: Then, 5 × SDS loading buffer (Beyotime, China) was added at a ratio of 1:4 and mixed, and denatured at 98 °C for 10 min. Then, protein samples, after separation via polyacrylamide gel electrophoresis (PAGE), were transferred onto a polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% skim milk powder (Beyotime, China), incubated with primary antibodies (VP6, Cat. No. ab181695, Abcam, UK; NCOA4, Cat. No. sc-373739, Santa Cruz, USA; FTH1, Cat. No. R23306 , Zenbio, China; FTL, Cat. No. A18051, ABclonal, China; LC3, Cat. No. 381,544, Zenbio, China; TFRC, Cat. No. 66180-1-Ig, Proteintech, USA; FPN, Cat. No. R23306 , Zenbio, China; HMOX1, Cat. No. R24541 , Zenbio, China; β-ACTIN, Cat. No. 380,624, Zenbio, China) at 4 °C overnight, and then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Knockdown, Control, Infection, Incubation, Virus, Expressing, Western Blot, Quantitative RT-PCR, Transfection, Biomarker Discovery, Immunofluorescence, Fluorescence

Journal: Journal of Animal Science and Biotechnology

Article Title: Rotavirus infection induces ferroptosis through NCOA4-mediated ferritinophagy in IPEC-J2 cells

doi: 10.1186/s40104-026-01457-0

Figure Lengend Snippet: NCOA4 overexpression promotes ferroptosis and enhances rotavirus replication in IPEC-J2 cells. IPEC-J2 cells were pretreated with control vector or NCOA4-overexpression plasmid before being infected with RV at 10 MOI. Cells were co-incubated with RV at 37 °C for 1 h, washed with PBS to remove unbound virus, and then incubated for another 24 h in fresh medium. A Relative mRNA expression of NCOA4 after NCOA4 overexpression. B and C Representative Western blot images and quantification of NCOA4 protein expression after NCOA4 overexpression. D Effect of NCOA4 overexpression on the viability of non-infected IPEC-J2 cells. E Effect of NCOA4 overexpression on cell viability in RV-infected IPEC-J2 cells. F Relative mRNA expression of VP6 detected by qRT-PCR. G and H Representative immunofluorescence images and quantification of VP6 protein expression. I and J Representative FerroOrange fluorescence images and quantification of intracellular Fe 2+ levels. K and L Representative DCFH-DA fluorescence images and quantification of intracellular ROS levels. M and N Representative immunofluorescence images and quantification showing the colocalization of NCOA4 and LC3. O and P Representative Western blot images and quantification of NCOA4, FTH1, FTL, VP6, LC3-I, and LC3-II protein levels. Data are presented as the mean ± SEM from at least three independent experiments. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001; ns, not significant. Scale bar = 50 μm

Article Snippet: Then, 5 × SDS loading buffer (Beyotime, China) was added at a ratio of 1:4 and mixed, and denatured at 98 °C for 10 min. Then, protein samples, after separation via polyacrylamide gel electrophoresis (PAGE), were transferred onto a polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% skim milk powder (Beyotime, China), incubated with primary antibodies (VP6, Cat. No. ab181695, Abcam, UK; NCOA4, Cat. No. sc-373739, Santa Cruz, USA; FTH1, Cat. No. R23306 , Zenbio, China; FTL, Cat. No. A18051, ABclonal, China; LC3, Cat. No. 381,544, Zenbio, China; TFRC, Cat. No. 66180-1-Ig, Proteintech, USA; FPN, Cat. No. R23306 , Zenbio, China; HMOX1, Cat. No. R24541 , Zenbio, China; β-ACTIN, Cat. No. 380,624, Zenbio, China) at 4 °C overnight, and then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Over Expression, Control, Plasmid Preparation, Infection, Incubation, Virus, Expressing, Western Blot, Quantitative RT-PCR, Immunofluorescence, Fluorescence

Journal: Journal of Animal Science and Biotechnology

Article Title: Rotavirus infection induces ferroptosis through NCOA4-mediated ferritinophagy in IPEC-J2 cells

doi: 10.1186/s40104-026-01457-0

Figure Lengend Snippet: Inhibition of the autophagy–lysosome pathway attenuates RV-induced ferroptosis and viral replication in IPEC-J2 cells. IPEC-J2 cells were pretreated with 3-methyladenine (3-MA) or chloroquine (CQ) before being infected with RV at 10 MOI. Cells were co-incubated with RV at 37 °C for 1 h, washed with PBS to remove unbound virus, and then incubated for another 24 h in fresh medium containing the corresponding compounds. A and B Cell viability of IPEC-J2 cells treated with different concentrations of CQ or 3-MA. C Effects of 3-MA and CQ treatment on cell viability in RV-infected IPEC-J2 cells. D Relative mRNA expression of VP6 detected by qRT-PCR. E and F Representative immunofluorescence images and quantification of VP6 protein expression. G and H Representative FerroOrange fluorescence images and quantification of intracellular Fe 2+ levels. I and J Representative DCFH-DA fluorescence images and quantification of intracellular ROS levels. K and L Representative immunofluorescence images and quantification of NCOA4 and LC3 fluorescence signals. M and N Representative Western blot images and quantification of FTH1, FTL, LC3-II, and VP6 protein levels. Data are presented as the mean ± SEM from at least three independent experiments. * P < 0.05, ** P < 0.01, **** P < 0.0001; ns, not significant. Scale bar = 50 μm

Article Snippet: Then, 5 × SDS loading buffer (Beyotime, China) was added at a ratio of 1:4 and mixed, and denatured at 98 °C for 10 min. Then, protein samples, after separation via polyacrylamide gel electrophoresis (PAGE), were transferred onto a polyvinylidene fluoride (PVDF) membranes, which were blocked with 5% skim milk powder (Beyotime, China), incubated with primary antibodies (VP6, Cat. No. ab181695, Abcam, UK; NCOA4, Cat. No. sc-373739, Santa Cruz, USA; FTH1, Cat. No. R23306 , Zenbio, China; FTL, Cat. No. A18051, ABclonal, China; LC3, Cat. No. 381,544, Zenbio, China; TFRC, Cat. No. 66180-1-Ig, Proteintech, USA; FPN, Cat. No. R23306 , Zenbio, China; HMOX1, Cat. No. R24541 , Zenbio, China; β-ACTIN, Cat. No. 380,624, Zenbio, China) at 4 °C overnight, and then incubated with corresponding horseradish peroxidase (HRP)-conjugated secondary antibodies.

Techniques: Inhibition, Infection, Incubation, Virus, Expressing, Quantitative RT-PCR, Immunofluorescence, Fluorescence, Western Blot