Fig 1: Downregulation of Atg5 impeded the hepatic differentiation of HPCs in vitro. a, b Expression of Atg5 mRNA and protein was detected by RT-PCR and WB (*P < 0.05 compared with scramble group, data are the mean ± SD, repeated in three experiments, n = 3 per experiment). c, d Relative expression of autophagy-related protein was detected by WB (*P < 0.05 compared with scramble group, data are the mean ± SD, repeated in three experiments, n = 3 per experiment). e Representative images of autophagy of HPCs transfected with Ad-mCherry-GFP-LC3B adenovirus in different groups (scale bar = 50 µm, n = 3). f, g Autophagosomes (arrowheads) were observed under electron microscopy (*P < 0.05 compared with scramble group, data are the mean ± SD, scale bar = 0.5 µm, n = 3). h, i WB analysis showed expression of HPC (CK19 and SOX9)- and hepatocyte (ALB and HNF4a)-related proteins in different groups (*P < 0.05 compared with scramble group, data are the mean ± SD, repeated in three experiments, n = 3 per experiment). j Immunofluorescence staining for CK19, ALB and HNF4a was performed in different groups; endogenous expression and distribution of ALB, CK19 and HNF4a were observed under a confocal microscope (scale bar = 25 µm, n = 3). k, l ICG uptake assay. HPCs with a green-stained nucleus were considered positive, which indicated differentiated HPCs (*P < 0.05 compared with scramble group, data are the mean ± SD, scale bar = 100 µm, n = 3). m, n ALB and urea production in the supernatant were detected by ELISA kits in different groups (*P < 0.05 compared with scramble group, n = 3)
Fig 2: PCR and western blot analysis results of ALB, ANGPTL7, BLOC1S5-TXNDC5, IL6, and NGB. (A) ALB was down-regulated in the BC tumor tissues. (B) ANGPTL7 was down-regulated in the BC tumor tissues. (C) BLOC1S5-TXNDC5 was up-regulated in the BC tumor tissues. (D) IL6 was down-regulated in the BC tumor tissues. (E) NGB was down-regulated in the BC tumor tissues. (F) Representative western blot images of ALB, ANGPTL7, BLOC1S5-TXNDC5, IL6, and NGB expressions in MCF-10A, MCF-7, and MDA-MB-231 cells.
Fig 3: HNF4a induction resulted in improved hepatocytic differentiation of Hep-3B cells after CLA treatment. (a) Immunofluorescence staining revealed the remarkable expression of ALB in the CLA treated Hep-3B cells compared to the BIM treated and control group. (b) CLA treatment increased ALB secretion from Hep-3Bcells. (c) The relative mRNA expression of the ALB gene was increased after treatment with CLA in Hep-3B cells compared to the control group. (d) PAS staining showed glycogen accumulation in CLA treated cells as compared to BIM treated group. PAS: periodic acid-Schiff staining. Data are presented as the mean ± SD, n = 3 (*p < 0.05 and **p < 0.01).
Fig 4: Glibenclamide enters EAE lesions in the spinal cord. a–c Sections of lumbar spinal cords from control (CTR) and EAE mice administered bodipy-glibenclamide, immunolabeled for MBP (a) or albumin (b), using an Alexa Fluor 488-conjugated secondary antibody (green signal, with signal shown as white for clarity), or imaged for intrinsic fluorescence due to bodipy (red) (c); nuclei labeled with DAPI (blue); scale bar 100 µm. d Quantification of bodipy signal in control (non-EAE) and in albumin-negative (ALB–) and albumin-positive (ALB+) regions of interest (ROI) from EAE; **P < 0.01 with respect to control (CTR). e–g Sections of lumbar spinal cords from EAE mice administered bodipy-glibenclamide, stained with H&E, illustrating a demyelinating lesion at low (e) and higher f magnification, and a non-demyelinating lesion with immune cell infiltrates (g), along with the associated intrinsic fluorescence due to bodipy (red); scale bars 200 µm. h Double immunolabeling of demyelinating lesion (3 left panels) and control tissue (right panel) for CD31 (endothelium) (green) and SUR1 (red) illustrates that SUR1 is not upregulated in endothelium but predominantly in perivascular structures typical of astrocytic endfeet; merged images are indicated; scale bars 25 µm
Fig 5: Expression of 19 ferroptosis-related DEGs in BC and their effects on the survival of BC patients. (A) Hazard ratio of the 19 genes by univariate Cox regression analysis. (B) Expression of AKR1C2 between BC and normal samples. (C) Expression of ALB between BC and normal samples. (D) Expression of IL6 between BC and normal samples. (E) Expression of TFR2 between BC and normal samples. (F) Kaplan–Meier survival curves for patients classified into high and low AKR1C2 expression groups (log-rank, P = 0.042). (G) Kaplan–Meier survival curves for patients classified into high and low ALB expression groups (log-rank, P = 0.038). (H) Kaplan–Meier survival curves for patients classified into high and low IL6 expression groups (log-rank, P = 0.013). (I) Kaplan–Meier survival curves for patients classified into high and low TFR2 expression groups (log-rank, P = 0.0019).
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