Journal: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: HSPA8 is a target of LACTB. A. qRT-PCR testing the effects of LACTB overexpression and knockout on HSPA8 mRNA levels. B. Western blot testing the indicated protein levels after LACTB overexpression in SK-HEP-1 cells. C. Western blot testing the indicated protein levels in LACTB −/− cells reexpressing LACTB. D. Western blot testing the indicated protein levels in LACTB-expressing cells transfected with HSPA8-expressing plasmid. E. Western blot testing the indicated protein levels in LACTB −/− cells transfected with si-HSPA8. F–I. Detection of effects of LACTB overexpression or in combination with HSPA8 overexpression on Fe 2+ , lipid peroxidation, MDA levels and GSH/GSSG ratio in cells. Scale bar = 25 μm. J,K. Detection of effects of LACTB knockout or in combination with HSPA8 silencing on Fe 2+ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells. Scale bar = 25 μm. L. Kaplan-Meier plotter database showing the overall survival rate of liver cancer patients with high or low HSPA8 expression. M. Xenograft tumour model testing the in vivo effects of LACTB or LACTB + HSPA8 on SK-HEP-1 cell growth. Scale bar = 1 cm. Student's t -test was used for A (left panel), one-way ANOVA with Tukey post-hoc test was used for A (right panel), F–K, M (right panel), and two-way ANOVA with Tukey post-hoc test was used for M (middle panel).

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Quantitative RT-PCR, Over Expression, Knock-Out, Western Blot, Expressing, Transfection, Plasmid Preparation, In Vivo

Journal: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: LACTB drives ferroptosis via regulating the p53/HSPA8 axis. A. qRT-PCR testing HSPA8 mRNA levels in LACTB-expressing Huh7 and Hep3B cells. B, C. qRT-PCR and Western blot testing HSPA8 mRNA and protein levels in LACTB-expressing cells transfected with si-p53. D. Western blot testing p53 protein expression in LACTB-expressing SK-HEP-1 cells treated with 100 μg/mL cycloheximide for the indicated time. E. Western blot testing p53 ubiquitination levels in LACTB-expressing SK-HEP-1 cells. F. Western blot testing HSPA8 protein expression in LACTB-expressing p53 +/+ Hep3B cells. G. Luciferase reporter assay testing HSPA8 promoter activity in LACTB-expressing cells transfected with si-p53. H. p53 binding sites on HSPA8 promoter among different species. I. The schema showing the wild-type or mutant p53 binding motifs on HSPA8 or p21 promoter, and primer design positions for ChIP assay. J. ChIP assay testing the binding of p53 on the indicated regions of HSPA8 promoter. K. DNA pull-down assay using wild-type or mutant HSPA8 promoter probe, followed by Western blot analysis of p53 protein expression. L. Generation of SK-HEP-1 cells with endogenous mutation of p53 binding site in HSPA8 promoter using CRISPR/Cas9 gene editing technology with the indicated ssODN and gRNA. M. CCK-8 testing the viability of wild-type or mutant SK-HEP-1 cells treated with erastin and RSL3. N, O. Liperfluo and FerroOrange staining testing lipid peroxidation and Fe 2+ levels in wild-type or mutant SK-HEP-1 cells. Scale bar = 25 μm. P. Western blot testing HSPA8 protein levels in wild-type or mutant SK-HEP-1 cells with LACTB or p53 overexpression. Q. IHC staining of LACTB, wild-type (wt) p53 and HSPA8 in liver cancer tissue microarray, followed by analysis of their expression correlations. Scale bar = 50 μm. One-way ANOVA with Tukey post-hoc test was used for B and G. Student's t -test was used for J, N, O.

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Quantitative RT-PCR, Expressing, Western Blot, Transfection, Ubiquitin Proteomics, Luciferase, Reporter Assay, Activity Assay, Binding Assay, Mutagenesis, Pull Down Assay, CRISPR, CCK-8 Assay, Staining, Over Expression, Immunohistochemistry, Microarray

Journal: Redox Biology

Article Title: LACTB suppresses liver cancer progression through regulation of ferroptosis

doi: 10.1016/j.redox.2024.103270

Figure Lengend Snippet: LACTB potentiates the response of liver cancer to lenvatinib. A, B. FerroOrange and liperfluo staining testing Fe 2+ and lipid peroxidation levels in HepG2 and SK-HEP-1 cells treated with lenvatinib, respectively. Scale bar = 25 μm. C. Western blot testing LACTB protein expression in HepG2 and SK-HEP-1 cells treated with lenvatinib. D-G. FerroOrange, liperfluo staining, CCK-8 and colony formation assays respectively testing Fe 2+ , lipid peroxidation levels, viability and cloning ability in lenvatinib-treated cells with LACTB overexpression or knockout. H, I. Xenograft tumour model testing the in vivo effects of LACTB overexpression or knockout on the anti-tumour effect of lenvatinib. Scale bar = 1 cm. J. Western blot testing the indicated protein expression in the indicated groups. K. The proposed model showing that LACTB induced by lenvatinib promotes ferroptosis by increasing p53 protein stability and inhibiting HSPA8-mediated anti -ferroptosis pathway. One-way ANOVA with Tukey post-hoc test was used for A, B, D-G, I.

Article Snippet: The sections were then incubated with anti -LACTB (#18195-1-AP, Proteintech), anti -p53 (wild-type) (#MABE339, Sigma-Aldrich), and anti -HSPA8 (#M01024-1, BOSTER) primary antibodies and a DAKO peroxidase-labelled secondary antibody.

Techniques: Staining, Western Blot, Expressing, CCK-8 Assay, Cloning, Over Expression, Knock-Out, In Vivo