Extended Data Fig. 6: THY1 signalling promote ROS-enriched mitochondria extrusion by neutrophils.
a, THY1+ Huh-7 cells were left untreated, co-cultured with neutrophils directly or in a transwell system. HIF1α protein level was analysed (n = 5). b,c, THY1+ Huh-7 cells were left untreated, treated with Co-CM or THY1-CM in the absence (b,c) or presence (c) of the proteasome inhibitor MG132. mRNA (b) and protein (c) level of HIF1α were analysed (each n = 5). d, Schematic overview of achieving different Co-CM fractions. e, Effects of blocking antibodies on mitochondrial proteins in the 18,000g pellet from Co-CM derived from cocultures of THY1+ Huh-7 cells and neutrophils (n = 5). f, Effects of THY1-Fc on mitochondrial proteins in the 18,000g pellet from neutrophil-conditioned medium (n = 5). g,h, Mitochondrial content (g) and mitochondrial membrane potential (h) in untreated or THY1-Fc-treated neutrophils were analysed (each n = 5). i, Schematic representation of isolating neutrophil mitochondria. j, Intracellular mitochondria were extracted from fresh, 12-hour-cultured untreated or THY1-Fc-treated neutrophils. Mitochondrial membrane potential of these mitochondria was analysed (n = 5). k, Selection of stable mitochondrial content indicator genes for Fig. 4n, o. The selected genes are highlighted in red, with variation coefficient less than 1.0 in both blood and tumour samples from patients with HCC. l, Gating strategies for FACS of neutrophils from HCC tumours. m, Mitochondrial content in neutrophils from HCC tumour and paired blood was analysed in the original HCC cohort by flow cytometry (n = 17 patients). Data are from five (a,c,e,f,j) independent experiments. Data are presented as mean ± s.e.m. of five independent experiments (b,g,h,m). Statistical significance was determined using unpaired two-tailed Student’s t-test (g,h), paired two-tailed Student’s t-test (m), or one-way ANOVA with Tukey’s post hoc test (b). Panels d,i and l created with BioRender.com.
