Journal: bioRxiv
Article Title: A PERK-FOXO1 AXIS LINKS DNA DAMAGE TO FIBROBLAST SURVIVAL IN DIFFUSE CUTANEOUS SYSTEMIC SCLEROSIS
doi: 10.64898/2026.02.17.706443
Figure Lengend Snippet: A) We reanalyzed spatial RNA sequencing data from our recent publication and focused on transcripts that are altered in the unfolded protein response. B) Relative PERK phosphorylation (activation) and downstream ATF4 translation C ) was determined using IB and FB from independent biological replicates of HC (n=4), dcSSc (n=5), or post-ASCT (n=4), then plotted the relative frequency of pPERK/PERK or ATF4 levels normalized to beta-actin. D ) FOXO1 mRNA levels were determined via qRT-PCR in FB from independent biological replicates of HC (n=5), dcSSc (n=7) and post-ASCT (n=5). E) Representative fluorescence microscope image (60x objective) of FOXO1 (green) and nucleus (DAPI, blue). FOXO1 activation was determined by either quantifying the relative fluorescence intensity of nuclear FOXO1 in independent biological replicates of primary FB from HC (n=6), dcSSc (n=7), and post-ASCT (n=5); F) or by quantifying the abundance of FOXO1/p-Ser257-FOXO1 (normalized to tubulin as a loading control) in independent biological replicates of primary FB from HC (n=6), dcSSc (n=8), and post-ASCT (n=6). G) We confirmed FOXO1 activation by quantifying the mRNA levels of its downstream target SOD2 in independent biological replicates of primary FB from HC (n=5), dcSSc (n=8) and post-ASCT (n=6). All data were presented as the means ± SEM. Statistical significance was determined by one -way ANOVA. Using GraphPad Prism, statistical significance was determined when *p<0.05, **p<0.005 and ***p<0.0005.
Article Snippet: The Taqman probes (Thermo Fisher Scientific) used for qRT-PCR (with targets) included: Hs02596861_s1 (D-loop), Hs02596876_g1 (ND4), Hs02596867_s1 (CytB), Hs00231106_m1 (FOXO1), Hs02758991_g1 (GAPDH), Hs00188166_m1 (Succinate dehydrogenase or SDHA), Hs00167309_m1 (SOD2), Hs01595220_g1 (COX7C), Hs00176875_m1 (PDK4), Hs01561847_m1 (PDK1), Hs00176865_m1 (PDK2).
Techniques: RNA Sequencing, Phospho-proteomics, Activation Assay, Quantitative RT-PCR, Fluorescence, Microscopy, Control