Extended Data Fig. 4: Apoptosis contributes to NICDiOE-EC WAT loss in male mice.
From: Endothelial Notch1 signaling in white adipose tissue promotes cancer cachexia
a, Representative images of isolectin B4 whole mount stainings of NICDiOE-EC vWAT and sWAT at week 7 post-recombination. b,c, Isolectin B4+ endothelial coverage area was quantified (n = 5 animals per group). d, Enrichment plot of ‘Hallmark_apoptosis’ in cachectic compared to healthy vWAT (GSE131835). e, Adipose stromal cells (CD45-CD31-CD140+Sca1+) assessed in sWAT of KPC mice compared to non-tumour bearing controls for early apoptotic (Annexin V+PI−), late apoptotic (Annexin V+PI+) and necrotic (Annexin V−PI+) markers by flow cytometry (n = 5-6 animals per group). f–i, Quantified percentages of apoptotic progenitors (CD45-CD31−CD34+; f and g) and AT-ECs (CD45−CD31+; h and i) in vWAT (g and i) and sWAT (f and h) of NICDiOE-EC mice at week 7 analysed by flow cytometry (n = 6-8 animals per group). j, Gating strategy of CD45−CD31−CD34+ adipose progenitors to assess early apoptosis (Annexin V+PI−), late apoptosis (Annexin V+PI+) and necrosis (Annexin V−PI+) by flow cytometry. k, Western blots of cleaved caspase-3 from control and NICDiOE-EC vWAT and sWAT. l,m, Quantifications of vWAT (l) and sWAT (m) cleaved caspase-3 levels normalized to VCP (n = 3-4 animals per group). n, Enrichment plot of ‘Hallmark_adipogenesis’ in healthy vWAT compared to cachectic patient samples (GSE131835). o, Western blot analyses of whole vWAT and sWAT ATGL protein levels normalized to the housekeeping protein VCP (n = 4 animals per group). p,q, Ser565 and Ser660 HSL phosphorylation and total HSL were quantified from whole sWAT and vWAT by Western blot analysis and normalized to the housekeeping protein VCP. The ratios of pHSL:HSL in vWAT (p) and sWAT (q) of NICDiOE-EC mice at week 5 post-recombination were quantified (n = 4 animals per group). Data shown represent mean ± SEM, Mann –Whitney test (b, c) or unpaired two-sided t-test with Welch correction (e-q).
