Fig 2: Differential baseline expression of genes of the autophagy pathway in adult, pediatric, and fetal OL-lineage cells.a Relative expression of lysosome formation genes of the autophagy pathway in total OL lineage cells from adult, pediatric, and fetal samples. Scale is z-scores of averaged normalized gene expression across the age groups; percent expressed indicates the percentage of OL lineage cells in the age group expressing the gene. Arrows indicate up- (orange) or down- (blue) regulation (adj. p < 0.05) against all other groups where one arrow is |logFC | > 0.1, two arrows |logFC | > 0.25, three arrows |logFC | > 0.99, by Wilcoxon rank sum differential expression testing with Bonferroni correction. Number of independent biological samples by age group: 4 adult, 7 pediatric and 4 fetal. b–e Relative expression of genes involved in nucleation of autophagosomes (b), elongation of autophagosomes (c), fusion of autophagosomes and lysosomes (d) and lysosome formation (e) in optimal conditions (N1) and low glucose (LG) in adult OLs. mRNA expression was measured by microarray analysis with N = 3 independent biological samples from adult donors. Mean ± SEM for each condition in the figure. Statistical significance was verified by Student’s t-test: *(<0.05), **(<0.01), ***(<0.001). SQSTM1 encodes for p62 and MAP1LC3B encodes for LC3-II.

Fig 3: Effect of luteolin treatment on doxorubicin-induced change in cardiomyocyte mitochondrial autophagy. (A) Representative Western gel blots depicting protein levels of mitochondrial autophagy, including LC3B and P62 in cardiomyocytes after doxorubicin and luteolin treatment. (B) LC3B; (C) P62; (D) mitochondrial LC3B; (E) Representative fluorescence images of LC3B co-localized with mitochondria (COXIV) from 9 to 10 fields per group. (F) Quantitative analysis of LC3 dots co-localized with mitochondria. (G) Representative Western gel blots of pink1, parkin and Bnip3; (H) pink1; (I) parkin; (J) Bnip3. Scale bars = 25 µm. Mean ± SEM, n = 3 independent experiment. * p < 0.05 vs. CON group, # p < 0.05 vs. DOX group.

Fig 4: Nox4 promotes survival of CAFs via an up regulation of Nrf2 pathway. (A) Western blot analysis showing expression levels of Nrf2 and KEAP1 in total lysates of CAFs versus RMFs. Relative signal intensity of Keap1 (normalized to the actin control) was shown in numerical numbers. (B) Total and phosphorylated (Ser349) levels of p62 in CAFs. (C) Levels of p62 in CAFs after 24 h of siNox4 transfection. (D) Starvation induced autophagy resulted in an increase in p62 accumulation in CAFs versus RMF. Fibroblasts were nutrient starved (HBSS buffer) for 6 h with or without treatment with the autophagy inhibitor, bafilomycin A1 (Baf; 100 nM). (E)Targeting Nrf2 impaired viability of CAFs compared to RMF. Confluent fibroblasts were treated with varying concentrations of brusatol and viability of cells were determined after 48 h of treatment. * represents p < 0.05 vs untreated CAFs, # represents p < 0.01 vs untreated CAFs. (F) Survival of CAFs after 48 h of siNox4 transfection in the presence or absence of DMF (30 uM). (G) Viability of CAFs after 48 h of sip62 transfection. # represents p < 0.01 vs untreated CAFs. (H) Oncomine microarray analysis of p62 mRNA expression in breast stroma vs normal stroma in the Finak et al. dataset. 1: Normal stroma, N = 6; 2: Tumor stroma, N = 53. (I) Inhibition of Nrf2 with brusatol induced caspase-3 activation in CAFs. T-Casp3 = total caspase-3; C-casp3 = cleaved caspase-3. (J) Bar graph—Relative mRNA expression of Birc5 in CAFs after 48 h of siNrf2 transfection. N = 3, * represents p < 0.05 vs siCon transfected CAFs. Right panel -Western blot analysis of Birc5 expression after 48 h of siNrf2 transfection. (K) Suppressing the expression of Keap1, a negative regulator of Nrf2, also resulted in an induction of Birc5 protein expression in CAFs. (L) Chemical induction of Nrf2 with DMF (ug) in RMF (left panel) or upstream overexpression of Nox4 with doxycycline (ng/mL) in the inducible RMF (iRMF.Nox4) (right panel) both increased the expression levels of Birc5. (M) Knocking down expression of Nox4 in CAFs inhibited expression of Birc5 in CAFs. Viability of CAFs when Birc5 was targeted either with 200 nM of YM155 treatment (N) or with siBirc5 transfection (O), for 48 h. N = 3, * represents p < 0.05 vs DMSO treated CAFs. (P) Collagen contraction activity of CAFs after 48 h of siBirc5 transfection. (Q) RT-PCR analysis of CAF markers, FAP and aSMA after 48 h of YM155 treatment. (R) Western blot analysis of Birc5 expression in primary breast CAFs vs. RMFs. All Western blots are representative of n = 3 separate experiments. Error bars are SD of n = 3

Fig 5: Downregulation of circ-ADAM9 in EPCs promotes angiogenesis in hind limbs of diabetic mice.a Non-diabetic and diabetic nude mice with hind limb ischemia were injected with human umbilical vein EPCs transfected with lentivirus sh-circ-ADAM9or sh-NC. Blood flow in hind limbs was measured using a laser Doppler imaging system on days 0, 4, 7, and 14 after injection. Representative images and blood flow ratios are shown. b CD31-specific immunofluorescence staining (red) in ischemic muscle tissues of nude mice 14 days after injection of the cells. c Western blot analysis of BCL-2, BAX, cleaved-CASP3, LC3B-II/LC3B-II, and SQSTM1/p62 levels. d Circ-ADAM9 promotes the expression of PTEN and decreases the phosphorylation of AKT/mTOR, and circ-ADAM9 directly induces the expression of the autophagy-related gene ATG7, leading to the enhancement of autophagy and apoptosis of EPCs in diabetes. *P < 0.05, **P < 0.01; n = 6.