Fig 1: Injured Young Keratinocytes Secrete SDF1 to Promote Scar Formation(A) A circulating factor promotes scar formation in aged mice. Shown is a schematic of parabiosis pairs, photographs, H&E staining, and trichrome staining. The trichome images are taken from the red squares. Dotted areas mark scars. The black arrow marks chondrocyte proliferation. Horizontal lines indicate the distance between cartilage end plates. n = 5.(B) Ear hole measurements of individual parabionts. n = 5. ***p < 0.001, comparing aged: aged with young: young or either parabiont of young: aged.(C) Mice exhibit age-dependent SDF1 induction in ear and back wounds. Shown is the relative SDF1 transcript in ear wound edge tissue of young or aged WT mice. n = 24. *p < 0.03.(D) Relative SDF1 transcript in back skin wound edge tissue of young or aged WT mice. n = 6. **p = 0.004.(E) Ear injury induces SDF1 blood serum levels in young but not aged mice. Shown are SDF1 blood serum levels at baseline and 1 week after ear punch injury. n = 6. **p = 0.002.(F) Ear hole injury induces SDF1 expression in injured keratinocytes. Shown is SDF1 (green) immunostaining of ears from young and aged mice. Dotted lines identify the epidermal-dermal border. The hole is located to the right of the section. n = 6.(G) Quantification of SDF1 immunostaining. n = 6. **p < 0.01.(H) Relative SDF1 transcript in fluorescence-activated cell sorting (FACS)-isolated keratinocytes and dermally based cells in young mice. n = 6. ***p < 0.001.N, biological replicates per group. Error bars are SEM. Scale bars, 100 μM (histology) and 2 mm (photographs). Nuclei are counterstained blue. See also Figure S2.
Fig 2: Mouse and Human Skin Exhibit Age-Dependent EZH2-Mediated SDF1 Induction(A) Age-dependent epigenetic regulation of SDF1. Shown are H3K27me3, H3K4me3, and EZH2 chromatin immune precipitation of ear wound edge tissue at baseline and 1 week post-injury at 3 different locations on the SDF1 gene. n = 6. *p < 0.05.(B–E) Injury-induced EZH2 increases with age. Shown are EZH2 transcript (B, n = 24), protein (C, n = 4), and immune staining (D, brown cells, black asterisks mark background staining, n = 4)(E) immunostaining quantification in ear wound edge tissue at baseline (BL) and 1 week post-injury. *p < 0.01.(F) Pharmacologic inhibition of EZH2 (DZNep) in aged mice rescues SDF1 induction and blocks tissue regeneration. Shown are SDF1 blood serum levels and ear hole measurements. n = 3. ***p = 0.00065.(G) Human skin exhibits age-dependent SDF1 induction. Shown are SDF1 transcript level sat baseline and 1 week post-injury in human skin of different ages. n = 5 patients < 46 years old (black bars) and n = 4 patients > 61 years old (gray bars). *p < 0.05.(H) EZH2 knockdown rescues SDF1 induction in aged human keratinocytes. Left: western blot analysis of EZH2 and actin in WT, control (scramble) knockdown (KD), and EZH2 knockdown keratinocytes. n = 4. Right: SDF1 transcript levels at baseline (BL) and after nutrient deprivation in WT, control knockdown, and EZH2 knockdown young and aged primary human keratinocytes. n = 4. **p < 0.01.(I) Young and aged human skin organoids exhibit age-dependent SDF1 induction. Shown are SDF1 immunostaining (brown cells) and quantification 3 days after hole punch. n = 3. *p < 0.05.(J) EZH2 inhibition rescues SDF1 induction in aged human skin organoids. Shown are SDF1 (left) and EZH2 (center) transcript levels in young (blue bars) and aged (red bars) human skin organoids and SDF1 transcript in aged human skin organoids with and without DZNep (right). n = 6. *p < 0.02.N, biological replicates per group. Error bars are SEM. Scale bars, 100 μM. Nuclei are counterstained blue. See also Figure S4.
Fig 3: The role and mechanism of SDF‐1 in hepatic IR were displayed. Up‐regulated SDF1 binds to its receptor CXCR4 and CXCR7 on hepatocytes following PA treatment. SDF‐1/CXCR4 signalling, not SDF‐1/CXCR7 signalling, inhibits lipophagy in hepatocytes via activating the phosphorylation of AKT and mTOR1 to promote PA‐induced IR. The blockade of SDF‐1/CXCR4/AKT/mTOR signalling‐induced lipophagy alleviates IR in PA‐treated hepatocytes.
Fig 4: Exosomes derived from M1 microglia promote angiogenesis in vivo, and suppressing the activation of retinal microglia reverses the effects. (A) Representative images of immunofluorescence staining for DAPI (blue), Sdf1 (red, left panel) and Cxcr4 (red, right panel). Sdf1 and Cxcr4 were enormously increased after the intravitreal injection of M1-EXO compared to the other groups. Scale bar, 50 μm. (B) Representative images of double-staining for Pecam1 (green) and Ki67 (red) in PBS-injected, SHAM, M0-EXO, or M1-EXO groups. Scale bar, 50 μm. (C) Double-staining for Pecam1 (green) and Iba1 (red) in PBS-injected, M0-EXO, M1-EXO, or Minocycline plus M1-EXO groups. Minocycline treatment before M1-EXO administration effectively suppressed M1-EXO induced microglia activation and abnormal EC proliferation (white arrowheads). Bar = 50 μm. (D) Quantification of fluorescence intensity showed that M1-EXO induced microglia activation, and Minocycline pretreatment alleviated this upregulation (n ≥ 6, ***P<0.001). (E) Quantification of the number of abnormal EC in various groups (n ≥ 6, **P<0.01). EXO: exosomes; GCL: ganglion cell layer; INL: inner nuclear layer; ONL: outer nuclear layer; EC: endothelial cell; Mino: minocycline.
Fig 5: Exosomes derived from M1 microglia elevate the proangiogenic ability of microglia in vitro. (A) HUVECs proliferation was assessed using CCK8 assays after pPMG-CM, rPMG-CM, or aPMG-CM treatment (n = 3, ***P<0.001). (B) Upper panel: representative microscopic images of tube formation on Matrigel; lower panel: the quantification of branch points and total line length (×200). There was a significant increase in HUVECs tubule connections in the presence of aPMG-CM compared with that in the presence of pPMG-CM or rPMG-CM (n = 6, **P<0.01). (C) Wound-healing assay. HUVECs were treated with pPMG-CM, rPMG-CM, or aPMG-CM, and cell monolayers were scratched with 200 μl yellow tips. Images were captured at 0, 24, and 48 h after the wound scratch. (D) The ratio of the residual scratch area was measured by comparing the area at each time point with the initial area (n = 6, ***P<0.001, *P<0.05). (E) ELISA results show that the concentration of Sdf1 was increased after aPMG-CM stimulation compared with the other groups (n = 3, *P<0.05). (F)Western blot images showing the protein levels of Hif1α, Cxcr4, and Vegf were increased in HUVECs after aPMG-CM stimulation compared with the other groups. (G) Relative quantification of protein expression in HUVECs (n = 3, **P<0.01, *P<0.05). HUVECs: human umbilical vein endothelial cells; PMGs: primary microglia; pPMG-CM: PBS-treated PMG-conditioned medium; rPMG-CM: resting exosomes-treated PMG-conditioned medium; aPMG-CM: activated exosomes-treated PMG-conditioned medium.
Supplier Page from Abcam for Anti-SDF1 antibody