Recombinant human sdf 1α cxcl12

All mouse experiments were performed according to UK Home Office Regulations, with the approval of the Ethics Committee at Cambridge University. CD-1 nude mice (Charles River) were maintained under standard pathogen-free conditions. Six- to eight-week-old male mice were anaesthetized with isoflurane (0.5–2%). LSCs WT or LSCs overexpressing a shRNA to knockdown SDF-1/CXCL12 were disassociated with accutase to generate a single-cell suspension (0.5–1 × 10⁵ cells in 10 μl PBS), and this suspension was injected under the kidney capsule. Mice were killed 2, 3 and 4 weeks later and the kidneys were collected to examine in vivo differentiation of the injected cells or to analyse the recruitment of stromal cells. Grafts were removed and prepared for immunofluorescent microscopy.
For rescue experiments, LSCs overexpressing a shRNA to knockdown SDF-1/CXCL12 (LSC-KD) were co-injected with recombinant human SDF-1α/CXCL12 (40–50 ng μl⁻¹) (R&D Systems, #350-NS-010) and matrigel under the kidney capsule. Alternatively, LSC-KD cells expressing a shSDF-1 resistant mRNA were injected under the renal capsule. Grafts were removed after 2 and 4 weeks and prepared for immunofluorescent microscopy.

Western analysis of protein from cell lysates was performed as previously described [26 (link)]. Rat anti-podoplanin NZ-1 (AngiobioCo., San Diego, CA) and mouse anti-FAP (Santa Cruz Biotechnology, INC) were employed as primary antibodies. Mouse anti-β-tubulin mAb E7 (DSHB; http://dshb.biology.uiowa.edu) was used as a loading control. To assess the release of SDF-1α/CXCL12, media were concentrated with Amicon Ultra 3K centrifugal filter devices (Merck Millipore Ltd.) according to the supplier’s protocol. Recombinant human SDF-1α/CXCL12 (R&D Systems; https://www.rndsystems.com) was used as a positive control. Rabbit anti-human SDF-1α/CXCL12 antibody (Thermo Fisher Scientific) was used to detect the cytokine. IRDye 800-conjugated goat anti-rat, goat anti-mouse or goat anti-rabbit antibody (Li-Cor Biosciences, Lincoln, NE) were used as secondary antibodies. Odyssey scanner and software were used for detection and quantification of immunoblots (Li-Cor Biosciences).

To analyse SDF-1 expression, LSCs were treated with recombinant human TGFβ1 (10 ng/ml) (Cell Signaling, #8915) for 6 h or with recombinant human TNFα (100 ng/ml) (Cell Signaling, #8902) for 12 h. For western blotting, LSCs were treated with recombinant human TNFα (10 ng ml⁻¹) for 15 min. For inhibition experiments, LSCs were pre-incubated with a JNK inhibitor SP600125 (5–50 μM) 60 min before TNFα treatment.
For JNK and p38αMAPK activation, fibroblasts were seeded in six-well plates 24 h before the start of the assay. Then, the medium was changed to DMEM containing 2% FBS. After 6 h the medium was replaced with DMEM containing 0.5% FBS and recombinant human SDF-1α/CXCL12 (200 ng ml⁻¹) (R&D Systems, #350-NS-010) was added to each well. The same samples were processed for real-time qPCR to measure TNFα, VEGF and IL8 expression.