Mice and diabetes induction
All mice were maintained in the pathogen-free animal facility and all animal studies have been approved by the Institutional Animal Care and Use Committee at The Affiliated Hospital of Southwest Medical University. Healthy male Kunming mice (3-5 weeks, 13-18 g, n=140) were provided by Laboratory Animal Center of The Affiliated Hospital of Southwest Medical University. Totally 15 mice received a saline injection and fed with normal diet as control, while 125 mice (n=125) received streptozotocin (45 mg/kg, i.p., 0.1 mmol/L sterile sodium citrate buffer, STZ, Sigma-Aldrich, St. Louis, MO, USA) after 6 weeks of high-sugar and high-fat diet to establish diabetic model. Seventy-two hours post STZ injection, blood glucose level was measured by a blood glucose monitoring system (Glucotrend 2, Roche Diagnostics, Mannheim, Germany). Mice with fasting blood glucose concentration over 16.7 mmol/L for at least 10 days were considered as diabetic mice. Five mice died, and therefore, success rate was 96.43%.
Excisional wound preparation
Two weeks after the establishment of diabetes model, mice were anesthetized with pentobarbital sodium (Sigma-Aldrich, St. Louis, MO, USA) at 50 mg/kg body weight. A 10 ×10 mm punch wound (excision of the skin and the underlying panniculus carnosus) were made on the instep of the mice. This wound was disinfected daily with 0.5% iodine. Control mice were defined as normal ulcer (NU, n=15). Diabetic mice further received different treatments (n=15/treatment): DFU, DFU + vehicle (veh), DFU + TGF-β, DFU + TGF-β + LY2109761, DFU + overexpression (oe)-negative control (NC), DFU + oe-WDR74, DFU + oe-WDR74 + veh, DFU + and oe-WDR74 + LY2109761 groups. Both oe-NC and oe-WDR74 were achieved by lentivirus injection (1 × 108 Tu/mL). Wound size was measured by tracking the edge of the wound on a transparent plastic film. Wound healing was observed under a light microscope during the experiment. On the 7th day after the wounding, 5 mice from each group were randomly selected and processed for hematoxylin and eosin (HE) staining, and 5 mice for immunohistochemistry and immunofluorescence assays while the rest were used for further studies.
Immunofluorescence
Paraffin-embedded tissue was cut into 5-μm sections. Samples were then deparaffinized and quenched with 3% H2O2 for 10 min. For M2 macrophage immunofluorescence, sections were incubated with mouse primary antibody against F4/80 (1:100, Cat# 14-4801-82; Thermo Fisher Scientific, Waltham, MA, USA) and CD206 (1:100, Cat# MA5-16871; Thermo Fisher Scientific, Waltham, MA, USA) overnight at 4°C. Samples were then probed with goat anti-mouse secondary antibody (1:200, ab150165 or ab150167; Abcam, Cambridge, UK). For colocalization between CD206 and WDR74, sections were incubated with primary mouse antibody against WDR74 (1:100, Cat# PA5-20924; Thermo Fisher Scientific, Waltham, MA, USA) and CD206 (1:100, Cat# MA5-16871; Thermo Fisher Scientific, Waltham, MA, USA) at 4°C overnight. Samples were then stained with goat anti-mouse immunoglobulin G (IgG) secondary antibody (ab150167, 1:200, Abcam, Cambridge, UK) or goat anti-rabbit IgG secondary antibody (1:200; ab150077; Abcam, Cambridge, UK). For angiogenesis, sections were incubated with primary antibody against CD31 (1:100, ab9498; Abcam, Cambridge, UK) overnight at 4°C. Samples were then incubated with goat anti-mouse secondary antibody (1:200, ab150167; Abcam, Cambridge, UK), followed by nuclear DAPI (Sigma-Aldrich, St. Louis, MO, USA) staining. Tissues were observed under an Olympus BX51 fluorescence microscope.
For immunofluorescence in cells, cells were permeabilized with 2% Triton X-100 for 15 min and with 2 M HCL for 20 min. Cells were blocked by 2% bovine serum albumin (BSA) for 45 min and then incubated with primary antibody against collagen 1A1 (COL1A1, 1:100, ab21286; Abcam, Cambridge, UK; rabbit), vascular endothelial growth factor (VEGF, 1:50, Cat#MA5-13182; Thermo Fisher Scientific, Waltham, MA, USA) or fibronectin (FN, 1:250, ab45688, Abcam, Cambridge, UK) overnight at 4°C. Cells were then incubated with goat anti-rabbit IgG H&L fluorescent secondary antibody (1:200, ab150165, 1:200; Abcam, Cambridge, UK), goat anti-mouse IgG H&L fluorescent secondary antibody (1:200, ab150117; Abcam, Cambridge, UK) or goat anti-mouse IgG H&L fluorescent secondary antibody (1:200, ab150115; Abcam, Cambridge, UK) for 2 h at room temperature, followed by DAPI (2 μg/mL) staining. Cells were mounted on a slide and observed under an Olympus BX51 fluorescence microscope.
Immunohistochemistry
Sections were deparaffinized with xylene and rehydrated by serial of gradient alcohol (100%, 100%, 95%, 85%, and 75%, 3 min each). Sections were then treated at high temperature and pressure for 2 min and allowed to cool down at room temperature. Sections were incubated with avidin for 10 min at room temperature, followed by 3 phosphate buffer saline (PBS)-washes with PBS. Sections were further incubated with d-biotin solution for 10 min at room temperature and washed 3 times with PBS. Endogenous peroxidase was eliminated by incubating with hydrogen peroxide for 15 min at room temperature. After washing with PBS the sections were blocked with 10% goat serum for 10 min at room temperature. Sections were incubated with primary antibodies (Abcam, Cambridge, UK) against TGF-β (1:100, ab215715, 1:100), Smad2/3 (1:100, ab217553) and phosphorylated Smad2/3 (1:100, ab63399) overnight at 4°C. After washing 3 times with PBS, sections were incubated with anti-biotin-labeled goat anti-rabbit IgG (1:1,000, ab150077; Abcam, Cambridge, UK) for 10 min at room temperature. Sections were then treated with streptomycin avidin-peroxidase solution for 10 min at 37°C. Freshly prepared DAB were added and color change was observed under a microscope. After 20 s, sections were rinsed with water, counterstained with hematoxylin, dehydrated, and observed under a microscope.
Hematoxylin and eosin (HE) staining
Tissue sections were deparaffinized and rehydrated as mentioned above. Sections were then immersed in hematoxylin solution for 5 min, and washed with running water for 1 min. The sections were then immersed in 1% hydrochloric acid alcohol for 30 s and washed with running water for 1 min. After that, the sections were stained in 0.5% eosin solution for 3 min and washed with running water for 1min. Then, the sections were dehydrated, mounted, and observed under a microscope.
Mouse primary macrophage culture
In order to isolate primary bone marrow-derived mouse macrophages, the tibias of mice were isolated and disinfected with 75% alcohol, then washed with precooling medium. The two ends of tibia were cut off, the bone marrow cells in tibia were washed out after absorbing culture medium, and red bone marrow could be seen. Bone marrow was slowly titrated into a single-cell suspension. The cell suspension was centrifuged at 4°C and 1600 rpm for 5 min with the supernatant discarded. The red blood cell lysis was used to lyse red blood cells for no more than one minute. Then, the red blood cells were washed twice with the pre-cooled RPMI 1640 medium with 5 times of the volume of red blood cell lysis at 4°C and 1600 rpm. After that, the cells were resuspended in RPMI 1640 containing 10% heat-inactivated fetal bovine serum (FBS) and 20ng/ml Recombinant Murine(M-CSF)and seeded into 6-well plate in the incubator at the concentration of 5 × 105 cells/well. After 3 days incubation, the medium and the unattached cells were removed, and the attached macrophages were cultured for 3 days for different treatments. Control macrophages were treated with interleukin (IL)-13 (10 ng/mL), IL-13 + TGF-β (5 ng/mL), TGF-β, over-Expression (oe)-WDR74, oe-NC, short hairpin (sh)-WDR74, sh-NC, IL-13 + oe-WDR74, or IL-13 + sh-WDR74. oe-NC, sh-NC, sh-WDR74 and oe-WDR74 were infected into macrophages by lentivirus at 2 × 106 TU/mL.
Effect of macrophage-derived conditioned medium on fibroblast
Mouse NIH3T3 fibroblasts were seeded onto culture plates with a density of 15,000 cells/cm2 in DMEM containing 10 % FBS and high glucose. The next day, culture medium was replaced with conditioned medium (CM) derived from macrophages. CM was collected from primary macrophages (Mo-CM), IL-13-stimulated macrophages (M2-CM), TGF-β-stimulated macrophages (TGF-β-CM) ,IL-13 and TGF-β co-stimulated macrophages (TGF-β-M2-CM), IL-13-stimulated macrophages infected with oe-NC (oe-NC-M2-CM), IL-13-stimulated macrophages infected with oe-WDR74 (oe-WDR74-M2-CM), IL-13-stimulated macrophages infected with sh-NC (sh-NC-M2-CM), or IL-13-stimulated macrophages infected with sh-WDR74 (sh-WDR74-M2-CM). After cultured for 72 h, COL1A1, FN and VEGF expression in fibroblasts were detected by Western blot analysis.
Reverse transcription quantitative polymerase chain reaction (RT-qPCR)
Total RNA was extracted by Trizol RNA (Invitrogen, Carlsbad, CA, USA). Reverse transcription was performed using Primescript RT reagent kit (RRO37A, TaKaRa, Dalian, China). Target genes (primers are shown in Table 1) were amplified by a real-time PCR system (ABI 7500, Applied Biosystems, Foster City, CA, USA). PCR reaction system consisted of 2.5 μL 10 × PCR Buffer, 2.5 μL 25 mmol/L MgCl2, 1.5 μL 10 mmol/L dNTP, 0.5 μL 10 mmol/L primer, 0.25 μL Taq, 2.5 μL cDNA and 15.25 μL sterile distilled water. Reaction conditions were as follows: 40 cycles of denaturation at 94 °C for 5 min, at 94 °C for 30 s, at 58 °C for 45 s and at 72 °C for 30 s, and extension at 72 °C for 10 min. Each sample was tested in triplicates. β-actin was used as an internal reference. mRNA expression level of target genes was calculated by the 2-ΔΔCt method [24].
Western blot analysis
Wound tissues or cells were treated with lysis buffer containing phenylmethylsulfonyl fluoride and phosphatase inhibitor followed by centrifugation at 12,000 r/min for 30 min at 4°C. Total protein concentration in the supernatant was measured by a BCA kit. Protein (50 μg) was dissolved in 2 × SDS loading buffer and boiled at 100°C for 5 min. Proteins were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to polyvinylidene difluoride membrane. Membrane was blocked by skim milk (5%) at room temperature for 1 h and rinsed 3 times with tris-buffered saline with Tween 20 (TBST). Membrane was incubated with primary antibodies against COL1A1 (1:1,000, ab34710), FN (1:1,000, ab45688), VEGF (1:1,000, ab69479), CD31 (1:1,000, ab9498), Smad2/3 (1:500, ab217553), phosphorylated Smad2/3 (1:5,000, ab63399), arginase-1 (1:1,000; #93668; Cell Signaling, Danvers, MA, USA), YM1 (1:10,000; ab192029), inducible nitric oxide synthase (iNOS; 1:500, ab178945) and β-actin (1:1,000, ab8226). After washing 3 times with TBST, membranes were incubated with horseradish peroxidase-labeled secondary antibodies (1:10000, ab6721 and ab205719) for 1 h. The above antibodies except for arginase-1 were purchased from Abcam (Cambridge, UK). After rinsing 3 times with TBST, the membrane was developed by electrogenerated chemiluminescence (ECL) kit (BB-3501, Amersham, Buckinghamshire, UK) in Bio-Rad Image Analysis System (Bio-Rad, Hercules, CA USA), and analyzed by Quantity One v4.6.2 software. Relative protein expression was expressed by gray intensity of protein bands in target protein normalized by that of β-actin protein band. Each sample was tested in triplicates.
Co-immunoprecipitation
Cells were harvested and lysed in cell lysis containing protease (Roche, Basel, Switzerland) and phosphatase inhibitor (Thermo Fisher Scientific, Waltham, MA, USA) for 30 min at 4°C and cleared cell lysates were collected by centrifugation at 4°C (16,000 g, 30 min). Supernatant was immunoprecipitated overnight at 4°C with WDR74 antibody (1:50, Cat# PA5-83390; Thermo Fisher Scientific, Waltham, MA, USA). Lysate was then incubated with 30 μL of protein G plus-sepharose (Amersham, Buckinghamshire, UK) for 2 h at 4°C. Beads were precipitated and washed three times with immunoprecipitation buffer. Western blot analysis was performed to determine Smad2, Smad3, and WDR74 protein expression.
Statistical analysis
All data were analyzed using SPSS 21.0 (IBM Corp. Armonk, NY, USA). Measurement data were expressed as mean ± standard deviation. Unpaired t-test was used to compare two groups of unpaired data obeying normal distribution and homogeneity of variance. One-way analysis of variance (ANOVA) and Tukey's post hoc test was used for comparing data among multiple groups. Differences were considered significant when p < 0.05.