Friday, September 22, 2023

Suppressed Akt/GSK-3β/β-catenin signaling contributes to excessive adipogenesis of fibro-adipogenic progenitors after rotator cuff tears – Cell Death Discovery


Male C57BL/6 mice (twelve-week-old) were purchased from Animal Model Research Center of Nanjing University. The RCT and ATT animal models were established as previously described [28,29,30]. Briefly, a complete transverse incision of Achilles tendon approximately 3 mm from the calcaneus was performed for ATT animal model, and a complete transverse incision of supraspinatus and infraspinatus tendons near tendon insertion was performed for RCT animal model. The experiments were approved by ethical committee in local institution (Approval NO. XHEC-F-2022-064).

Muscle harvest and fluorescence-activated cell sorting

Supraspinatus, infraspinatus, and gastrocnemius muscles were dissected one week after tendon injury. The muscles were cut into pasty and digested by collagenase II (Worthington biochemical, 700–800 U/ml, cat#LS004177) for 45 min. Then they were further digested with the mixtures of dispase (Life Technologies, 11 U/ml, cat#17105041) and collagenase II for 30 min. The single-cell suspension was stained with APC-CD31 (Biolegend, cat#102510), APC-CD45 (Biolegend, cat#103112), PE-PDGFRα (Thermo Fisher Scientific, cat#12-1401-81) for 40 min at 4 °C. Finally, CD45-CD31-PDGFRα+ murine FAPs were obtained by BD Influx sorter (BD Biosciences) [31].

Cell culture, treatment, and adipogenic differentiation

The FAPs were cultured in growth medium on Matrigel-coated (Biocoat, cat#354277) plates. The growth medium contains high-glucose DMEM (Gibco, cat#12100-046), 20% FBS (Gibco, cat#10-013-CV), 2.5 ng/ml bFGF (R&D systems, cat#233-FB-025), and 1% Penicillin-Streptomycin (Gibco, cat#15140-122). 10 μM SC-79 (Beyotime, cat#SF2730), 1 μM BML-284 (Medchemexpress, cat#HY-19987) and 1 μM KYA1797K (Medchemexpress, cat#HY-101090) were applied during the specific experiment. Adipogenic induction medium (AIM) was used to induce the adipogenic differentiation for 7 days [32]. The AIM contains 20% FBS, 1 μg/mL insulin (Sigma-Aldrich, cat#I2643), 0.25 µM dexamethasone (Sigma-Aldrich, cat#D4902), 0.5 mM 3-isobutyl-1-methylxanthine (Sigma-Aldrich, cat#I5879), and 1% penicillin-streptomycin.

Immunohistology and Immunofluorescent staining

Cultured cells or frozen tissue slices were fixed in 4% paraformaldehyde for 20 min, blocked with 1% BSA for 1 h and permeabilized by 0.5% Triton X-100 for 30 min. Then they were incubated with anti-PDGFRα (Abcam, cat#ab203491), anti-Laminin (Abcam, cat#ab44941), and anti-perilipin A/B (Millipore, cat#P1873) at 4 °C overnight. Subsequently, Alexa 488- or Alexa 594-labeled anti-mouse or rabbit secondary antibodies (Invitrogen) were used at room temperature for 1 h. The nuclei were stained by 4, 6-diamidino-2-phenylindole (Sigma-Aldrich, cat#D9542) for 5 min. The images were acquired by BX53 or IX73 fluorescence microscope (Olympus). All the images were analyzed by image J or image-pro plus 6.0 software.

Oil red staining and triglycerides quantification analysis

Tissue slices or cultured cells were first fixed in 4% paraformaldehyde for 10 min, followed by oil red (Solarbio, cat#G1260) treatment for 10 min. Then samples were stained with hematoxylin for 5 min and mounted with 10% glycerol. For quantification analysis of lipid after adipogenic differentiation, the oil red was extracted by isopropanol for 5 min, and the value at 496 nm absorbance was detected by a microplate reader (Thermo Fisher Scientific).

Triglycerides colorimetric assay kit (Elabscience, cat#E-BC-K261-M) was employed to quantify the muscular fatty infiltration after tendon injury. Briefly, the muscle was first grounded with isopropanol (ACMEC, cat#I86620). Then tissue fragments were discarded by centrifugal machine at 10000 × g for 3 min. The supernatant was used to detect triglycerides by microplate reader at 510 nm absorbance.

Gait analysis

Noldus Catwalk system was administrated to perform gait analysis after RCT. Weights were measured to avoid interference of body size. All mice walked through the tunnel of catwalk system and then the stride length, stance width, stance time, and paw area at the peak stance were recorded [33, 34].

Treadmill test

The treadmill test was performed by treadmill apparatus (ZII-PT/5 S). In endurance test, mice run on the treadmill at a constant speed of 20 m/min. In exhaustion test, the speed began at 16 m/min and was increased 2 m/min per 3 min. The situation that mice were incapable of running for 10 s would be identified as exhaustion.

Western blotting

Lysed proteins with loading buffer were separated and transferred to nitrocellulose membranes according to the standard methods. Full membranes were blocked by 5% nonfat milk in TBST for 1 h at room temperature. Primary antibodies were incubated overnight at 4 °C: anti-GAPDH (CST, cat#2118 L), anti-Akt (CST, cat#4865), anti-phospho-Akt (CST, cat#4060), anti-GSK-3β (Santa Cruz, cat#sc-53931), anti-phospho-GSK-3β (CST, cat#93336), anti-β-catenin (CST, cat#8480), anti-PPARγ (CST, cat#2435). Then the sample was incubated by HRP-conjugated secondary anti-rabbit or anti-mouse IgG antibodies (Ray antibody biotech, cat#RM3004) for one hour at room temperature. Lumi Q ECL luminescent liquid was used to capture the target strips.

Transfection of siRNA

In total, 2 μl lipofectamine 2000 (Invitrogen, cat#11668019) was mixed with 200 μl Opti-MEM™ I reduced serum medium (Thermo Fisher, cat#31985062) and 1 μl 100 μM β-catenin siRNA at room temperature for 20 min. The mixture was then equably added into the 12-well plate cultured with FAPs. The sequence of siRNA was listed as follow: β-catenin siRNA 5ʹ-GCAGAATACAAATGATGTA-3ʹ.

Gene expression analysis

Total RNA was extracted by EZ-press RNA Purification Kit (EZ Bioscience, cat#B0004DP) according to manufacturer’s instruction. RNA quantification was measured by NanoDrop 2000 (Thermo Fisher Scientific). One microgram total RNA was reverse transcribed into cDNA with M-MuLV Reverse Transcriptase (NEB, cat#M0253L). The products of reverse transcription were used for Real-Time qPCR. Quantitative reverse transcription PCR was performed by FastStart Universal SYBR Green Master (Roche, cat#4913914001) in the ABI Quant Studio 6 real-time qPCR system. The primers of RT-qPCR were shown in Table 1.

Table 1 Primer sequences for real-time PCR.

Bulk RNA-seq and analysis

RNA sequencing libraries were obtained by NEBNext Ultra RNA Library Prep Kit for Illumina (NEB, cat#E7530L). The paired-end RNA-seq sequencing library was sequenced by the Illumina NovaSeq 6000 sequencer (2×150 bp read length). The raw paired-end reads were embellished and quality controlled by fastp ( with default parameters [35]. Differential expression analysis with P-adjust ≤ 0.001 (DEGseq) were considered significantly different expressed genes. GO and KEGG analyses were performed by Goatools ( and KOBAS ( [36].

Statistical analysis

Statical analysis was conducted by GraphPad Prism 8 (San Diego, USA) software. Student t test was used to compare quantitative parameters between two groups. The histological evaluation was used as primary outcome. The equation, n = 2(Zα/2 + Zβ)2σ2/(μct)2, was used to calculate sample size [37], and the alpha value was set as 0.05 with a power of 0.80. P < 0.05 was considered statistically significant. All the experiment results were presented by mean ± standard deviation. Technical replicates or biological repeats were introduced in the figure legend. All the experiments were assessed and analyzed by two independent investigators (H.Z. and X.S.) in a blinded manner.

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