Cell lysis buffer

The cellular proteins of eMSCs were extracted with cell lysis buffer (Ambion, Grandisland, NY, USA). The proteins (5 μg) were mixed with 5X SDS loading buffer (60 mM Tris-HCl, pH 6.8, 2% SDS, 0.1% bromophenol blue, 25% glycerol and 14.4 mM β-mercaptoethanol), denatured at 95 °C for 10 min, subjected to sodium dodecyl sulphate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Immobilon™-P, Milllipore). The membranes were blocked with 5% skim milk in PBS containing 0.1% Tween-20 for 30 min, incubated with primary antibodies at appropriate concentrations (Additional file: Table S3) overnight at 4 °C and stained with appropriate horseradish peroxidase-conjugated secondary antibodies (Additional file: Table S3) for 1 h at room temperature. The protein bands were visualized by the WesternBright ECL Kit (Advansta, CA, USA). The intensities of the protein bands were quantified densitometry, and the values were normalized to β-actin using the ImageJ software (US National Institutes of Health, USA).

Total protein extraction was performed using cell lysis buffer (Ambion), and protein quality was determined using a bicinchoninic acid (BCA) protein assay (Pierce, USA). Briefly, 20 μg of protein was denatured at 95°C for 10 min in 5% β-mercaptoethanol solution and 95% Laemmli sample buffer (Bio-Rad). After separation by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE), the proteins were transferred to nitrocellulose membranes (Schleicher & Schuell, Germany) and blocked for 1 h at 25°C using 0.5% skimmed milk. Thereafter, the membrane was incubated with ALP (ab229126; Abcam, USA), RUNX2 (ab236639; Abcam), and GAPDH (ab8245; Abcam) at 25°C for 1 h, followed by secondary antibodies (Bethyl, USA) at 25°C for 1 h. Membrane-bound antibodies were assessed by chemiluminescence after rinsing with PBS [32 (link)].

Proteins from in vitro differentiated FPC and non-FPC were extracted with cell lysis buffer (Ambion, Grand Island, NY, USA). The protein samples (5 μg) were mixed with 5X SDS loading buffer (60 mM Tris–HCl pH 6.8, 2% SDS, 0.1% bromophenol blue, 25% glycerol and 14.4 mM β-mercaptoethanol) and denatured at 95°C for 10 minutes. Protein samples were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and transferred to polyvinylidene difluoride (PVDF) membranes. Membranes were blocked with 5% skim milk (Nestle, Vevey, Switzerland) in PBS containing 0.1% Tween-20 (PBST) for 30 minutes and incubated with primary antibodies at appropriate concentrations (Additional file 5: Table S3) overnight at 4°C. The membranes were stained with appropriate horseradish peroxidase-conjugated secondary antibodies (Additional file 5: Table S3) for one hour at room temperature. The protein bands were visualized by enhanced luminal-based chemiluminescence (Westsave UP™; AbFrontier, Seoul, Korea). Endometrial stromal cells cultured in serum containing medium were used as the control. Non-immune immunoglobulin of the same isotype as the primary antibody was used as negative control. The scanned Western blot bands were quantified densitometrically and the values were normalized to the amount of β actin using Image J software (US National Institutes of Health, USA).

Cells in 6-well plates were transfected with 50 nM biotin-labeled miR-3173-5p or miR-NC for 48 h. About 5 million cells were lysed using 1000 μL cell lysis buffer (Ambion, Austin, TX, USA), followed by the centrifugation at 14,000 rpm for 10 min. 100 μL supernatant was take out as the input, and the remaining supernatant was incubated with 100 μL M-280 streptavidin magnetic beads (Sigma) pre-blocked by yeast tRNA and RNase-free BSA (Sigma) at 4 °C overnight. The magnetic beads were rinsed 4 times with lysis buffer, and RNA samples was extracted and analyzed by RT-qPCR [45 (link)]. Three independent assays were conducted for RNA pull-down assay.

Cultured eMSC were lysed in cell lysis buffer (Ambion, Grandisland, NY, USA) in the presence of protease inhibitors. Then, 5 μg of the denatured protein samples were separated on 10% SDS-PAGE and transferred to polyvinylidene difluoride membranes (Immobilon™-P, Milllipore). After blocking with 5% skim milk for 1 h at room temperature, the membranes were incubated with appropriate primary antibodies overnight at 4 °C followed by horseradish peroxidase-conjugated secondary antibodies for 1 h at room temperature (Supplementary Table S4). The protein expression was detected by the Western Bright ECL Kit (Advansta, CA, USA). The relative expression of the target proteins was normalized to housekeeping protein β-actin.

BCa cells were transfected with wild type (WT) and mutant (MUT) biotinylated circ_0000658 (50 nM each). After 48 h of transfection, the cells were collected, vortexed, and incubated with cell lysis buffer (Ambion, Austin, Texas) for 10 min. Next, 50 mL of sample cell lysate was aliquoted. The remaining lysate was incubated with M-280 streptavidin magnetic beads (Sigma) pre-coated with RNase-free and yeast tRNA (Sigma) for 3 h at 4 °C, then wash twice with cold lysis buffer, three times with low-salt buffer, and once with high-salt buffer. Finally, the total protein was extracted.

For verification of indel mutations, injected embryos were collected at the blastocyst stage. Genomic DNA was extracted from a single blastocyst with a cell lysis buffer (Ambion) at 75 °C for 15 min and 95 °C for 10 min. The sgRNA target sites were amplified by using high-fidelity Golden Star T6 DNA polymerase (TsingKe, Beijing, China). PCR primers used for mutation detection were as follows: F, 5′-ACAGCCTCCCCCAGTCCT-3′; R, 5′-GCACCTCCTTGAGCGTCC-3′. The genomic DNA from MC1R KO and WT newborn rabbits were extracted from a small sample of ear tissue using Tris-phenol/chloroform. Primers MC1R-F/R were used for PCR genotyping. After gel purification, the PCR products were cloned into a pEASY-blunt simple vector (TransGen, Beijing, China) and then subjected to Sanger sequencing. Mutations were identified via alignment of the sequenced alleles to the WT.