Dgcr8

MM cells were fixed with 4% formaldehyde and permeabilized with 0.3% Triton X-100 in 1 × PBS. After blocking with 2% goat serum, the cells were stained with antibodies against hnRNPA2B1 (Santa Cruz) or DGCR8 (Abcam) at 4°C overnight, followed by incubation with Alexa 594- or Alexa 488-conjugated secondary antibodies (Abclonal) for 30 min at room temperature and the cell nuclei were stained with DAPI and mounted with antifade reagent (Molecular Probes). Immunofluorescent images were acquired with an IX71 confocal microscope system (Olympus).

Proteins in endplate chondrocytes were lysed and collected, and the protein concentration was determined by the bicinchoninic acid quantitative method. Denatured protein was separated by sodium dodecyl sulphate‐polyacrylamide gel electrophoresis at 20 g per pore and then transferred onto a nitrocellulose membrane. The membrane was blocked with 5% bovine serum albumin for 1 hour, and primary antibodies against METTL3 (1:1000; Abcam), IL‐1β (1:100, Abcam), cleaved caspase 3 (1:100, Abcam), cleaved caspase 9 (1:100, Abcam), cleaved poly(ADP‐ribose) polymerase (PARP; 1:500, Abcam), ACAN (1:100, Abcam), COL2A1 (1:5000, Abcam), GAPDH (1:5000, Abcam), PIK3R2 (1:5000, Abcam), pAKT (1:5000, Abcam), AKT (1:500, Abcam) or DGCR8 (1:1000, Abcam) were added. The membrane was then incubated at 4°C overnight. The following day, a secondary antibody (1:5000; Cell Signaling Technology, Danvers, MA) was added and the membrane was incubated again for 1 hour at room temperature on a shaker. After washing the membrane three times with Tris‐buffered saline containing Tween 20, images were acquired with a gel imaging system.

For co‐immunoprecipitation, cells were lysed using RIPA protein extraction reagent (Beyotime) supplemented with a protease inhibitor cocktail (Roche) and PMSF (Roche). The protein concentration was measured using the Bio‐Rad protein assay kit. The supernatants were collected and incubated with anti‐pStat3, Drosha at 4°C for 12 hours. Protein A Sepharose CL‐4B beads (GE) were incubated with the mixture at 4°C for 2 hours. Then, the beads were washed three times with RIPA buffer. The bound proteins were eluted with SDS‐PAGE loading buffer and used for Western blot. For Western blot assay, approximately 40 μg of protein extract was electrophoresed on a 10% SDS‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto 0.22‐μm nitrocellulose membrane (Sigma) and incubated with specific antibodies. The ECL chromogenic substrate was used to visualize the bands. The intensity of the protein bands was quantified by Quantity One software (Bio‐Rad ChemiDoc XRS). GAPDH was used as a control. Antibodies for Jak1, p‐Jak1, Stat3, p‐Stat3, Ago, IL‐6, Drosha, DGCR8 and GAPDH were purchased from Abcam.