Ab34710

HuR-silenced and control NP cells were plated on glass coverslips and cultured in hypoxia for 72 h. After treatment, cells were fixed with 4% paraformaldehyde at room temperature for 15 minutes, washed with PBS and then blocked with 5% normal goat serum in PBS with 0.3% Triton X-100 (Sigma Aldrich, T8787) for 1 h at room temperature. Cells were then incubated with anti-Collagen I antibody (1:500, Abcam, ab34710), anti-Collagen II antibody (1:100, Fitzgerald, 70R-CR008), or anti-Aggrecan antibody (1:100, EMD Millipore, AB1031) in blocking buffer at 4°C overnight. Cells were washed with PBS and incubated with Alexa-fluor-594-conjugated anti-rabbit secondary antibody (1:800, Jackson ImmunoResearch Lab, 711–586-152) for 1 h at room temperature. After washing with PBS, cells were mounted with ProLong Gold Antifade Mountant with DAPI (Thermo Fisher Scientific, P36934) and visualized using a Zeiss AxioImager A2 (Carl Zeiss, Germany).

Caudal spines from 8 and 14-month old WT and MCT4 KO mice were harvested and fixed in 4% PFA for 24 hours and decalcified in 12.5% EDTA at 4^oC for 15 days prior to paraffin embedding. Mid-coronal IVD sections (Ca5–8) were stained with Safranin O/Fast Green/Hematoxylin or Picrosirius red, then visualized using a light microscope (AxioImager 2, Carl Zeiss) or a polarizing microscope (Eclipse LV100 POL, Nikon). Histopathological scores were collected from n = 4 mice/genotype with 3 discs per mouse (total 12 discs/genotype) at 8 months; n = 8 mice/genotype with 3 discs per mouse (total 24 discs/genotype) at 14 months. Modified Thompson Grading was used to score NP and AF compartments (Table S2) and Boos Grading Scale was used to score the CEP compartment ^{(5 (link),36 (link))}. Histological sections were incubated with antibody against KRT19 (1:3, DSHB, TROMA-III/supernatant), CA3 (1:150, Santa Cruz, sc-50715), MMP13 (1:200, Abcam, ab39012), Aggrecan (1:50, Millipore, AB1031), Collagen I (1:100, Abcam, ab34710), Collagen II (1:400, Fitzgerald, 70R-CR008), Collagen X (1:500, Abcam, ab58632), LDHA (1:200, Novus, NBP2–19320), CA9 (1:200, Novus, NB100–417), MCT1 (1:200, Santa Cruz), GLUT-1 (1:200, Abcam, ab40084) and ARGxx (1:200, Abcam, ab3773). See Supplemental Methods.