Passive buffer

RB1-null C33A cells were cotransfected with indicated quantities of pCMV-HA-RB1, pLL-FLAG-BCOR, pCMV-V5-ESRRG-WT or pCMV-V5-ESRRG-MUT, and pERRE-LUC using jetPRIME reagent (Polyplus). Stoichiometric quantities of empty vectors pCMV-V5-EV, pCMV-HA-EV, and pLL-FLAG-EV were used for C33A cell transfection normalization. HEK293 cells expressing TET-shRB1 (HEK293-TET-shRB1) were induced with doxycycline for 48 hours and then cotransfected with indicated quantities of pCMV-HA-RB1, pCMV-V5-ESRRG-WT or pCMV-V5-ESRRG-MUT, and pERRE-LUC. Cells not treated with doxycycline and stoichiometric quantities of EVs pCMV-V5-EV and pLL-FLAG-EV were used for HEK293 cell transfection normalization. Luciferase activity was measured 24 hours after plasmid transduction by lysing the cells in the passive buffer (Promega) and combining equal volumes of cell lysates with the Bright-Glo reagent (Promega). Cotransfection with a CMV-beta-gal plasmid and bicinchoninic acid protein quantification assays were used for luciferase signal normalization.

To evaluate the differentiation potential of MSCs in gels 1 d after encapsulation, they were cultured in medium supplemented with either an osteogenic chemical cocktail (No. CCM009) alone or both osteogenic and adipogenic (No. CCM011) cocktails for 7 or 10 d, respectively. All reagents for MSC differentiation were purchased from R&D Systems. One half of each sample was used to quantify an absolute number of viable cells by flow cytometry as described previously, while the other half was used to evaluate ALP activity. To quantify ALP activity, samples were lysed with 100 μL passive buffer (No. E1941, Promega) for at least 10 min at 4 °C. Each lysate was then added to a black 96‐well plate preloaded with 100 μL 4‐methylbelliferyl phosphate (4‐MUP) substrate (No. M3168, Sigma). Signals were acquired with excitation at 360 nm and emission at 450 nm using a plate reader. Recombinant mouse ALP protein (Novus Biologicals) was used to generate a standard curve for calibration. ALP activity of each sample was then normalized to the number of viable cells.

For the dicistronic expression assay, RD cells were transfected with UGGT1 siRNA, and after 3 days, a dicistronic construct, pRHF-EVA71, was cotransfected with UGGT1 siRNA to RD cells. After 2 days, cell lysates were prepared in a passive buffer (Promega) and examined for Renilla luciferase (RLuc) and Firefly luciferase (FLuc) activities with a Lumat LB 9507 bioluminometer (EG&G Berthold, Wildbad, Germany), using dual-luciferase reporter assays (Promega) conducted in accordance with the manufacturer’s instructions.

MSCs in gels were cultured for 1 day in a basal medium (No. CCM007), followed by the medium supplemented with both osteogenic (No. CCM007) and adipogenic (No. CCM11) cocktails for 10 days. The medium was refreshed on day 5. All reagents for MSC differentiation were purchased from R&D Systems. One‐half of each sample was used to quantify an absolute number of viable cells by calcein staining, while the other half was used to evaluate early osteogenesis or adipogenesis by ALP activity or lipid droplet staining, respectively. To quantify ALP activity, cells were lysed with 200 µl passive buffer (No. E1941, Promega) for 15 min at 4 °C. Each lysate was then added to a black 96‐well plate preloaded with 100 µl 4‐methylumbelliferyl phosphate (4‐MUP) substrate (No. M3168, Sigma). Signals were acquired with excitation at 360 nm and emission at 450 nm using a plate reader. Recombinant mouse ALP protein (Novus Biologicals) was used to generate a standard curve. To quantify lipid droplets in cells, cells were incubated with the LipidSpot™ 610 Lipid Droplet Stain (Biotium) at 37 °C for 30 min. Signals were acquired with excitation at 592 nm and emission at 638 nm using a plate reader.

Proteasome activity was examined using fluorogenic peptides N-Suc-LLVY-AMC, Z-LRR-AMC, and Z-LLE-AMC as substrates to measure chymotrypsin-like, trypsin-like, and caspase-like peptidase activities, respectively. Briefly, the cells were washed twice with PBS and homogenized in a passive buffer (Promega Corporation, Madison, WI, USA). After sonication for 20 s, the supernatant was collected by centrifugation at 16,000 × g for 10 min. The aliquots were diluted in an assay buffer containing 250 mM sucrose, 10 mM NaCl, 1.5 mM MgCl₂, 1 mM EDTA, 1 mM EGTA, 2 mM ATP, 5 mM dithiothreitol, 50 mM Hepes (pH 7.8), and 10 μM proteasome substrates, and then the reaction mixture was incubated for 1 h at 37 °C. The proteolytic activity was measured by monitoring the formation of the fluorescent AMC (excitation wavelength 380 nm and emission wavelength 460 nm).