![Figure 4. Expression of the P+C domains of calreticulin reduces adipogenesis. (A) A schematic of the structural and functional calreticulin domains as well as Western blot analysis (with anti-HA antibodies) of G45crt−/− ES cells expressing the N+P and P+C domain of calreticulin. (B) Oil red O staining in WT, G45crt−/−, and G45crt−/− ES cells expressing P+C and N+P domains. G45crt−/−cells expressing the P+C domain had decreased staining compared with the G45crt−/− cells. When the cells were treated with 50 nM BAPTA-AM, both the P+C domain–expressing and WT ES cells showed a dramatic increase in oil red O staining. The N+P domain–expressing ES cells had oil red O staining similar to that of the G45crt−/− ES cells. When the cells were treated with 500 nM ionomycin, both the N+P domain–containing and G45crt−/− ES cells showed a decrease in oil red O staining. (C) Expression of the P+C domain in 3T3-L1 cells (3T3-L1 + [P+C]) reduced oil red O staining. Because 3T3-L1 is used as a control, it is always counted as 1. Expression of the P+C domain was confirmed using HA antibody. (D) Adipogenic marker expression was decreased in the P+C domain–containing G45crt−/− cells when compared with the G45crt−/− cells (P < 0.01; n = 6) and was comparable to that in WT cell levels (P > 0.05). BAPTA-AM treatment increased adipogenic marker expression in all cell lines (n = 6). (E) Adipogenic marker expression was higher in the N+P domain–expressing G45crt−/− cells than in the WT ES cells (P < 0.01; n = 6) and was similar to that in G45crt−/− cell levels (P > 0.05). Ionomycin treatment reduced adipogenic marker expression in all cell lines (n = 6). (F) 45Ca2+-loaded ES cells were treated with thapsigargin or ionomycin to measure ER-releasable and total [Ca2+], respectively. WT and P+C domain–expressing G45crt−/− cells had higher [Ca2+]ER and [Ca2+]Tot than the G45crt−/− cells (n = 3). (G) Fura-2-AM–loaded ES cells were treated with thapsigargin to measure ER-releasable and cytosolic [Ca2+]. WT and P+C domain–expressing G45crt−/− cells had higher [Ca2+]ER and higher cytosolic [Ca2+] than the G45crt−/− cells (n = 3). (H) 45Ca2+-loaded 3T3-L1 cells were treated with thapsigargin or ionomycin to measure [Ca2+]ER and [Ca2+]Tot levels. 3T3-L1 + CRT cells (overexpressing calreticulin) and 3T3-L1 expressing the P+C domain had higher [Ca2+]Tot and [Ca2+]ER than the control 3T3-L1 cells (n = 3). Error bars represent SD.](https://cdn.rupress.org/rup/content_public/journal/jcb/182/1/10.1083_jcb.200712078/8/jcb_200712078_rgb_fig4.jpeg?Expires=1767712023&Signature=3TS9O4McTttDSKFk0I8qmo15nukZe7pOiQWyMdvtEGmTQt2bV5Ko2OET2iaH8KkrgT2x0iYpWDX0-1AUn1hRQP9jd42fPyf84ele84qqgugI7TYgMpZtnB7hqw5Ty51f0dMoSwLHK8kDlaIAwkLj6FIrzqCxXItt5RSVbbVakPeXfY4C7eOncPPZ3Qb7E19lE1eSZePxuGGh10KsyUAHHVz0KRN01O1soJs0SRliEU8kMQPkgcp0U1CT~2OAhtCMVmeM5AmL2NOLcoeWSWFg~7A2knJd0Dw6bb6nqXzquLO2P9jl~HHisJ0crUrAlL3WYzDFHftdU4N181Fdu3~kHg__&Key-Pair-Id=APKAIE5G5CRDK6RD3PGA)
Expression of the P+C domains of calreticulin reduces adipogenesis. (A) A schematic of the structural and functional calreticulin domains as well as Western blot analysis (with anti-HA antibodies) of G45crt−/− ES cells expressing the N+P and P+C domain of calreticulin. (B) Oil red O staining in WT, G45crt−/−, and G45crt−/− ES cells expressing P+C and N+P domains. G45crt−/−cells expressing the P+C domain had decreased staining compared with the G45crt−/− cells. When the cells were treated with 50 nM BAPTA-AM, both the P+C domain–expressing and WT ES cells showed a dramatic increase in oil red O staining. The N+P domain–expressing ES cells had oil red O staining similar to that of the G45crt−/− ES cells. When the cells were treated with 500 nM ionomycin, both the N+P domain–containing and G45crt−/− ES cells showed a decrease in oil red O staining. (C) Expression of the P+C domain in 3T3-L1 cells (3T3-L1 + [P+C]) reduced oil red O staining. Because 3T3-L1 is used as a control, it is always counted as 1. Expression of the P+C domain was confirmed using HA antibody. (D) Adipogenic marker expression was decreased in the P+C domain–containing G45crt−/− cells when compared with the G45crt−/− cells (P < 0.01; n = 6) and was comparable to that in WT cell levels (P > 0.05). BAPTA-AM treatment increased adipogenic marker expression in all cell lines (n = 6). (E) Adipogenic marker expression was higher in the N+P domain–expressing G45crt−/− cells than in the WT ES cells (P < 0.01; n = 6) and was similar to that in G45crt−/− cell levels (P > 0.05). Ionomycin treatment reduced adipogenic marker expression in all cell lines (n = 6). (F) 45Ca2+-loaded ES cells were treated with thapsigargin or ionomycin to measure ER-releasable and total [Ca2+], respectively. WT and P+C domain–expressing G45crt−/− cells had higher [Ca2+]ER and [Ca2+]Tot than the G45crt−/− cells (n = 3). (G) Fura-2-AM–loaded ES cells were treated with thapsigargin to measure ER-releasable and cytosolic [Ca2+]. WT and P+C domain–expressing G45crt−/− cells had higher [Ca2+]ER and higher cytosolic [Ca2+] than the G45crt−/− cells (n = 3). (H) 45Ca2+-loaded 3T3-L1 cells were treated with thapsigargin or ionomycin to measure [Ca2+]ER and [Ca2+]Tot levels. 3T3-L1 + CRT cells (overexpressing calreticulin) and 3T3-L1 expressing the P+C domain had higher [Ca2+]Tot and [Ca2+]ER than the control 3T3-L1 cells (n = 3). Error bars represent SD.
