Figure 2.

Increased Intracellular Ca²⁺ Affects HPCA-PKCα Activation during Neuronal Differentiation of NSCs

(A and B) NSCs were used for fluo-3/AM Ca²⁺ imaging 1 day after plating on poly-L-lysine-coated glass coverslips (see Experimental Procedures). We collected images every 1 s at 470 nm excitation/514 nm emission using Metafluor software. The threshold of increased intracellular Ca²⁺ was defined as a 20% increase from the basal level.

(C) Cells were depleted with bFGF for 5 min, fixed, and immunostained with anti-HPCA (red). The boxed area is magnified. Scale bar, 5 μm.

(D and E) Cells were pretreated with 500 μM EGTA for 1 day and incubated for 3 days after removal of bFGF. After immunostaining with anti-TUJ1, TUJ1-positive cells were counted under fluorescence microscopy. The proportion of TUJ1-positive cells and total cells was determined in random areas from at least three slides of each condition. Data are shown as mean ± SD (n = 3). N.S, not significant (p = 0.245). Scale bar, 20 μm.

(F) Schematic of Hpca (wild-type) and its deletion mutants.

(G and H) NSCs were transfected with pMSCV-IRES-EGFP, pMSCV-IRES-EGFP-Hpca-Myc, pMSCV-IRES-EGFP-Hip_1-72-Myc, or pMSCV-IRES-EGFP-Hip_65-193-Myc for 48 hr and induced to differentiate for 24 hr. mRNA levels of neuronal factors were analyzed by RT-PCR (G) and real-time RT-PCR (H). ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3).

(I and J) G2 is a key residue for myristoylation of HPCA. NSCs were transfected with pMSCV-IRES-EGFP, pMSCV-IRES-EGFP-Hpca-Myc, or pMSCV-IRES-EGFP-Hpca G2A-Myc for 48 hr and induced to differentiate for 24 hr. mRNA levels of neuronal factors were analyzed by RT-PCR (I) and real-time RT-PCR (J). ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3).

(K) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and induced to differentiate by withdrawal of bFGF for 24 hr. Cells were then lysed and centrifuged at 100,000 × g to separate cytosolic and membrane fractions. Samples of protein (30 μg) from each fraction were analyzed by western blotting with anti-PKCα, anti-MYC, anti-integrin α2, and anti-GAPDH. Data are shown as mean ± SD (n = 3).

(L) Cells were transfected with p-MSCV-IRES-EGFP-Hpca-Myc for 48 hr, and induced differentiate for 24 hr. Immunoprecipitates (IP) from the cell lysates with anti-MYC were analyzed by western blotting with anti-PKCα and anti-MYC.

(M) Cells were transiently transfected with control siRNA or Hpca siRNA for 48 hr, then incubated for 1 day after removal of bFGF. Proteins were analyzed by western blotting with anti-p-PKCα, anti-PKCα, anti-HPCA, and anti-GAPDH. ^∗p < 0.05 compared with the −bFGF/control siRNA (mean ± SD; n = 3).

(N) Cells were transfected with pMSCV-IRES-EGFP or pMSCV-IRES-EGFP-Hpca-Myc for 48 hr and then treated with 10 μM RO320432 for 30 min before differentiation for 1 day. Cells were lysed and analyzed by western blotting with anti-TUJ1, anti-GFAP, anti-MYC, and anti-GAPDH. ^∗p < 0.05 compared with −bFGF/Hpca (mean ± SD; n = 3).