Figure 5

Increased Aβ ₄₂ /Aβ ₄₀ ratio reduced responsiveness of SH-SY5Y cells to RA and the knock down of up-regulated CRABP1 rescued their differentiation potential. Phase contrast images showing living human neuroblastoma cells (SH-SY5Y), grown on collagen coated glass cover slips and treated with 1 nM retinoic acid (RA). Differentiation was evaluated by the number, shape and length of outgrowing protrusions: (1A) C99I45F (Aβ₄₂/Aβ₄₀↑); (2A) C99V50F (Aβ₄₂/Aβ₄₀↓). Differentiation was evaluated after RA-treatment for 6 days. Both cultures were 50% confluent when RA was added (day zero). C99I45F reached 90–100% confluency after 4–6 days without any signs of differentiation, whereas C99V50F did not exceed more than 60–70% confluency (after 6–10 days) but showed strong differentiation. C99I45F was also evaluated at 60–70% of confluency showing no signs of differentiation (data not shown), thus strong confluency of C99I45F (shown here) does not conceal putative signs of differentiation. (B) C99I45F (Aβ₄₂/Aβ₄₀↑): 30 nM siRNA was administered to the cells for 24 hours in combination with a treatment of 1 nM RA for 2.5 days. After 2.5 days, the effects of more than 50% knockdown of CRABP1 (2B) was compared with a nonsense sequence (negative control, (1B)). (C) C99I45F: same conditions as in (B) except that RA was administered for 4 days. Differentiation was evaluated after 4 days. Knockdown of CRABP1 (2C) was compared with a nonsense sequence (negative control, (1C)). (D) C99I45F: same conditions as in (C), but with another preparation from the same experiment as in (C). (B) and (C) show preparations from different experiments. Experiments were repeated three times with consistent results.