Figure 7.

Prolonged effect of ATF3 inhibition on neurite outgrowth. (A1–A4) Cortical neurons derived from P5 opossum. (A1,A3) represent control neuronal cultures treated with PBS only. At DIV10, neurons were first treated with SP inhibitor for 24 h, followed by scratch and washout for the next 24 h (A2), or incubated with SP immediately after scratch over the next 24 h and followed by 24 h washout (A4). (B1–B4) Cortical neurons derived from P16 opossum. (B1,B3) represent control neuronal cultures treated with PBS only. At DIV10, neurons were first treated with SP inhibitor for 24 h, followed by scratch and washout for the next 24 h (B2), or incubated with SP immediately after scratch over the next 24 h and followed by 24 h washout (B4). Neurons were fixed and immunostained for TUJ1. The scratch area is defined by the red dashed lines. Scale bar is 50 μm. (C) The scatter plot shows P5 average TUJ1 pixel intensity. Data are shown as mean ± SD. For each condition, 2 ROIs of 3 random fields per sample from 3 culture preparations were analyzed. Welch’s t-test, SHAM CUT 24H vs. SP + CUT 24H p < 0.001 ***. Unpaired t-test, SHAM CUT 48H vs. CUT 48H + SP p < 0.001 ***. (D) The scatter plot shows P16 average TUJ1 pixel intensity. Data are shown as mean ± SD. For each condition, 2 ROIs of 3 random fields per sample from 3 culture preparations were analyzed. Unpaired t-test, SHAM CUT 24H vs. SP + CUT 24H p < 0.001 ***. Unpaired t-test, SHAM CUT 48H vs. CUT 48H + SP p < 0.001 ***. (E) Schematic representation of the time course of SP inhibitor application. (1) represents the application of SP for experimental conditions (A2,B2). (2) represents the application of SP inhibitor for experimental conditions (A4,B4).