Fig. 2
FRET measurements of Casp3 activity in fixed OCCs and specificity controls. a calculation of FRETeff of the SCAT3 probes by acceptor photobleaching (experiment #3). There is a statistically significant difference in the ECFPem/Venusem ratio before and after photobleaching of pSCAT3-DEVG. The mean value of FRETeff was 0.22 ± 0.02 (see text and Additional file 1 for further information). b A comparison of the ECFPem/Venusem mean values after transfection with pSCAT3-DEVD (FRET probe) or pSCAT3-DEVG (control probe) shows a reduction of about 56 % in ECFPem/Venusem in cells transfected with the control probe. As in the control probe cellular Casp3 cannot cleave the FRET pair, this experiment demonstrates that the enzyme is constitutively active in CGCs in the absence of any pharmacological treatment. After transfection, OCCs were maintained in medium 2, containing Neurobasal and B27 supplement. c Specificity of the pSCAT3-DEVD probe for Casp3 is demonstrated after RNAi and Ac-DEVD-CMK inhibition. After multiple transfection with pSCAT3-DEVD and the mix of the four shRNA plasmids targeting the Casp3 gene or with pSCAT3-DEVD in the presence of the caspase inhibitor Ac-DEVD-CMK (100 μM) there is a significant reduction in the mean value of ECFPem/Venusem in comparison to that measured from OCCs transfected with pSCAT3-DEVD alone (pastel blue bars). There are notably no differences in mean values of ECFPem/Venusem when the pSCAT3-DEVG control probe was employed (blue bars), with the exception of OCCs transfected with a combination of pSCAT3-DEVG and the RNAi control clone [pSCAT3-DEVG 0.33 ± 0.01 (294 cells); pSCAT3-DEVG + RNAi control 0.27 ± 0.02 (100 cells); P = 0.01]. This result is somewhat puzzling as pSCAT3-DEVG is insensitive to Casp3 cleavage. It is also worth noting that after co-transfection with pSCAT3-DEVD + RNAi control values of ECFPem/Venusem displayed higher variance (0.21) and, consequently, SEM was much higher (0.15 – see bars in graph) than in all other tested conditions. To explain these data we can only speculate that transfection with a Casp3-unrelated RNAi control clone of unknown specificity (such as that provided by manufacturer) may somehow interfere with protein synthesis machinery in cells. d TCD after RNAi and Ac-DEVD-CMK inhibition of Casp3. There is a statistically significant increase of TCD after multiple transfection with shRNA plasmids that epigenetically inhibit the casp3 gene together with pSCAT3-DEVD (pSCAT3-DEVD 13.13 ± 1.80, n. cells = 584; pSCAT3-DEVD + RNAi 33.89 ± 4.09, n. cells = 840; P = 0.000926) or pSCAT3-DEVG (pSCAT3-DEVG 18.84 ± 3.72, n. cells = 941; pSCAT3-DEVG + RNAi 27.37 ± 3.91, n. cells = 1391; P = 0.04279), but not in RNAi control experiments and after Ac-DEVD-CMK [n. cells = 213 (pSCAT3-DEVD + RNAiCONTR), 585 (pSCAT3-DEVD+ Ac-DEVD-CMK), 133 (pSCAT3-DEVG + RNAiCONTR), 794 (pSCAT3-DEVG+ Ac-DEVD-CMK)]. In these experiments, we have counted a total of 5,841 transfected cells and measured their areas. Mean area was 165.71 ± 2.16 μ2, corresponding to a mean diameter of 15 μm for a circular object. e Transfection with pSCAT3-DEVD or pSCAT3-DEVG demonstrates the existence of a subpopulation of CGCs resistant to induction of apoptosis by ionotropic glutamate receptor agonists and A23187 after measurement of mean values of ECFPem/Venusem (pSCAT3-DEVD 0.59 ± 0.04; n. cells = 251; pSCAT3-DEVD + KA 0.30 ± 0.01; n. cells = 103; pSCAT3-DEVD + NMDA 0.34 ± 0.01; n. cells = 105; pSCAT3-DEVD + KA + NMDA 0.28 ± 0.01; n. cells = 62; pSCAT3-DEVD + A23187 0.27 ± 0.01; n. cells = 132; pSCAT3-DEVG 0.33 ± 0.01 n. cells = 294; pSCAT3-DEVG + KA 0.29 ± 0.01; n. cells = 98; pSCAT3-DEVG + NMDA 0.33 ± 0.01; n. cells = 80; pSCAT3-DEVG + KA + NMDA 0.29 ± 0.01; n. cells = 68; pSCAT3-DEVG + A23187 0.27 ± 0.005; n. cells = 7). Error bars = SEM. *P-value 0.05–0.01 ** P-value < 0.01–0.001 % *** P-value < 0.001 %