Fig. 2: Loss of Cdk5 causes increased mitochondrial Ca²⁺ level.

MEFs were isolated from wt and Cdk5^−/− embryos from Cdk5^+/− pregnant female mice. Cells loaded with Fluo-4 AM (5 µM) were subjected to single-cell Ca²⁺ imaging in Ca²⁺ free buffer. Carbonyl cyanide-p-trifluoromethoxyphenylhydrazone (FCCP, 1 µM) was added where indicated. a FCCP-induced Ca²⁺ flux to the cytoplasm is greater in Cdk5^−/− MEFs than in wt MEFs. Mean values of Ca²⁺ signals from 15 randomly selected cells from each genotype are shown. Data represent results from one of four independent experiments (n = 4) showing a similar staining patterns. Analysis of results from all four independent experiments, each with data from 15 randomly selected cells from wt and Cdk5^−/− MEFs, revealed that: b the peak amplitudes (F/F₀) of Ca²⁺ response to FCCP is higher in Cdk5^−/− MEFs than in wt, and (c) the integrated Ca²⁺ signals (area under the curve from 180 to 600 s) in response to FCCP is also higher in Cdk5^−/− MEFs than in wt, indicating greater Ca²flux to the cytoplasm due to increased stored [Ca²⁺]_mt in Cdk5^−/− MEFs compared with wt. MEFs obtained from two different sets of wt and Cdk5^−/− embryos were used at passage 2–7. Values are means ± SEM from the four independent experiments. *p < 0.05 by unpaired Student’s t test.