CriticalAppraisaloftheMTTAssayinthePresenceofRottlerin4

Table 1 MTT reduction normalized to cell number

The MTT (% control)/cell number (% control) ratio was calculated in the samples treated with 5 and 20 μM Rottlerin (R5 and R20, respectively) at the indicated time points

As shown in Fig. 3 , an overestimation of the MTT assay with respect to cell number was also observed in HMVEC, after a 24-h treatment with 20 μM Rottlerin.

Fig. 3 Effect of Rottlerin on HMVEC cell number and viability assessed by direct cell counting and MTT assay, respectively. HMVEC were treated with 20 μM Rottlerin (R20) before adding MTT. a Twenty-four hours later, cell number was determined, and b cell viability was measured as described in “Materials and methods”. Results are expressed as % of the control (100%). Values are the average of three separate experiments in quadruplicate and are expressed as mean ± SD. *p < 0.05.

LDH assay in the presence of Rottlerin

As shown in Table 2 , the kinetic of NADH oxidation by LDH is unchanged in the presence of 5 (not shown) and 20 μM Rottlerin with respect to the control, as it results from the very similar ΔA at the different times, although the absolute optical densities (OD; unchanged in the presence of 5 μM Rottlerin) are higher in the presence of 20 μM Rottlerin due to its intrinsic absorbance at 340 nm. Anyway, LDH activity and quantification are not altered by Rottlerin, the spectrophotometric interference of which does not invalidate LDH quantification.

Table 2 LDH absorbance changes at 340 nm as a function of time in the presence or absence of Rottlerin

The values reported in the table are representative of two LDH assays in the same sample and conditions

Rottlerin effect on the energy charge