Sigmaplot 14

Data are shown as mean ± SD, number, or rates. Data are graphed versus month and year, or versus year. The categorical data from the time after full NICU implementation is compared to the 3 quarters of 2015 prior to the full NICU implementation using Fisher’s Exact Test (Sigmaplot 14.0, Carlsbad, CA). Linear regression was used to compare 2 variables, for example, NICU survival versus number of NICU admissions (Sigmaplot 14.0). The slopes of NMR versus year for the time period 2006–2015 and 2016–2019 were compared using analysis of covariance (GraphPad Prism 9.0). A p-value of <0.05 is considered significant.
Details of the Development and Implementation of the Level III NICU at GPHC:

The MDCKII cells were seeded as described in the WST-1 assay section, and treated with increasing concentrations of mitoxantrone (0.01 µM up to 50 µM) or solvent (0.1% DMSO) for 48 h. In order to detect a reversal of the ABCG2-mediated chemoresistance, novel ABCG2 interacting compounds (QCc, DMQCa, DMQCb, DMQCc, and DMQCd) were added to mitoxantrone in 1.0 µM for 48 h. Afterwards, the WST-1 assay was performed as described [37 (link)]. The left-shift factor was calculated from IC₅₀ minus SEM from the mitoxantrone alone treatment by IC₅₀ plus SEM from combined use of mitoxantrone and carborane-based derivatives (QCc, DMQCa, DMQCb, DMQCc, and DMQCd). Data were tested for normality by the Shapiro Wilk test and significant differences between the mitoxantrone treatment groups were determined by two-way ANOVA with the Holm-Šidák post hoc test using SigmaPlot 14.5. The IC₅₀ values were defined as 50% reduced cell viability and calculated with SigmaPlot 14.5 by nonlinear regression. All data were normalized against the vehicle control and were expressed as mean ± SEM, calculated from at least three independent experiments.

The parameters measured were the latency to the onset of activity, the duration of activity, the calculated cocaine level at the onset of activity (the satiety threshold), and the calculated cocaine level at the cessation of activity (the remission threshold). These data were collected from multiple rats over multiple sessions, however, as rats lost catheter patency the overall number of animals used fluctuated. All the parameters were analyzed using SigmaPlot 14.5. ANOVA was conducted to compare SCH23390 doses and statistical significance was determined by comparing the values to the vehicle.
Following administration of h2E2 the same parameters were collected each day for 21 days, and statistical significance of these values were compared to the baseline using a One Way ANOVA. Baseline values were collapsed across days. The mean of 3–5 sessions for each of the 7 rats was calculated. These values were averaged. The ANOVA was conducted using SigmaPlot 14.5.

The autofluorescence of all compounds was investigated with a modified Hoechst 33342 accumulation assay. The MDCKII-hABCG2 and MDCKII-WT cells were seeded and treated as previously described for the Hoechst accumulation assay. Afterwards, the cells were washed twice with ice-cold PBS buffer, lyzed with 100 µL/well 0.1% (v/v) SDS/PBS, and, subsequently, the intracellular fluorescence was measured by spectrofluorometer (360 nm excitation/465 nm emission wavelengths, Tecan Infinite F200 Pro, Crailsheim, Germany). The protein amounts were quantified by BCA assay. For this approach, the Hoechst 33342 dye was not added. The background of non-treated cells was subtracted from the measured intracellular fluorescence and was correlated to the protein amount for MDCKII-hABCG2 or MDCKII-WT cells, respectively. All data were expressed as mean ± SEM and tested for normality by the Shapiro Wilk test using SigmaPlot 14.5. An autofluorescence was defined as the significant increase in total intracellular relative fluorescence unit (RFU) in comparison to the solvent-treated control and was determined by one-way ANOVA with Holm-Šidák post hoc test using SigmaPlot 14.5. As the negative control, the ABCG2-Inhibitor Ko143 (1 µM) was added.

GraphPad Prism 8 software (San Diego, CA) was used for statistical analyses. For comparing two groups, unpaired t tests were used. For comparisons between three or more groups, one-way analysis of variance (ANOVA) followed by Tukey’s post-hoc test for multiple comparisons between treatment groups was conducted. For analyzing the nest building behavior, a two-way repeated measures ANOVA was performed using the Sigma Plot 14 (San Jose, CA) software. Differences were accepted as significant at p < 0.05. Statistical details of each experiment are provided within the relevant result and legend sections.