Aida image analyzer 4

IHC sections were stained with anti-APP antibody 6E10 (Covance) as described previously [28 (link)]. For the densitometric analysis, two sections per mouse were used (total magnification of 40×). Five areas were determined to be measured. All areas were corrected for the value of the background area. For cortical tissue, two distinct areas were analyzed and mean value of both measures was used. Experimenters were blinded for the treatment of the mice during the analysis. Microscopic pictures of the IHCs were acquired by an EVOS XL microscope (Life Technologies, Darmstadt, Germany). AIDA image analyzer 4.26 software (Raytest, Straubenhardt, Germany) was used for quantitative analysis.

THP‐1 macrophages (0.8 × 10⁶/well) were seeded in 6‐well plates and first treated with the indicated concentrations of piperine and pioglitazone for 24 h in FBS‐free RPMI‐1640 medium supplemented with 0.1% BSA and 10 μg/mL unesterified cholesterol. After incubation, cells were lysed with NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40; 1% protease inhibitor Complete (Roche); 1% PMSF; 0.5% Na₃VO₄; 0.5% NaF) for 30 min at 4°C. The lysed cells were collected and centrifuged at 16 060 g for 20 min to collect the supernatant. Protein concentration was assessed with Bradford assay using Roti^®‐Quant from Carl Roth (#K015.1, Karlsruhe, Germany). Total protein (20 μg per sample) was loaded and separated via SDS‐PAGE. Expression levels of specific proteins were analyzed using antibodies against the indicated proteins and visualized with ECL reagent and a LAS‐3000 luminescent image analyzer (Fujifilm) with AIDA image analyzer 4.06 software (Raytest). A representative whole‐blot picture resulting from the detection of ABCA1 in the presence and absence of piperine is presented in the Supporting Information Fig. 1.

THP-1 cells were seeded in 6-well plates and differentiated into macrophages with 200 nM PMA for 72 h. The macrophages were treated with the indicated compounds at the corresponding time points, as described in detail in each figure legend. Cellular proteins were extracted using NP40 buffer (150 mM NaCl; 50 mM HEPES (pH 7.4); 1% NP40; 1% protease inhibitor Complete™ (Roche); 1% phenylmethylsulfonyl fluoride (PMSF); 0.5% Na₃VO₄; 0.5% NaF) and quantified with the Bradford method. For western blot analysis, an equal amount of protein (20 μg) was separated via SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked for 1 h at room temperature with 5% non-fat milk, and then incubated at 4°C overnight with primary polyclonal antibody against ABCA1, ABCG1 or SR-B1 (1:500) or actin (1:10,000). After being washed for three times (15 min each time), the membranes were incubated with a secondary antibody [horseradish peroxidase-conjugated goat anti-rabbit (1:500) or anti-mouse (1:10,000)] for 1 h at room temperature. The signals were detected and visualized using a LAS-3000 luminescent image analyzer (Fujifilm, Düsseldorf, Germany) using AIDA image analyzer 4.06 software (Raytest, Sprockhövel, Germany). The indicated protein band intensity was normalized to that of actin.