Elx808 absorbance reader

The total calcium concentration in all solutions was determined using an atomic absorption spectrometer (AAS; AAnalyst 400, Perkin Elmer Analytical Instruments, Waltham, MA, USA). Lanthanum nitrate (0.5%, lanthanum nitrate hexahydrate: La(NO₃)₃·6H₂O) was added to the solution to eliminate the interference of other ions^{14 (link)}.
The inorganic phosphate concentration was analysed photometrically using the method reported by Chen et al.^{15 (link)}. From the test solution, an aliquot was diluted with ultra-pure water, and 2 ml of this dilution was mixed with 2 ml of a phosphate reagent (2% ascorbic acid, 0.5% ammonium heptamolybdate, 0.6 M H₂SO₄). This mixture was stored for 90 min at 37 °C, allowed to cool to 24 °C, and then absorbance was measured at 820 nm using a spectrophotometer.
The total protein concentration was determined colorimetrically using a Pierce^TM BCA Protein Assay Kit (Thermo Scientific) with bovine serum albumin (BSA) as a standard. The assay was performed in a 96-well micro plate using triplicates of 25 µl of each sample and standard. The plates were read at 570 nm using an ELx808 Absorbance Reader (BioTek).

CMT-1026 was diluted to different concentrations to provide a normal curve diagram (34 (link)). Cells were seeded in triplicate in 96 well plates. After 24 h of cultivation, CMT-1026 cells were treated in triplicate with a Cell Counting Kit for 4 h (CCK-8, Beyotime, Shanghai, China) and then detected at 24 h intervals for 9 days. Then the different concentrations were determined with a 450 nm filter and a microplate reader (ELx808 Absorbance Reader, BioTek, USA). When cell growth was in the exponential phase, the CMT-1026 doubling time was calculated with GraphPad Prism 8 software (GraphPad Software, Inc. USA).

Mouse β-defensin-3 were measured by ELISA (MyBioSource, MBS034940, San Diego, CA, US) according to manufacturer’s instructions. Briefly, each sample was separately weighted, and PBS was added accordingly (10 µl/mg of sample). Then, samples were mechanically homogenized with 3.0 mm ceramic beads (Sigma, USA) in a bullet homogenizer (Next Advance, NY USA). The samples were homogenized for 10 min at maximum speed, followed by centrifugation at 3000 rpm for 20 min. The supernatants were then collected and loaded on the ELISA plate with blanks and standards. Each sample was run in duplicate. Optical density was read at 450 nm using ELx808 absorbance reader (BioTek, Winooski, VT, USA), and the results were expressed in pg/ml.

A 96-well microtiter plate, containing 200 μl MHBII per well, was inoculated with cells in exponential growth phase using a 96-pin replicator. All seven isolated colonies per lineages were included in the growth measurement. OD₆₀₀ was measured in a ELx808 Absorbance Reader (BioTek) every 5 min for 10 h at 37°C and 650 r.p.m. The data was analyzed with R (Team R Core, 2014 ). The growth rate was calculated based on the steepest part of the growth curve during exponential growth. The doubling time was normalized to the wild type and plotted grouped by drug with the package ggplot (Wickham, 2009 ). To test whether the observed differences in growth rate were significant (P < 0.05) the non-parametric Kruskal-Wallis one-way analysis of variance was applied in R.

CCK-8 assay was used to detect the effect of Tan IIA, miR-497-5p mimic or miR-497-5p inhibitor transfection, or the combination on the proliferation of AML cells. In brief, the transfected cells were seeded in 96-well plates at a density of 5 × 10⁴ cells/well in 200 µl culture medium. At appointed time, 10 µl of CCK-8 assay solution (Dojindo Molecular Technologies, Japan) was added to each well, and incubation at 37 °C with 5% CO₂ for 2 h, the optical density at 450 nm wavelength was selected for the measurement using an ELx808 absorbance reader (BioTek Instruments, USA).