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Differentiation efficiency also did not show significant differences when all four clones were treated with CO or AO88 (Fig. 4d).

In parallel with non-targeting sgRNA clones, validated knockout clones were treated with PMA to induce macrophagic differentiation, subjected to iMiD treatment, and then stimulated with LPS.

Stable cell clones were treated with Dox to allow specific downregulation of the individual components of the CCR4-NOT complex as assessed at the mRNA level (Fig. 1A).

After 3 4 wks, single-cell derived clones were treated with 0.25% trypsin-EDTA and seeded into 24-well plates for further expansion.

Transgene expression proved optimal when clones were treated with 1 µg/ml doxycycline.

These clones were treated with E2 and RA alone or in combination before exposure to etoposide.

Each of the cell clones was treated with the proinflammatory cytokines TNF- α and IL-1 β and was transiently transfected with Tat101.

To determine if the NE and IE phenotypes observed within the U-937 cell clones could be induced into expression, each of the cell clones was treated with IL-1 β for 24 hours.

To determine if the NE and IE phenotypes observed within the U-937 cell clones could be induced into expression, each of the cell clones was treated with the various concentrations of TNF- α mentioned previously.

ICR2 DNA methylation was determined by COBRA: 1.5 µg of DNA extracted from single-cell clones was treated with sodium bisulfite, PCR-amplified, the PCR product digested with a restriction enzyme containing a CpG dinucleotide in its target sequence and the fragments separated on a polyacrylamide gel.

To confirm that the effects observed for these cell lines were consistent with each selected VDR genotype, two additional single clones along with the initially characterized clone, were treated with 1,25D3 for 7 days and subjected to the crystal violet assay.