Endo porter delivery reagent

For Figures 3b and 5d, a modified, non-radioactive version of our chromium-based assay was conducted using the CytoTox-ONE Homogenous Membrane Integrity assay (Promega), which measures LDH release as a measure of cell death. Assay setup was similar to the chromium assay, excluding the chromium loading step and a 50:1 E:T ratio of PBMCs to tumor cells (because of the higher sensitivity of this assay). After 4 h incubation of cells at 37 °C, supernatant (50 μL) was collected and mixed with 50 μL CytoTox-ONE reagent. Luminescence was measured using a BMG FLUOstar OPTIMA (BMG Labtech, Cary, NC, USA) and percent lysis solely due to ADCC was calculated by: %specific lysis (PBMC+antibody)-%specific lysis (PBMC alone). For reversal experiments, antioxidant SOD mimetic MnTBAP (Santa Cruz Biotechnology, Dallas, TX, USA), or caspase inhibitor qVD-OPh (EMD Millipore, Billerica, MA, USA), alone and in combination, were added during antibody incubation. For inhibition of NF-κB activation, NRAGE peptide (described above) was added with 6 μM EndoPorter delivery reagent (GeneTools, LLC, Philomath, OR, USA) to facilitate intracellular transport.

Mononuclear or HL60 cells were transfected with 10 μM of VDR siRNA or Bim siRNA or scrambled Control siRNA, using Endo-Porter delivery reagent from Gene Tools Inc (Philomath, OR) before exposure to other agents. The cells were allowed to recover in RPMI 1640 medium with 10% FCS for 24 h, and then were exposed to the indicated compounds for the indicated times. The reduction of target protein or mRNA by siVDR or siBim transfection in the enhancement group was approximately 40%, an efficiency consistent with other reports in the literature for AML cells which are difficult to transfect.

We delivered 1 mM MOs; Genetools, Philomath, OR, USA) into the cells using the Endo Porter delivery reagent. The sequence of antisense MOs was as follows: 5′-CATGGCTAAGTTATCTGTTAGGTCA-3′. Standard Control Oligo, Classic was used as a negative control. The sequence of control MOs was as follows: 5′-CCTCTTACCTCAGTTACAATTTATA-3′.

HL-60, Molm13, HL-60 R, and Molm13 R cells were seeded in a 6-well plate at a density of 3 × 10⁵ cells/well. When reaching 50% confluence, the cells were transfected according to Endo-Porter delivery reagent from Gene Tools Inc (Philomath, OR). AraC was dissolved in PBS to the required concentration for culturing cells. The culture medium was changed after 6 h, and the cells were collected 48 h after transfection.
HL-60 and Molm13 cells were transfected with short hairpin RNA targeting negative control (sh-NC), sh-MEG3-1, sh-MEG3-2, inhibitor NC, miR-493-5p inhibitor, overexpression (oe)-NC, oe-METTL3, oe-MYC, AraC, mimic NC, miR-493-5p mimic, sh-METTL3, or sh-MYC.
HL-60 R and Molm13 R cells were treated with oe-NC, oe-MEG3, mimic NC, miR-493-5p mimic, sh-NC, sh-METTL3-1, sh-METTL3-2, sh-MYC-1, sh-MYC-2, AraC, oe-NC, inhibitor NC, miR-493-5p inhibitor, oe-METTL3, or oe-MYC.
The required plasmids or sequences were chemically synthesized by Shanghai GenePharma Co. Ltd. (Shanghai, China). The cells were collected after transfection for 48 h, then pre-incubated with AraC for 2 h, and finally used for subsequent experimentations. The silencing sequences are listed in Additional file 2: Table S2.