Optiplate 96 hb

MM001 and MM047 were seeded in 24-well plates and transfected with 400 ng of pGL4.23-enhancer vector + 40 ng of pRL-TK Renilla vector (Promega) with Lipofectamine 2000 (Thermo Fisher Scientific). As positive controls, the previously published enhancers MLANA_5-I, IRF4_4-I and TYR_−9-D or ABCC3_11-I and GPR39_23-I were used for MM001 and MM047, respectively^{77 (link)}. One day after transfection, luciferase activity was measured through the Dual-Luciferase Reporter Assay System (Promega) by following the manufacturer’s protocol. Briefly, cells were lysed with 100 µl of passive lysis buffer for 15 min at 500 rpm. A total 20 µl of the lysate was transferred in duplicate in a well of an OptiPlate-96 HB (PerkinElmer) and 100 µl of luciferase assay reagent II was added in each well. Luciferase-generated luminescence was measured on a Victor X luminometer (PerkinElmer). A total of 100 µl of the Stop & Glo Reagent was added to each well and the luminescence was measured again to record Renilla activity. Luciferase activity was estimated by calculating the ratio luciferase/Renilla; this value was normalized by the ratio calculated on blank wells containing only reagents. Three biological replicates were done per condition for MM001 and two biological replicates for MM047.

Cytotoxicity was determined either by staining for cell surface accumulation of CD107a as above where indicated, or by four-hour chromium (⁵¹Cr)-release assay. Briefly, 1x10⁶ target cells were labelled with 20 µL ⁵¹Cr amounting to 3.7 MBq (PerkinElmer) for 60 minutes at 37°C. Following this, target cells were co-cultured with effector CAR T cells at range of effector: target (E:T) ratios (10:1, 5:1, 2.5:1 and 1.25:1) for four hours at 37°C in 96 well U bottom plates (Grenier). After incubation, the plates were centrifuged at 1500RPM for 5 minutes and 50 µL of the supernatant was transferred to 96 well OptiPlate-96 HB (PerkinElmer). 150 µL of scintillation fluid was added per well and the plates were sealed and incubated at room temperature overnight. ⁵¹Cr release from lysed target cells was counted on 1450 MicroBeta Trilux Scintillation Counter (PerkinElmer). The scintillation counts from wells with only targets (without effectors) were used as spontaneous release controls and target cells lysed with 1% Triton X-100 (Thermofisher) were used as a maximum ⁵¹Cr release control.

To estimate the interaction of mycobacterial PPE51 with the T-6 compound, independent wells of 96-well plates (OptiPlate—96 HB; Perkin Elmer) were coated with 100 µL of recombinant PPE51_Mtb or MBP-PPE51_Mtb protein, and, as a control, recombinant NAD-dependent DNA ligase LigA_Mtb [45 (link)] and MBP-MtrA_Mtb at concentrations of 20 µg/mL in 0.1 M sodium carbonate buffer, pH = 9.5. The optimal concentration of the coating protein was evaluated in the preliminary experiments. After 90 min of incubation at 37 °C, the wells were extensively washed with PBS/0.05% Tween 20 (Sigma, Darmstadt, Germany) and blocked with 1% skim milk in PBS for 1 h, at room temperature. In the next step, all samples were washed with PBS/0.05% Tween 20, and 5 µCi/well [³H]T-6 in DMSO, corresponding to 1 µg of T-6, was applied to each sample for an additional 2 h, at room temperature. Unbound isotope-labeled T-6 was removed by intensively washing with PBS, and the amounts of the bound compound were determined by liquid scintillation counting. All samples and experiments were performed in triplicate. The wells coated with the recombinant proteins and incubated with PBS instead of [³H]T-6 served as negative controls. The protein-uncoated/blocked samples incubated with [³H]T-6 were used to calculate the background radioactivity.