Monochrome camera

For obtaining the EEFs, the HepG2 cells were cultured in 24 well culture plates (Corning). When HepG2 cells reached 60–70% confluency, 2 × 10⁴ sporozoites were added per well. Cultures were fixed at different time points of EEF development and IFA was performed as described earlier59 (link). Briefly, the cultures were incubated for 1 h with UIS4 antibody and immunoreactivity reveled by anti-rabbit antibody conjugated to Alexa Fluor 594 (Life Technologies). The HepG2 and parasite nuclei were stained with DAPI. After final wash the coverslips were mounted using antifade reagent (Life Technologies). To reveal the stage specific expression and localization of the reporter in the pbspeldmCherry transgenics, the oocyst and salivary gland sporozoites were fixed and stained with 3D11 monoclonal antibody38 (link) which reacted with the CSP, a major surface protein of sporozoite. The CSP immunoreactivity was revealed by using antimouse antibody conjugated to Alexa Fluor 488. To monitor the expression of mCherry in the developing liver stages, 2 × 10⁴pbspeldmCherry transgenic sporozoites were added to HepG2 cultures and harvested at different time points. All processed slides were visualised using a Nikon (Ni-E AR) upright fluorescent microscope. Images were procured using monochrome camera (Andor) and captured images were processed using software NIS elements AR.

For the detection of phospho GR and total GR localization, MM.1S cells were treated according to the study design and washed with 1XPBS. Cells were plated to coverslips using cytospin. Cells were fixed with 100% ice-cold methanol (MeOH) and permeabilized/ blocked with 0.1% Tween 20, 0.3 M glycine, and 1% BSA in 1XPBS. The cells were then probed with the appropriate primary antibody for 1 hour and washed 3 times. The rabbit secondary antibody, Alexa Fluor 488 (Invitrogen, A11008) was used for all the staining, while nuclei were stained with DAPI (Invitrogen). Protein localization was visualized with a Nikon Eclipse Ti inverted fluorescence microscope (Nikon) and monochrome camera (ANDOR) at 20× magnification.

HT1080 parental and resistant cells were plated on glass coverslips (BioCoat, BD Biosciences) at 500,000 cells/well in 6 well plates and grown overnight. Cells were treated with 1 μM KPT −185 for either 4 hours to detect p53 and IkB or for 24 hours to detect p21, p27, FOXO-1, and PP2A. After treatment, coverslips were washed with 1× PBS (phosphate buffered saline) then fixed in either 3 % paraformaldehyde buffer (3 % paraformaldehyde/2 % sucrose/1× PBS) or 100 % ice-cold methanol for 15 min then washed with 1× PBS. Cells were permeabilized with 0.1 % Triton X-100/1 % BSA/1× PBS (PFA fixation) or 0.1 % Tween 20/0.3 M glycine/1 % BSA/1× PBS (Methanol fixation) for at least 30 minutes. After washing 3 times with 1× PBS, cells were stained overnight with the corresponding antibodies listed above diluted in 1%BSA/1× PBS. Protein signal was detected with species specific Alexa Fluor 488 secondary antibodies (Invitrogen) while DNA was stained with DAPI (Invitrogen). Protein localization was visualized with a Nikon Eclipse Ti inverted fluorescence microscope (Nikon) and monochrome camera (ANDOR).