Fig 2: PKAc cKO parasites develop and egress normally in the liver.(A) To determine if PKAc cKO parasites develop and egress from the liver, infected mice livers were harvested at the indicated time points. The parasite burden in the liver was checked by amplifying the parasite 18S rRNA. Mice were treated with a combination of chloroquine, beginning 24 h after sporozoite injection, and artesunate, beginning 39 h after sporozoite injection, to prevent the growth of blood stage parasites. Therefore, the decrease of copy number between 55 and 72 h indicates a loss of liver-specific signal. This experiment was performed twice with similar results, and data from one experiment are presented. Unpaired two-tailed t test was used for statistical analysis for 36 h (P = 0.824), 55 h (P = 0.623), and 72 h (P = 0.493). (B) Development of PKAc cKO and TRAP/FlpL EEFs in vitro. HepG2 cells were infected with SG sporozoites, and the culture was harvested at different time points. Cultures removed at 36 h p.i. were fixed and parasites were detected using anti-Hsp70 and anti-UIS4 antibodies. Scale bar, 5 µm. (C) Quantification of EEF numbers at 36 h p.i.; no significant difference was observed in three independent experiments. One-way ANOVA was used for statistical analysis (P = 0.693). (D) Cultures removed at 62 h p.i. were probed with UIS4 and MSP1 to visualize PV membrane and formation of merozoites, respectively. Scale bar, 5 µm. (E) Quantitation of EEF area at 36 and 62 h p.i. The EEF size was similar in both PKAc cKO and TRAP/FlpL parasites. Unpaired two-tailed t test was used for statistical analysis for 36 (P = 0.406), and 62 h (P = 0.977). Error bars represent SD. To determine the size of the EEFs, the perimeter was delineated using the “region of interest” tool and the area was calculated using Nikon NIS elements BR imaging software.

Fig 3: SSA does not influence the nuclear translocation of NF-?B in MDA-MB-231 cellsa. The immunofluorescence imaging results show that SS can block NF-?B to enter the nucleus by attenuating TNFa simulative effect in MDA-MB-231 cells, but SSA doesn't. The treatments include the vehicle control (0.1% DMSO), TNFa (25 ng/ml), TNFa+SS (50 µM), and TNFa+ SSA (4 µM). Red: NF-?B antibody; Blue: DAPI. Images were captured by using Nikon Eclipse Ti Laser Confocal Scanning Microscopy.b. Quantitative analysis of NF-?B in the nucleus. The relative fluorescent intensity of NF-?B was analyzed by using NIS-Elements AR imaging software. Data are presented as the mean of thirty measurements ± standard deviation. *P < 0.05.

Fig 4: MCL-1 targeting induces changes in actomyosin structure organization in a DRP-1-dependent manner.A Mitochondrial fission was detected by TOM20 staining in CAFs treated or not with S63845 500 nM or A1331852 100 nM for 18 h in the presence or not of Mdivi-1 50 µM. Scale bar = 50 µm. B Quantification of mitochondrial network length was performed by measuring the size of TOM-20 labeled structures on deconvolved images in 10 cells/condition in 3 independent experiments. Objects smaller than 0.5 µm were not considered. C Representative co-immunoprecipitation of MCL-1 and DRP-1 and western blotting for MCL-1, DRP1, or NOXA in CAFs treated or not with S63845 in presence or not of Mdivi-1 (n = 3). FT: Flow Through. D qRT-PCR of MYH10, MYH2, ACTA2, and ITGA11 mRNA in CAFs treated or not by S63845 500 nM for 18 h in the presence or not of Mdivi-1. Mean and SEM of three independent experiments are represented as fold-change of mRNA level related to untreated control. Two-way ANOVA, *P < 0.1, **P < 0.01, ***P < 0.001, ****P < 0.0001, ns: not significant. E Quantification (percentage of cells positive for nuclear YAP or nuclear and cytosolic YAP) (Bottom) and confocal image (Top) of YAP staining (red) and FITC-phalloidin (green) in CAFs treated or not with S63845. Randomly generated images set names were randomized for analysis. Around sixty to one-hundred cells were analyzed per condition. Counting events were done manually through NIS-Elements software (Nikon software). Fluorescence intensity and positivity staining were determined by positive and negative control comparison, dependent on the experiment. The nuclei were counterstained with DAPI (blue). Scale bar = 50 µm. Two-way ANOVA, *P < 0.05. F Quantification (Bottom) and confocal image (Top) of Myosin IIB staining (green) and immunofluorescence of TOM-20 (red) in CAFs treated or not with different BH3 mimetics (as indicated) in the presence or not of Mdivi-1. Randomly generated images set names were randomized for analysis. Around sixty to one-hundred cells were analyzed per condition. Counting events were done manually through NIS-Elements software (Nikon software). Fluorescence intensity and positivity staining were determined by positive and negative control comparison, dependent on the experiment. The nuclei were counterstained with DAPI (blue). Scale bar = 50 µm, Two-way ANOVA, ###P < 0.001, ##P < 0.01, #P < 0.05 for myosin IIB-, **P < 0.01; *P < 0.05 for fragmented mitochondria.

Fig 5: In vitro transcribed dsRNA, viral dsRNA, and polyinosinic:polycytidylic acid (poly I:C) bind the same class A scavenger receptor in RTgutGC cells. RTgutGC cells were pretreated for 3 h with high-molecular weight (HMW) poly I:C, low-molecular weight (LMW) poly I:C, poly I, or poly C at 100 µg/mL, or total chum salmon reovirus genome (v-dsRNA) at 40 µg/mL, or control media. Cells then received 1.25 µg (5 µg/mL) of Alexa Fluor 546 labeled CSVseg1 dsRNA (ivt-dsRNA) with 50 µg/mL of DEAE-dextran. 6 h posttreatment cells were formalin fixed, and nuclei were counterstained with diamidino-2-phenylindole; slides were visualized using an inverted fluorescence microscope (Nikon Eclipse TiE with Qi1 camera). (A) Representative images of cells with or without pretreatment, dsRNA = red, nuclei = blue. (B) Fluorescence intensity per cell was quantified using Nikon NIS-elements software and is presented as percent of the ivt-dsRNA only control, no pretreatment. Data represent three independent replicates and were analyzed statistically by one-way ANOVA, alpha = 0.05; a P value < 0.05 considered significant.