Fig 2: Effect of 2b inhibitor (20 and 80 µM) on the Vero-76-infected monolayers. Control (A), treated cells with 20 µM (B), treated cells with 80 µM (C), infected cells (D), infected treated cells (20 µM) (E), and infected treated cells (80 µM) (F). Pictures of cell morphology were taken at 72 h post-infection using ZOE Fluorescent cell imager (Bio-Rad) (bar size = 100 µm, magnification, 20×).

Fig 3: Cell imaging with blue and green staining. Human dermal fibroblasts in the concentration of 1 × 105 cells per well were seeded on 96-well plates without disks (control), UV-sterilized and media-soaked disks for 2 h and 48 h, respectively, and 2 h UV-sterilized disks only. Staining was carried out according to the manufacturer’s guidelines for 30 min and plates were centrifuged before imaging via ZOE Fluorescent Cell Imager (Bio-Rad Laboratories) at 4× magnification. DAPI (blue dye) represents the nuclei of all the cells and GFP (green dye) represents the nuclei of damaged cells. The colocalization was analyzed from a minimum of 3 representative images from three independent experiments after 2.5 h incubation. Data are represented individually and as average ± SEM. Scale bar 100 µm.

Fig 4: PAMAM-DENCYS decreases P. aeruginosa infection in CF cells.(A, B) IB3-1 cells were seeded on a 6-well plate and treated with PAMAM-DENCYS for 12 hours followed by Pseudomonas aeruginosa-GFP (PA01-GFP) infection at MOI of 10 (A) and 1 (B) for 180 minutes. Cells were washed thoroughly with PBS (1x) to remove all extracellular bacteria. Bio-Rad ZOE™ Fluorescent Cell Imager was used to capture images. Representative bright field (left) and fluorescent images (right) show cell number and the number of intracellular bacteria respectively. (C) We quantified the number of fluorescent bacteria and found that PAMAM-DENCYS significantly decreased the PaO1 counts at both MOI of 1 and 10 (**p<0.01), verifying the efficacy of PAMAM-DENCYS as an autophagy-inducing antibacterial nano-formulation. (D) IB3-1 cells were transfected with WT-CFTR and infected with PA01 at MOI of 1 as a positive control. (E) In a parallel experiment, IB3-1 cells were infected with PA01-GFP (MOI of 1) for 180 minutes. Post infection, the media was removed and the cells were washed with PBS (1x) and used to quantify the number of intracellular bacteria using flow cytometry based analysis. Analysis of the flow cytometry data shows a significant (*p<0.05) decrease in the number of intracellular bacteria, and provide further evidence substantiating autophagy-mediated bacterial clearance by PAMAM-DENCYS formulation.

Fig 5: Intracellular distribution of DOX in 4T1 cells. Under the Bio-Rad Zoe cell imager, blue: nucleus after being stained with Hoechst 33258. Green: DOX because of its intrinsic fluorescence. Cyan: merged florescence signal.