Functional Autophagic Flux Regulates AgNP Uptake And The Internalized Nanoparticles Determine Tumor Cell Fate By Temporally Regulating Flux

Figures & data

Figure 1 Synthesis and characterization of AgNPs. (A) UV-Visible spectra analysis confirming the synthesis of AgNPs. Inset showing test tubes after the formation of AgNPs at different concentration of silver nitrate solution. (B, C) TEM images of ss-AgNPs and ls-AgNPs, respectively. (D, E) Particle size distribution histogram of AgNPs determined using TEM images.

Figure 2 Analysis of internalization of ss-AgNPs and ls-AgNPs. (A) Phase-contrast images of cells after treatment with 25 µM of AgNPs for 24 h. “Ctrl” represents un-treated cells. Scale bar- 100 µm (B) MTT assay analyzing cell viability after 24 h of treatment with AgNPs in MCF-7 cells. [C (i) and (ii)] Fluorescent microscopic images of MCF-7 cells after treatment with IC₅₀ dose of C6 tagged AgNPs for 6 h and 24 h. Scale bar- 200 µm. (D) A comparative fold change between C6 tagged ss-AgNP and ls-AgNp uptake (25 µM) measured through flow cytometry at different time points. (E) A comparative difference in uptake of C6 tagged AgNPs (IC₅₀) at different time points (1 h, 6 h & 24 h) as measured through flow cytometry. The symbol (*) represents a significant difference between different treatments.

Figure 3 Analysis of intra-cellular uptake mechanism of ss-AgNPs and ls-AgNPs. Flow cytometric analysis of the uptake of ss-AgNPs (A) and ls-AgNPs (B) (at IC₅₀ dose) 1 h after treatment with NPs. The cells were exposed for 30 min to the different endocytic pathway inhibitors prior to AgNP exposure. The inhibitors used were- Nocodazole (Noc; 0.5 µM), Genistein (Gen; 10 µM), Chlorpromazine (CPZ; 5 µM) and Dynasore (Dyn; 50 µM). (C) Immunofluorescence images of cells treated with IC₅₀ dose of ss-AgNPs (green) with and without Dyn. Cells were stained with anti-rabbit TR conjugated secondary antibody against clathrin (red). Scale bar- 200 µm. The symbol (*) represents a significant difference with respect to AgNP-treated cells.

Figure 4 Analysis of early induction of autophagy and JNK after AgNP exposure. (A) Immunoblot showing expression of LC3B-II and p62 after 1 h of treatment with AgNPs. (B) Immunoblot showing expression of LC3B-II, p62 and JNK after treatment with AgNPs for 6 h. (C) Immunoblot showing expression of LC3B-II upon AgNPs treatment for 6 h after inhibiting JNK by SP600125. Wherever mentioned, SP600125 (25 µM) was added 24 h before AgNP treatment. GAPDH served as a loading control. (D) Fluorescent microscopic images showing co-localization of LC3 (red) with lysosomes (Lyso Tracker green) after 6 h of AgNP treatment. Scale bar- 200 µm. [Symbol (*), (#) and ($) represent statistically significant difference with respect to untreated cells, AgNP-treated cells, and AgNP with inhibitor/inducer respectively].

Figure 5 Analysis of correlation of JNK and autophagy signaling with NP uptake. (A) Immunoblot showing expression of Rab7 after treatment with IC₅₀ dose of AgNPs for 6 h in presence or absence of CQ. [Symbol *@ and # represent significant difference with respect to untreated cells, CQ-treated cells and CQ plus NP treatment, respectively]. CQ (20 µM) was added 24 h before NP treatment. (B) Flow cytometric analysis of the uptake of AgNPs post inhibition of autophagy and JNK signaling. CQ (20 µM) or SP600125 (25 µM) were added 24 h before NP treatment (C) Flow cytometric analysis of the uptake of AgNPs post treatment with Rapa (500 nM). Rapa was added 24 h before NP treatment [$ represents significant difference with respect to AgNP treatment].

Figure 6 Analysis of AgNP accumulation over time on trafficking and autophagic flux. (A) Immunoblots showing expression of LC3B-II and p62 after AgNP exposure for 24 h. (B) Immunoblots showing expression of EEA1 and Rab7 at different time points post exposure to IC₅₀ dose of AgNPs. (C) A comparative analysis of GFP-ub punctae green fluorescence after 6 h and 24 h of AgNP exposure. Scale bar- 200 µm. (D) Immunoblot showing total ubiquitinated protein after exposure to AgNPs. Rapa was added 24 h before NP treatment. [Symbol *#, @ and $ represent statistically significant difference with respect to untreated cells, AgNP-treated, 1 h AgNP treatment & 6/24 h of AgNP treatment respectively].

Figure 7 Analysis of prolonged AgNP exposure on lysosomal function. (A) Immunoblot showing comparative LAMP1 expression at different time points post-AgNP exposure. (B) A comparative analysis of LysoTracker Red fluorescence between 6 h and 24 h, post exposure to AgNPs. (C) A comparative analysis of AO red fluorescence at different time points as analyzed through flow cytometer post-exposure to AgNPs. [Significant difference between 6 h and 24 h of AgNP treatment with respect to 1 h is represented as (*), whereas as ($) represents a significant difference between 6 h and 24 h]. (D) A comparative analysis of LysoTracker Red fluorescence with or without Rapa, post exposure to AgNPs at IC₅₀ dose for 24 h. (E) A comparative analysis of AO red fluorescence, in presence or absence of Rapa, post exposure to AgNPs at IC₅₀ dose for 24 h. Rapa was added 24 h before NP treatment. [Symbol @ represents statistically significant difference with respect to AgNP-treated cells]. Scale bar- 200 µm.

Figure 8 Analysis of autophagy mediated intra-cellular ROS and cytotoxicity. [A(i)] Fold change in intracellular ROS levels post-exposure to AgNPs for 6 h & 24 h, (ii) MTT assay analyzing cell viability after 6 h & 24 h of AgNP treatment, in presence or absence of NAC (20 mM). NAC was added 1 h before AgNP treatment. [B(i)] Fold change in intracellular ROS levels post-exposure to AgNPs for 6 h in presence or absence of CQ (20 µM). (ii) MTT assay measuring cell viability post-exposure to AgNPs for 6 h in the presence or absence of CQ. (iii) Immunoblots showing expression of LC3B-II protein after 24 h of AgNP treatment with or without NAC. (iv) AnnexinV/PI staining representing percentage of dead cells post-exposure to IC₅₀ dose of AgNPs for 24 h, with or without silencing of ATG5, as analyzed through flow cytometry. (C) Fold change in intracellular ROS levels and cell viability analysis post-exposure to AgNPs for 24 h, in presence or absence of Rapa. (D) Flow cytometric analysis of MMP with JC-1 dye in cells treated with IC₅₀ dose of AgNPs for 24 h. [E (i)] Dot plots of AnnexinV/PI staining post-exposure to IC₅₀ dose of AgNPs for 24 h and 48 h, respectively, as analyzed through flow cytometry. (ii) Immunoblots showing expression of total PARP after exposure of IC₅₀ dose of AgNPs for 24 h. [Symbol (*) and (#) represent a significant difference in AgNP-treated cells with respect to untreated control and specific inhibitor respectively].

Figure 9 Schematic representation of the crosstalk between AgNP and autophagy.