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Research Paper

Kinetics of autophagic activity in nanoparticle-exposed lung adenocarcinoma (A549) cells

ORCID Icon, ORCID Icon, ORCID Icon & ORCID Icon
Article: 2186568 | Received 01 Jun 2022, Accepted 06 Feb 2023, Published online: 15 Mar 2023

Figures & data

Figure 1. Time-dependent activation of autophagy in A549 cells exposed apically to PNP (80 μg/mL), UFP (1 μg/mL) or Rapamycin (50 nM) as a single agent at t = 0. Autophagic activity increased over time as shown by increased fluorescence intensity of DAPRed (red). Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). Scale bars are 25 μm.

Figure 1. Time-dependent activation of autophagy in A549 cells exposed apically to PNP (80 μg/mL), UFP (1 μg/mL) or Rapamycin (50 nM) as a single agent at t = 0. Autophagic activity increased over time as shown by increased fluorescence intensity of DAPRed (red). Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). Scale bars are 25 μm.

Table 1. Kinetics of autophagy activation in A549 cells after NP and/or Rapamycin exposure. Significant differences among means at a given time point were determined by one-way ANOVA. *: p<0.05 compared to t = 0; **: p<0.01 compared to t = 0; #: p<0.05 compared to PNP exposure alone at given time; $: p<0.05 compared to UFP exposure alone at given time; γ: p<0.05 compared to PNP + Rapamycin (sequential) at given time; δ: p<0.05 compared to UFP + Rapamycin (sequential) and ε: p<0.01 compared to Rapamycin + UFP (concurrent) at given time. There were no significant differences between the two experiments for any condition at any time point.

Figure 2. Kinetics of autophagy activation in A549 cells exposed to a single agent using Rapamycin, PNP or UFP at t = 0 and monitored for up to 24 hrs. Apical exposure of A549 cells to PNP (80 μg/mL, red line) or UFP (1 μg/mL, grey line) led to time-dependent gradual increases in autophagic flux. When exposed to either PNP or UFP alone at t = 0, increased autophagic flux was detected at ~3-6 hrs post exposure, reaching a peak at ~8-10 hrs. Exposure to Rapamycin (50 nM, blue line) alone at t = 0 led to a rapid increase in autophagic flux, peaking at ~3 hrs post exposure. Data at each time point were collected from 26-69 single cells. Detailed data with statistical analyses are shown in .

Figure 2. Kinetics of autophagy activation in A549 cells exposed to a single agent using Rapamycin, PNP or UFP at t = 0 and monitored for up to 24 hrs. Apical exposure of A549 cells to PNP (80 μg/mL, red line) or UFP (1 μg/mL, grey line) led to time-dependent gradual increases in autophagic flux. When exposed to either PNP or UFP alone at t = 0, increased autophagic flux was detected at ~3-6 hrs post exposure, reaching a peak at ~8-10 hrs. Exposure to Rapamycin (50 nM, blue line) alone at t = 0 led to a rapid increase in autophagic flux, peaking at ~3 hrs post exposure. Data at each time point were collected from 26-69 single cells. Detailed data with statistical analyses are shown in Table 1.

Figure 3. Time-dependent activation of autophagy in A549 cells during concurrent apical exposure (at t = 0) to 50 nM Rapamycin and 80 μg/mL PNP or 1 μg/mL UFP. Autophagic activity increased over time as seen by fluorescence intensity of DAPRed (red). Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). Scale bars are 25 μm.

Figure 3. Time-dependent activation of autophagy in A549 cells during concurrent apical exposure (at t = 0) to 50 nM Rapamycin and 80 μg/mL PNP or 1 μg/mL UFP. Autophagic activity increased over time as seen by fluorescence intensity of DAPRed (red). Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). Scale bars are 25 μm.

Figure 4. Kinetics of autophagy activation in A549 cells concurrently exposed apically to Rapamycin and PNP or UFP at t = 0. Concurrent apical exposure at t = 0 of A549 cells to Rapamycin (50 nM) + PNP (80 μg/mL) or Rapamycin (50 nM) + UFP (1 μg/mL) resulted in more rapid activation of autophagy in comparison to exposure to PNP or UFP alone (). Data at each time point are from 31-49 single cells. Detailed data with statistical analyses are shown in .

Figure 4. Kinetics of autophagy activation in A549 cells concurrently exposed apically to Rapamycin and PNP or UFP at t = 0. Concurrent apical exposure at t = 0 of A549 cells to Rapamycin (50 nM) + PNP (80 μg/mL) or Rapamycin (50 nM) + UFP (1 μg/mL) resulted in more rapid activation of autophagy in comparison to exposure to PNP or UFP alone (Figure 2). Data at each time point are from 31-49 single cells. Detailed data with statistical analyses are shown in Table 1.

Figure 5. Representative images showing the time-dependent activation of autophagy in sequential exposure experiments. A549 cells exposed apically to a single agent (PNP, UFP or Rapamycin (Rapa)) for the first 5 hrs of the experiment, followed by exposure to the single agent plus a second agent (Rapamycin followed by PNP or UFP and PNP or UFP followed by Rapamycin) from 5 to 24 hrs. Autophagic activity increased in response to the different exposures over time as seen by fluorescence intensity of DAPRed. Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). C = control, cq = chloroquine. Scale bars are 25 μm.

Figure 5. Representative images showing the time-dependent activation of autophagy in sequential exposure experiments. A549 cells exposed apically to a single agent (PNP, UFP or Rapamycin (Rapa)) for the first 5 hrs of the experiment, followed by exposure to the single agent plus a second agent (Rapamycin followed by PNP or UFP and PNP or UFP followed by Rapamycin) from 5 to 24 hrs. Autophagic activity increased in response to the different exposures over time as seen by fluorescence intensity of DAPRed. Data were collected at each time point after 1 hr incubation with chloroquine (40 μM). Plasma membranes of A549 cells were labeled by Dylight 405-conjugated tomato lectin (blue). C = control, cq = chloroquine. Scale bars are 25 μm.

Figure 6. Kinetics of autophagic activation in A549 cells exposed to two agents sequentially (one from 0 to 24 hrs and the other from 5 to 24 hrs). Autophagic flux was assessed in A549 cells first by exposure to PNP (solid line in left panel) or UFP (solid line in right panel) at t = 0, followed by the addition of Rapamycin at t = 5 hrs. In a different set of experiments, A549 cells were exposed to Rapamycin at t = 0, followed by the addition of PNP (dotted line in left panel) or UFP (dotted line in right panel) at 5 hrs. The overall kinetics and steady state values of autophagic flux in these reverse order sequential double exposure models were not different from those measured in single () or concurrent double agent exposures (), suggesting that autophagic capacity in A549 cells is limited. Data at each time point are from 24-33 single cells. Detailed data with statistical analyses are shown in .

Figure 6. Kinetics of autophagic activation in A549 cells exposed to two agents sequentially (one from 0 to 24 hrs and the other from 5 to 24 hrs). Autophagic flux was assessed in A549 cells first by exposure to PNP (solid line in left panel) or UFP (solid line in right panel) at t = 0, followed by the addition of Rapamycin at t = 5 hrs. In a different set of experiments, A549 cells were exposed to Rapamycin at t = 0, followed by the addition of PNP (dotted line in left panel) or UFP (dotted line in right panel) at 5 hrs. The overall kinetics and steady state values of autophagic flux in these reverse order sequential double exposure models were not different from those measured in single (Figure 2) or concurrent double agent exposures (Figure 4), suggesting that autophagic capacity in A549 cells is limited. Data at each time point are from 24-33 single cells. Detailed data with statistical analyses are shown in Table 1.
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