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16HBE14o-cells were exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) for 24 hours. (A) Untreated 16HBE 2 hours into experiment (10x objective lens) (B) PFOB group 5 hours into experiment (20x objective lens) (C) PFOB group 24 hours into experiment (20x objective lens)

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: 16HBE14o-cells were exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) for 24 hours. (A) Untreated 16HBE 2 hours into experiment (10x objective lens) (B) PFOB group 5 hours into experiment (20x objective lens) (C) PFOB group 24 hours into experiment (20x objective lens)

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques:

(A) 16HBE14o-cells were exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) for 2, 5 and 24 hours, and PARP protein quantified via western blot. Membranes were probed for full length and cleaved PARP with β-actin as the loading control. Elevation in cleaved PARP was found to be positively associated with PFC exposure and therefore increased apoptotic activity. Three independent experimental replicates are presented. (B) Cleaved PARP blots were quantified and normalised to the β-actin loading control. Data are presented as bar graphs representing mean ± SEM (n = 3). Two-way ANOVA analysis revealed a significant effect of exposure time (P = 0.0166) and PFC exposure (0.0146) on PARP cleavage. These factors were also significant (0.0282), indicating the effect of exposure on PARP cleavage varied with exposure duration. However, post hoc test revealed no significant differences between PFC-exposed group and untreated group at all experiment timepoints (P>0.05 for all comparisons), which could be due to insufficient sample size.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) 16HBE14o-cells were exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) for 2, 5 and 24 hours, and PARP protein quantified via western blot. Membranes were probed for full length and cleaved PARP with β-actin as the loading control. Elevation in cleaved PARP was found to be positively associated with PFC exposure and therefore increased apoptotic activity. Three independent experimental replicates are presented. (B) Cleaved PARP blots were quantified and normalised to the β-actin loading control. Data are presented as bar graphs representing mean ± SEM (n = 3). Two-way ANOVA analysis revealed a significant effect of exposure time (P = 0.0166) and PFC exposure (0.0146) on PARP cleavage. These factors were also significant (0.0282), indicating the effect of exposure on PARP cleavage varied with exposure duration. However, post hoc test revealed no significant differences between PFC-exposed group and untreated group at all experiment timepoints (P>0.05 for all comparisons), which could be due to insufficient sample size.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Control, Activity Assay

(A) Untread/media exposed 16HBE (B) Perfluorooctyl bromide exposed 16HBE (C) Perfluorodecalin exposed 16HBE All pictures were taken at endpoint of experiment (after 24 hours), and at 20x objective lens.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Untread/media exposed 16HBE (B) Perfluorooctyl bromide exposed 16HBE (C) Perfluorodecalin exposed 16HBE All pictures were taken at endpoint of experiment (after 24 hours), and at 20x objective lens.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques:

(A) Western blot analysis of 16HBE14o-cells after 24 hours exposure of PFD (perfluorodecalin), and PFOB (Perfluorooctyl bromide) in transwell model. Sample groups from three replicates were arranged with untreated (NT), PFD, and PFOB conditions grouped adjacently for direct comparison. Blots were probed for full length and cleaved PARP (apoptosis marker), SQSTM1 (SEQ), LC3B-I/II (both autophagy markers), with β-actin as the loading control. (B) Quantification of blot intensities normalised to β-actin. Data are presented as bar graphs, with each representing mean ± SEM (n = 3). No significant differences (P>0.05) were observed between the no-exposure group and the perfluorocarbons groups

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Western blot analysis of 16HBE14o-cells after 24 hours exposure of PFD (perfluorodecalin), and PFOB (Perfluorooctyl bromide) in transwell model. Sample groups from three replicates were arranged with untreated (NT), PFD, and PFOB conditions grouped adjacently for direct comparison. Blots were probed for full length and cleaved PARP (apoptosis marker), SQSTM1 (SEQ), LC3B-I/II (both autophagy markers), with β-actin as the loading control. (B) Quantification of blot intensities normalised to β-actin. Data are presented as bar graphs, with each representing mean ± SEM (n = 3). No significant differences (P>0.05) were observed between the no-exposure group and the perfluorocarbons groups

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Comparison, Marker, Control

16HBE cells were cultured air-liquid interface in transwells. The apical compartment was filled with perfluorodecalin (PFD), perfluorooctyl bromide (PFOB), or PBS for 24 hours. The untreated group (NT) remained in the air-liquid interface. TEER values (Ω/cm2) were measured at the experiment endpoint, and data are presented as bar graphs of mean ± SEM (n = 3). One-way ANOVA was used to determine the significance of exposure conditions, with a post hoc test comparing differences between PFC and PBS groups to the untreated group. ****P<0.0001, *P<0.05.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: 16HBE cells were cultured air-liquid interface in transwells. The apical compartment was filled with perfluorodecalin (PFD), perfluorooctyl bromide (PFOB), or PBS for 24 hours. The untreated group (NT) remained in the air-liquid interface. TEER values (Ω/cm2) were measured at the experiment endpoint, and data are presented as bar graphs of mean ± SEM (n = 3). One-way ANOVA was used to determine the significance of exposure conditions, with a post hoc test comparing differences between PFC and PBS groups to the untreated group. ****P<0.0001, *P<0.05.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Cell Culture

Pictures were taken at 10x objective lens after 24 hours of exposure experiment. Increasing magnifications decrease the visibility of cell-cell connections and were not included. (D) Air-exposed hSABCi (untreated) (E) Perfluorooctyl bromide-exposed hSABCi

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: Pictures were taken at 10x objective lens after 24 hours of exposure experiment. Increasing magnifications decrease the visibility of cell-cell connections and were not included. (D) Air-exposed hSABCi (untreated) (E) Perfluorooctyl bromide-exposed hSABCi

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques:

(A) Western blot analysis of hSABCi-NS1.1 cells exposed to perfluorooctyl bromide (PFOB) for 24 hours. Blots were probed for full-length and cleaved PARP (apoptosis marker), SQSTM1 (SEQ), and LC3B-I/II (both autophagy markers). β-actin served as a loading control. Cleaved PARP was nearly indistinguishable from the background, indicating minimal apoptosis. Multiple bands were observed in the SEQ blot, likely representing post-translational modifications. The band with the strongest intensity was quantified. (B) Quantification of blot intensities (A) normalised to β-actin. Data are presented as a bar graph and mean ± SEM (n=3). There were no significant differences (P>0.05) between blot intensity across all biochemical analysis for untreated (NT) and PFOB group. The variance shown of autophagy markers could be attributed to biological variance, evident by overlapping error bars.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Western blot analysis of hSABCi-NS1.1 cells exposed to perfluorooctyl bromide (PFOB) for 24 hours. Blots were probed for full-length and cleaved PARP (apoptosis marker), SQSTM1 (SEQ), and LC3B-I/II (both autophagy markers). β-actin served as a loading control. Cleaved PARP was nearly indistinguishable from the background, indicating minimal apoptosis. Multiple bands were observed in the SEQ blot, likely representing post-translational modifications. The band with the strongest intensity was quantified. (B) Quantification of blot intensities (A) normalised to β-actin. Data are presented as a bar graph and mean ± SEM (n=3). There were no significant differences (P>0.05) between blot intensity across all biochemical analysis for untreated (NT) and PFOB group. The variance shown of autophagy markers could be attributed to biological variance, evident by overlapping error bars.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Marker, Control

Small airway epithelial cells hSABCi were cultured and differentiated in ALI. Transepithelial electrical resistance (TEER) values (Ω/cm2) were measured before perfluorooctyl bromide (PFOB) exposure at Day 0 and the 24-hour exposure endpoint (Day 1). Differentiated hSABCi remained in the air-liquid interface and was assigned to the untreated (NT) group. Data are represented as bar graphs indicating mean ± SD. The decrease of TEER value observed from untreated and PFOB-exposed groups was nonsignificant; no significant differences were found between the untreated and PFOB-exposed groups at either timepoints (P>0.05).

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: Small airway epithelial cells hSABCi were cultured and differentiated in ALI. Transepithelial electrical resistance (TEER) values (Ω/cm2) were measured before perfluorooctyl bromide (PFOB) exposure at Day 0 and the 24-hour exposure endpoint (Day 1). Differentiated hSABCi remained in the air-liquid interface and was assigned to the untreated (NT) group. Data are represented as bar graphs indicating mean ± SD. The decrease of TEER value observed from untreated and PFOB-exposed groups was nonsignificant; no significant differences were found between the untreated and PFOB-exposed groups at either timepoints (P>0.05).

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Cell Culture

(A) Western blot analysis of hSABCi-NS1.1 cells exposed to perfluorooctyl bromide for 24 hours. Blots were probed against tight junction protein Occludin-1 and claudin-1 with β-actin serving as a loading control. (B) Quantification of blot intensity from (A). Data are presented as a bar graph representing mean ± SEM (n=3). Both tight junction protein expressions were not significantly different (P>0.05) between untreated and perfluorooctyl bromide-exposed hSABCi-NS1.1 cells.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Western blot analysis of hSABCi-NS1.1 cells exposed to perfluorooctyl bromide for 24 hours. Blots were probed against tight junction protein Occludin-1 and claudin-1 with β-actin serving as a loading control. (B) Quantification of blot intensity from (A). Data are presented as a bar graph representing mean ± SEM (n=3). Both tight junction protein expressions were not significantly different (P>0.05) between untreated and perfluorooctyl bromide-exposed hSABCi-NS1.1 cells.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Control

(A) Western Blot analysis of hSABCi cells exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) and remained in the air-liquid interface (NT). The membrane was probed against PARP protein (full length and cleaved length), indicating apoptosis, SQSTM1 (SEQ), and LC3BI/II, both autophagy markers. No visible PARP cleavage can be observed, and all autophagy markers are identical in blot intensity between untreated and PFC-exposed groups. (B) Quantitative analysis of western blot (A). Data represents mean ± SEM (n = 3). No visible cleaved PARP protein was observed from (A) and thus was not included in the quantitative data. Untreated overall showed higher blot intensities in all autophagy markers compared to the PFC-exposed group. Further statistical analysis revealed differences were not significant (P>0.05).

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Western Blot analysis of hSABCi cells exposed to perfluorodecalin (PFD) and perfluorooctyl bromide (PFOB) and remained in the air-liquid interface (NT). The membrane was probed against PARP protein (full length and cleaved length), indicating apoptosis, SQSTM1 (SEQ), and LC3BI/II, both autophagy markers. No visible PARP cleavage can be observed, and all autophagy markers are identical in blot intensity between untreated and PFC-exposed groups. (B) Quantitative analysis of western blot (A). Data represents mean ± SEM (n = 3). No visible cleaved PARP protein was observed from (A) and thus was not included in the quantitative data. Untreated overall showed higher blot intensities in all autophagy markers compared to the PFC-exposed group. Further statistical analysis revealed differences were not significant (P>0.05).

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Membrane

Before PFC exposure, small airway epithelial cells hSABCi were cultured at an air-liquid interface. The cell line maintained barrier integrity as indicated by TEER measurement at the end of 72 hours of exposure. Both perfluorooctyl bromide (PFOB) and perfluorodecalin (PFD) maintained TEER levels comparable to the untreated group at both time points. Data are presented as bar graphs reflecting mean ± SD (n = 3). No statistically significant differences were observed between any groups.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: Before PFC exposure, small airway epithelial cells hSABCi were cultured at an air-liquid interface. The cell line maintained barrier integrity as indicated by TEER measurement at the end of 72 hours of exposure. Both perfluorooctyl bromide (PFOB) and perfluorodecalin (PFD) maintained TEER levels comparable to the untreated group at both time points. Data are presented as bar graphs reflecting mean ± SD (n = 3). No statistically significant differences were observed between any groups.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Cell Culture

(A) The expression of three tight junction proteins, ZO-1, Occludin-1 and Claudin-1, was assessed using western blot. hSABCi cells were exposed to perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), or air (NT). ZO-1 expression appeared to be upregulated in the PFC-exposed groups, while Occludin-1 and Claudin-1 blot intensities are similar across all groups. (B) Quantification of tight junction protein western blot (A). Blot intensities were presented as mean ± SEM (n = 3). One-way ANOVA analysis revealed PFC exposure had a significant effect (P = 0.0175) in ZO-1 blot intensities, and post-hoc indicated only the PFD group resulted in significant increases (P = 0.0114) in ZO-1 expression compared to the untreated group. No other significance was found in Occludin-1 or Claudin-1 (A) quantification. ZO-1 blot results show a pattern of increased expressions in PFC-exposed groups, while the others showed a more uniform expression.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) The expression of three tight junction proteins, ZO-1, Occludin-1 and Claudin-1, was assessed using western blot. hSABCi cells were exposed to perfluorooctyl bromide (PFOB), perfluorodecalin (PFD), or air (NT). ZO-1 expression appeared to be upregulated in the PFC-exposed groups, while Occludin-1 and Claudin-1 blot intensities are similar across all groups. (B) Quantification of tight junction protein western blot (A). Blot intensities were presented as mean ± SEM (n = 3). One-way ANOVA analysis revealed PFC exposure had a significant effect (P = 0.0175) in ZO-1 blot intensities, and post-hoc indicated only the PFD group resulted in significant increases (P = 0.0114) in ZO-1 expression compared to the untreated group. No other significance was found in Occludin-1 or Claudin-1 (A) quantification. ZO-1 blot results show a pattern of increased expressions in PFC-exposed groups, while the others showed a more uniform expression.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Expressing, Western Blot

The electrical impedance of differentiated nAEC cultured at an air-liquid interface was measured during 3-day exposure to perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS). PFOB-exposed nAEC maintained TEER levels comparable to the no-treatment (NT) control, while PBS significantly reduced the barrier integrity of nAEC. Data are presented as mean; error bars represent 95% confidence intervals (CI) from 3 independent experiments. Statistical significance was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test. The Dunnett’s test confirmed that PBS significantly reduced TEER compared to NT at all time points, while no significant differences were observed between PFOB and NT. Significant differences are indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: The electrical impedance of differentiated nAEC cultured at an air-liquid interface was measured during 3-day exposure to perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS). PFOB-exposed nAEC maintained TEER levels comparable to the no-treatment (NT) control, while PBS significantly reduced the barrier integrity of nAEC. Data are presented as mean; error bars represent 95% confidence intervals (CI) from 3 independent experiments. Statistical significance was determined by two-way ANOVA followed by Dunnett’s multiple comparisons test. The Dunnett’s test confirmed that PBS significantly reduced TEER compared to NT at all time points, while no significant differences were observed between PFOB and NT. Significant differences are indicated as *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Cell Culture, Saline, Control

Differentiated nAECs were exposed to perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS) for 72 hours. ELISA assessed IL-6 and IL-8 secretion in apical washes and basal media. Data are presented as mean ± SEM represented by bar graphs. (A) IL-6 levels between untreated and PFOB are comparable from apical wash or basal media. The PBS group shows low levels of IL-6 apically but substantially more IL-6 in basal media. Despite that, the differences between exposure groups (PFOB & PBS) and untreated reference were nonsignificant. (B) IL-8 levels appear uniform across all apical wash samples, and corresponding levels in basal media are all higher. While untreated control and PFOB show comparable IL-8 levels, IL-8 detected in the PBS group’s basal media is considerably increased, showing high significant differences (P<0.0001) in the untreated group.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: Differentiated nAECs were exposed to perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS) for 72 hours. ELISA assessed IL-6 and IL-8 secretion in apical washes and basal media. Data are presented as mean ± SEM represented by bar graphs. (A) IL-6 levels between untreated and PFOB are comparable from apical wash or basal media. The PBS group shows low levels of IL-6 apically but substantially more IL-6 in basal media. Despite that, the differences between exposure groups (PFOB & PBS) and untreated reference were nonsignificant. (B) IL-8 levels appear uniform across all apical wash samples, and corresponding levels in basal media are all higher. While untreated control and PFOB show comparable IL-8 levels, IL-8 detected in the PBS group’s basal media is considerably increased, showing high significant differences (P<0.0001) in the untreated group.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Saline, Enzyme-linked Immunosorbent Assay, Control

(A) Western blot analysis of tight junction protein markers in primary nasal airway epithelial (nAEC) cells after 72 hours of liquid exposure in transwells. Blots were probed for Occludin-1 and Claudin-1 with β-actin as a loading control. Exposure conditions were untreated (NT), perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS). (B) Quantification of tight junction protein blots intensity. Data are presented as mean ± SEM in a bar graph format. PFOB exposure significantly increased occludin-1 expression (P<0.01) but not claudin-1, compared to the untreated group. Conversely, PBS exposure also significantly increased Occludin-1 expression (P<0.05) but substantially decreased (P<0.0001) Claudin-1 expression compared to the untreated group. As the Claudin-1 blot was not detectable in (A) for the PBS group, it is represented as 0.

Journal: bioRxiv

Article Title: Organotypic tissue architecture is a requisite for predictive outcomes of perfluorocarbon exposure in airway models

doi: 10.1101/2024.10.29.620983

Figure Lengend Snippet: (A) Western blot analysis of tight junction protein markers in primary nasal airway epithelial (nAEC) cells after 72 hours of liquid exposure in transwells. Blots were probed for Occludin-1 and Claudin-1 with β-actin as a loading control. Exposure conditions were untreated (NT), perfluorooctyl bromide (PFOB) and phosphate-buffered saline (PBS). (B) Quantification of tight junction protein blots intensity. Data are presented as mean ± SEM in a bar graph format. PFOB exposure significantly increased occludin-1 expression (P<0.01) but not claudin-1, compared to the untreated group. Conversely, PBS exposure also significantly increased Occludin-1 expression (P<0.05) but substantially decreased (P<0.0001) Claudin-1 expression compared to the untreated group. As the Claudin-1 blot was not detectable in (A) for the PBS group, it is represented as 0.

Article Snippet: The PFCs used in this thesis are perfluorodecalin (PFD; Sigma-Aldrich) and perflubron/ perfluorooctyl bromide (PFOB; ThermoFisher).

Techniques: Western Blot, Control, Saline, Expressing