Fig 1.
The effects of forskolin on the viability of LPS-treated RT4-D6P2T cells.
Using the CellTiter-Glo 2.0 Assay (Promega), the immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin, for (A) 1, (B) 3, (C) 12, and (D) 24 hours. Relative luminescence units were read as an indicator of viability and are displayed as a mean percent control ± SEM. The dotted line indicates a percent control of 100%, with a percent control above 100% representing increased relative luminescence units (more viable cells) and a percent control below 100% representing decreased relative luminescence units (less viable cells) compared to the N2 control. Results from all experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. Mean percent controls with the same number of asterisks (*) are significantly different from each other (*p < 0.05, **p < 0.01, ***p < 0.001) (n = 3).
Table 1.
Pearson’s correlation coefficients and p-values.
Fig 2.
The effects of forskolin on NF-κB expression in LPS-treated RT4-D6P2T cells.
The immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin (+/- Fsk), for 3 hours. SDS-PAGE gel electrophoresis and Western blotting were performed using prepared cell lysates. NF-κB expression was visualized using enhanced chemiluminescence reagent and quantified via densitometry analysis using Bio Rad Image software. Actin was used as a loading control, and detected expression levels of actin were used to normalize all Western blots. The above blots are representative blots from three independent experiments that were spliced together, as indicated by the vertical black lines. Relative band densities are displayed as mean fold change (over the N2 control [no LPS]) ± SEM. The dotted line indicates a fold change of 1, with a fold change above 1 representing increased protein expression and a fold change below 1 representing decreased protein expression compared to the N2 control. Results from three independent experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. There was no significant difference in mean fold change between the various doses of LPS (F = 0.240, df = 1, p = 0.635) (n = 3).
Fig 3.
The effects of forskolin on TNF-α expression in LPS-treated RT4-D6P2T cells.
The immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin (+/- Fsk), for 3 hours. SDS-PAGE gel electrophoresis and Western blotting were performed using prepared cell lysates. TNF-α expression was visualized using enhanced chemiluminescence reagent and quantified via densitometry analysis using Bio Rad Image software. Actin was used as a loading control, and detected expression levels of actin were used to normalize all Western blots. The above blots are representative blots from three independent experiments that were spliced together, as indicated by the vertical black lines. Relative band densities are displayed as mean fold change (over the N2 control [no LPS]) ± SEM. The dotted line indicates a fold change of 1, with a fold change above 1 representing increased protein expression and a fold change below 1 representing decreased protein expression compared to the N2 control. Results from three independent experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. There was no significant difference in mean fold change between the various doses of LPS (F = 0.613, df = 1, p = 0.452) (n = 3).
Fig 4.
The effects of forskolin on AKAP95 expression in LPS-treated RT4-D6P2T cells.
The immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin (+/- Fsk), for 3 hours. SDS-PAGE gel electrophoresis and Western blotting were performed using prepared cell lysates. AKAP95 expression was visualized using enhanced chemiluminescence reagent and quantified via densitometry analysis using Bio Rad Image software. Actin was used as a loading control, and detected expression levels of actin were used to normalize all Western blots. The above blots are representative blots from three independent experiments that were spliced together, as indicated by the vertical black lines. Relative band densities are displayed as mean fold change (over the N2 control [no LPS]) ± SEM. The dotted line indicates a fold change of 1, with a fold change above 1 representing increased protein expression and a fold change below 1 representing decreased protein expression compared to the N2 control. Results from three independent experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. There was no significant difference in mean fold change between the various doses of LPS (F = 0.151, df = 1, p = 0.706) (n = 3).
Fig 5.
The effects of forskolin on cyclin D3 expression in LPS-treated RT4-D6P2T cells.
The immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin (+/- Fsk), for 3 hours. SDS-PAGE gel electrophoresis and Western blotting were performed using prepared cell lysates. Cyclin D3 expression was visualized using enhanced chemiluminescence reagent and quantified via densitometry analysis using Bio Rad Image software. Actin was used as a loading control, and detected expression levels of actin were used to normalize all Western blots. The above blots are representative blots from three independent experiments that were spliced together, as indicated by the vertical black lines. Relative band densities are displayed as mean fold change (over the N2 control [no LPS]) ± SEM. The dotted line indicates a fold change of 1, with a fold change above 1 representing increased protein expression and a fold change below 1 representing decreased protein expression compared to the N2 control. Results from three independent experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. There was no significant difference in mean fold change between the various doses of LPS (F = 0.438, df = 1, p = 0.523) (n = 3).
Fig 6.
The effects of forskolin on TNF-α secretion by LPS-treated RT4-D6P2T cells.
The immortalized rat RT4-D6P2T Schwann cell line (ATCC #CRL-2768) was treated with 0.1, 1, or 10 μg/mL of LPS in N2 media, with or without 2 μM of forskolin, for 3 hours. The Invitrogen Rat TNF alpha ELISA kit (RayBiotech) was used to quantify the TNF-α concentration (pg/mL) in media samples, which is displayed as mean fold change ± SEM. The dotted line indicates a fold change of 1, with a fold change above 1 representing increased TNF-α secretion and a fold change below 1 representing decreased TNF-α secretion compared to the N2 control. Results from three independent experiments were examined using one-way ANOVA and tested with Tukey’s and LSD post-hoc analysis in R Studio. There was no significant difference in mean fold change between the various doses of LPS (F = 2.437, df = 1, p = 0.147) (n = 3).
Fig 7.
Summary of the effects of forskolin-mediated cAMP activation on protein expression and viability in Schwann cells.
Cartoon illustration depicting the effects of forskolin-mediated cAMP activation on NF-κB, TNF-α, AKAP95, and cyclin D3 expression, and viability in Schwann cells treated with (A) 0.1 and (B) 10 μg/mL of LPS for 3 hours.