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. 2020 May 13;21(10):3449. doi: 10.3390/ijms21103449

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© 2020 by the authors.

Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (http://creativecommons.org/licenses/by/4.0/).

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Short-term nutrient deprivation triggers the degradation of AHR in HeLa cells when either p23 or HSP90 is down-regulated. (A) Zero to four hours’ treatment of HBSS (nutrient deprivation) in wild type (WT), p23 stable knockdown (p23KD), and HSP90 stable knockdown (HSP90KD) HeLa cells. Fifteen minutes of HBSS treatment decreased AHR protein levels in p23KD and HSP90 HeLa cells but not in WT HeLa cells. Longer nutrient deprivation of up to 4 h increased AHR protein levels in all three cell lines. The graph represents replicate data of means ± SD (upper error bars shown), n = 3 for all, except n = 4 for HSP90KD data from 0 to 1 h. Zero timepoints in each cell line were arbitrarily set as one for comparison. Data were analyzed by unpaired two-tailed t-test for comparisons between zero and 15 min timepoints in each individual cell line (significant data points marked with # symbol). Data were analyzed by two-way ANOVA (mixed-model) analysis corrected with the Dunnett test for multiple comparisons to determine the statistical significance between WT and p23KD or WT and HSP90 KD at each time point (significant data points marked with * symbol). WT HeLa data were set as a control group at each timepoint for comparison. The images above represent the replicate data. Note that the timepoints are intentionally presented not in chronological order to preserve the integrity of the original immunoblots. (B) A 40 μM amount of CQ pre-treatment for 6 h blocked the AHR decrease in p23KD HeLa cells treated with HBSS for 0–1 h. Zero time points in each condition were arbitrarily set as one for comparison. The graph represents replicate data of means ± SD (upper error bars shown), n = 3. Data were analyzed by multiple t-tests corrected with the Holm-Sidak method for multiple comparisons to determine the statistical significance. The images above represent the replicate data. (C) A 4 h HBSS treatment significantly increased the ahr message levels in WT HeLa cells. The graph represents replicate data of means ± SD, n = 3 of one experiment. This experiment was repeated once with similar results. Data were analyzed by unpaired t-test to determine statistical significance. (D) Treatment of 5 μg/mL of actinomycin D (ActD) for 4 h abolished the increase of AHR protein levels induced by HBSS (nutrient deprivation) in WT HeLa cells. The graph represents replicate data of means ± SD, n = 3 of one experiment. This experiment was repeated once with similar results. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test to determine statistical significance. For A to D, each Western lane contained 30 μg of whole-cell lysate. The intensity of all Western bands was normalized by total protein stain.

Short-term nutrient deprivation triggers the degradation of AHR in HeLa cells when either p23 or HSP90 is down-regulated. (A) Zero to four hours’ treatment of HBSS (nutrient deprivation) in wild type (WT), p23 stable knockdown (p23KD), and HSP90 stable knockdown (HSP90KD) HeLa cells. Fifteen minutes of HBSS treatment decreased AHR protein levels in p23KD and HSP90 HeLa cells but not in WT HeLa cells. Longer nutrient deprivation of up to 4 h increased AHR protein levels in all three cell lines. The graph represents replicate data of means ± SD (upper error bars shown), n = 3 for all, except n = 4 for HSP90KD data from 0 to 1 h. Zero timepoints in each cell line were arbitrarily set as one for comparison. Data were analyzed by unpaired two-tailed t-test for comparisons between zero and 15 min timepoints in each individual cell line (significant data points marked with # symbol). Data were analyzed by two-way ANOVA (mixed-model) analysis corrected with the Dunnett test for multiple comparisons to determine the statistical significance between WT and p23KD or WT and HSP90 KD at each time point (significant data points marked with * symbol). WT HeLa data were set as a control group at each timepoint for comparison. The images above represent the replicate data. Note that the timepoints are intentionally presented not in chronological order to preserve the integrity of the original immunoblots. (B) A 40 μM amount of CQ pre-treatment for 6 h blocked the AHR decrease in p23KD HeLa cells treated with HBSS for 0–1 h. Zero time points in each condition were arbitrarily set as one for comparison. The graph represents replicate data of means ± SD (upper error bars shown), n = 3. Data were analyzed by multiple t-tests corrected with the Holm-Sidak method for multiple comparisons to determine the statistical significance. The images above represent the replicate data. (C) A 4 h HBSS treatment significantly increased the ahr message levels in WT HeLa cells. The graph represents replicate data of means ± SD, n = 3 of one experiment. This experiment was repeated once with similar results. Data were analyzed by unpaired t-test to determine statistical significance. (D) Treatment of 5 μg/mL of actinomycin D (ActD) for 4 h abolished the increase of AHR protein levels induced by HBSS (nutrient deprivation) in WT HeLa cells. The graph represents replicate data of means ± SD, n = 3 of one experiment. This experiment was repeated once with similar results. Data were analyzed by one-way ANOVA with Tukey’s multiple comparisons test to determine statistical significance. For A to D, each Western lane contained 30 μg of whole-cell lysate. The intensity of all Western bands was normalized by total protein stain.