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. 2021 Jul 13;12:4285. doi: 10.1038/s41467-021-24505-x

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© The Author(s) 2021

Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.

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Fig. 2 — a Screen for neuronal subsets in which knocking down Nf1 elevates CO₂ production. CO₂ production from each Gal4 line is normalized to its own genetic controls. b Significant lines from a, showing RNAi and heterozygous controls. Sample sizes and p-values in Source data file for (a, b). *p < 0.05, **p < 0.01, ***p < 0.001 (Dunn’s test, two-sided; n = 7–14). c Normalized CO₂ production, knocking down Nf1 pan-neuronally with R57C10-Gal4, with and without the tsh-Gal80 repressor. ***p < 0.001 (Sidak, two-sided; n = 6). d Expression of GFP in neurons labeled by the PCB-Gal4 driver. Bruchpilot (brp) counterstains the neuropil. Representative image shown. e Normalized CO₂ production, knocking down Nf1 in neurons with the PCB-Gal4 driver (left) (**p < 0.01 ***p < 0.001 [Dunn’s test, two-sided; n = 7]), adding the elav-Gal80 repressor (middle) (experimental p = 0.0678 re: UAS-Nf1 RNAi/+, p > 0.999 re: PCB-Gal4/+ [Dunn’s test, two-sided; n = 5]), or adding the tsh-Gal80 repressor (right) (***p < 0.001 [Sidak, two-sided; n = 6]). The left data set is duplicated from (b) for comparison. f Normalized CO₂ production when PCB-Gal4 neurons were blocked using UAS-Shibire^ts at 31 °C (right) and control temperature (22 °C, left). At 22 °C, experimental p > 0.999 re: UAS-Shibire^ts/+, p = 0.147 re: PCB-Gal4/+. (Sidak, two-sided; n = 8 UAS-Shibire^ts/+, n = 10 PCB-Gal4/+, and n = 10 experimental). At 31 °C experimental p = 0.590 re: UAS-Shibire^ts/+, p = 0.903 re: PCB-Gal4/+. (Sidak, two-sided; n = 5 per genotype). g Normalized CO₂ production following activation of PCB-Gal4 neurons using UAS-TrpA1 at 29 °C (right) and control temperature (22 °C, left). At 22 °C experimental p = 0.269 re: UAS-TrpA1/+, p = 0.655 re: PCB-Gal4/+. (Sidak, two-sided; n = 10 per genotype). At 29 °C ***p < 0.001 (Sidak, two-sided; n = 10 UAS-TrpA1/+, n = 9 PCB-Gal4/+, n = 10 experimental). For each experiment, data were normalized to the mean of both controls at each temperature. Each “n” = single respirometer (containing 4 animals). Box plots—box: 1st to 3rd quartiles, median: line, whiskers: min–max, individual data points: circles. ns: not significant.