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. 2024 Mar 15;43(8):1653–1685. doi: 10.1038/s44318-024-00063-y

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© The Author(s) 2024

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 licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence 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 licence, visit http://creativecommons.org/licenses/by/4.0/. Creative Commons Public Domain Dedication waiver http://creativecommons.org/publicdomain/zero/1.0/ applies to the data associated with this article, unless otherwise stated in a credit line to the data, but does not extend to the graphical or creative elements of illustrations, charts, or figures. This waiver removes legal barriers to the re-use and mining of research data. According to standard scholarly practice, it is recommended to provide appropriate citation and attribution whenever technically possible.

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Figure EV3 — SCD_complete medium was inoculated with Rtn1-bait cells to an OD₆₀₀ of 0.003 from an overnight pre-culture and grown to an OD₆₀₀ of 1.2. Cells were washed with inositol-free medium and then cultivated for an additional 2 h in either inositol-free (inositol depletion) or SCD_complete medium (control) starting with an OD₆₀₀ of 0.6. Another perturbation of lipid metabolism was achieved by addition of choline. For ‘+choline’ conditions, SCD_complete medium was inoculated to an OD₆₀₀ of 0.1 using stationary overnight cultures. Cells were then cultivated to an OD₆₀₀ of 1.0 in the presence of 2 mM choline. (A) UPR activation was measured by determining the levels of spliced HAC1 mRNA. Data for relative HAC1 splicing was normalized to the inositol depletion Rtn1-bait condition (n = 8 biological replicates based on two technical replicates for Rtn1-bait control, Rtn1-bait inositol depletion, BY4741 control and BY4741 inositol depletion, but n = 4 biological replicates based on two technical replicates for Rtn1-bait + choline and BY4741 + choline). (B) mRNA upregulation of the downstream UPR target gene PDI. PDI mRNA fold change was calculated as 2^-ΔΔCt and normalized to Rtn1-bait control condition (n = 8 biological replicates based on two technical replicates for the Rtn1-bait control, Rtn1-bait inositol depletion, BY4741 control, and BY4741 inositol depletion, but n = 4 biological replicates based on two technical replicates for Rtn1-bait + choline and BY4741 + choline). (C) Upregulation of mRNA of the downstream UPR target gene KAR2 calculated as 2^-ΔΔCt and normalized to Rtn1-bait control condition (n = 8 biological replicates based each on two technical replicates for Rtn1-bait control, Rtn1-bait inositol depletion, BY4741 control, and BY4741 inositol depletion, but n = 4 biological replicates based on two technical replicates for Rtn1-bait + choline and BY4741 + choline). Data information: All data from biological replicates are presented in (A–C) as individual data points with the mean ± SD. ^nsP > 0.05, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001 (unpaired parametric t test with Welch’s correction). Nonsignificant comparisons are not highlighted. Source data are available online for this figure.