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. 2017 Jul 19;7:5941. doi: 10.1038/s41598-017-05734-x

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© The Author(s) 2017

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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Lipids in the absence of calreticulin. (A) Sudan black staining of neutral lipids in wild-type (N2) and crt-1 calreticulin deficient (jh101) C. elegans. Lipid droplets are indicated by the arrows. (B) BODIPY 505/515 staining of neutral lipids in wild-type and calreticulin-deficient (Calr ^−/−) cells. (C) Cholesteryl esters, triacyglycerols and unesterified cholesterol levels in wild-type and Calr ^−/− cells. *Indicates statistically significant differences: cholesteryl esters, p-value = 0.0374; triacylglycerols, p-value = 0.0451. NS, not significant. Representative of 3 biological replicates. Student’s t-test was used. (D) SwitchGear Cholesterol Biosynthesis Pathway assay for SREBP target genes in wild-type and Calr ^−/− cells; fatty acid synthase (FASN), isopentenyl-diphosphate delta isomerase (IDI1), insulin induced gene-1 (INSIG1), low density lipoprotein receptor (LDLR), lanosterol synthase (LSS), mevalonate pyrophosphate decarboxylase (MVD) and squalene epoxidase (SQLE) Representative of 2 biological replicates. (E) Q-PCR analysis of replication initiator 1 (RI1), squalene (SQLE), isopentenyl-diphosphate delta isomerase (IDI1), lanosterol synthase (LSS), LDL receptor (LDLR) and HMG-CoA reductase mRNA level in wild-type and Calr ^−/− cells. Results were normalized to 18S rRNA (internal control). **Indicates statistically significant differences: SQLE in wild-type vs. Calr ^−/− cells, p-value = 0.0041; LSS in wild-type vs. Calr ^−/− cells, p-value = 0.001. *Indicates statistically significant differences: IDI1 in wild-type vs. Calr ^−/− cells, p-value = 0.0133; LDLR in wild-type vs. Calr ^−/− cells, p-value = 0.0112; HMG-CoA Reductase in wild-type vs. Calr ^−/− cells, p-value = 0.0001. Representative of 4 biological replicates. NS, not significant (Student’s t-test). (F) Analysis of rates of synthesis of cholesterol and triacyglycerols in wild-type and Calr ^−/− cells. *Indicates statistically significant differences: cholesterol, p-value = 0.0012; triacylglycerols, p-value = 0.0006 (Student’s t-test). See “Experimental Procedures” for additional details.