Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo

doi: 10.1073/pnas.1318262111

Figure Lengend Snippet: mdt-15 inactivation alters membrane lipid composition and induces the UPRER. (A) Bar graphs represent the average fold change of xbp-1, spliced xpb-1 (xbp-1s), and hsp-4 mRNA levels in control(RNAi) and mdt-15(RNAi) worms (P = 0.098, 0.049, and 0.00049, respectively) (Left), WT and mdt-15(tm2182) worms (P = 0.19, 0.063, and 0.0064, respectively) (Center), or control(RNAi) worms treated with DMSO, 5 µg/mL tunicamycin (Tm 5; P = 0.16, 0.13, 0.04, respectively) or 10 µg/mL tunicamycin (Tm 10; P = 0.31, 0.18, 0.03, respectively) (Right). n = 3 for all experiments; error bars represent SEM; *P < 0.05 (two-tailed Student t test). (B) Micrographs depict control(RNAi) and mdt-15(RNAi) worms or WT and mdt-15(tm2182) worms expressing the hsp-4p::GFP transcriptional reporter; one of four independent experiments is shown. (C) Immunoblots depict the levels of phospho-Ser51 eIF2α (P-eIF2α) and actin protein levels in control(RNAi), mdt-15(RNAi), WT, and mdt-15(tm2182) worms. The numbers represent the intensity of the P-eIF2α bands relative to corresponding actin bands. One of four independent experiments is shown. (D) Micrographs show control(RNAi) and mdt-15(RNAi) worms or WT and mdt-15(tm2182) worms expressing the hsp-4p::GFP transcriptional reporter grown without dietary supplements (no supplement), with 300 μM C18:1n-9 (oleate), with 150 μM C18:2 and 150 μM C20:5 (PUFAs), or with 150 μM C18:1n-9 and 300 μM PUFAs (O+PUFAs). (E) Bars represent the relative abundance of individual FAs in PC. (F) Bars represent the average levels of individual lipid species relative to total PLs. For E and F, lipids were extracted from L4-stage control(RNAi) or mdt-15(RNAi) worms raised in the absence or presence of dietary unsaturated FAs (UFAs; C18:1n-9, C18:2, and C20:5). n = 3; error bars represent SEM; *P < 0.05 as determined by two-tailed Student t test. For P values, see Tables S1–S3.

Article Snippet: Protein concentrations were determined using the RC DC Protein Assay kit (no. 500–0121; Bio-Rad), and SDS/PAGE analysis and immunoblotting were performed as described ( 4 ) with antibodies against Ser51-Phospho-eIF2α (no. 9721; Cell Signaling Technology), pan-actin (no. 8456; Cell Signaling Technology), tubulin (DM1A, no. T9026; Sigma), GFP (to visualize CPL-1 W32A,Y35A ::YFP and hsp-4p –driven GFP; JL-8; BD Bioscience), and anti-rabbit HRP-conjugated (no. 7074; New England Biolabs) or donkey anti-mouse IR-Dye 800 (LI-COR) secondary antibody.

Techniques: Two Tailed Test, Expressing, Western Blot

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo

doi: 10.1073/pnas.1318262111

Figure Lengend Snippet: mdt-15 inactivation and defects in PL biosynthesis activate the UPRER without compromising ER protein quality control. (A) The plots show the survival of adult control(RNAi), mdt-15(RNAi), ire-1(RNAi), WT, mdt-15(tm2182), ire-1(zc14), SCD(RNAi), and sams-1(RNAi) worms on 30 μg/mL tunicamycin. The P values for mdt-15(tm2182) and sams-1(RNAi) worms indicate resistance. One of at least three independent experiments is shown; for additional replicates, see Fig. S4A. (B) Bar graphs represent the fractions of L1/L2, L3, or L4 stage control(RNAi), mdt-15(RNAi), and SCD(RNAi) larvae that are alive after 48 h on 0, 2, or 5 μg/mL tunicamycin (Upper) or on 0, 3, or 5 μg/mL thapsigargin (Lower). One of three independent experiments is shown. (C, Left) The average number of hatched F1 progeny per individual P0 WT or ire-1(zc14) worm grown on control, enpl-1, mdt-15, SCD, or sams-1 RNAi. (Right) The ratio of hatched progeny from ire-1(zc14) mutants over WT worms for each RNAi treatment. n = 4; error bars represent SEM; *P < 0.05 as determined by two-tailed unpaired Student t test. Micrographs below the bar graphs show WT or ire-1(zc14) worms carrying the hsp-4p::GFP reporter on control, enpl-1, mdt-15, SCD, or sams-1 RNAi. (D) Micrographs depict worms expressing the hsp-4p::GFP reporter on control or sams-1 RNAi with or without 30 mM ethanolamine or choline supplementation (Left and Center) and on SCD RNAi without supplementation (no supplement), with C18:1n-9 supplementation (oleate), with C18:2 and C20:5 supplementation (PUFAs), or with C18:1n-9, C18:2, and C20:5 supplementation (O+PUFAs) (Right). One of three independent experiments is shown.

Article Snippet: Protein concentrations were determined using the RC DC Protein Assay kit (no. 500–0121; Bio-Rad), and SDS/PAGE analysis and immunoblotting were performed as described ( 4 ) with antibodies against Ser51-Phospho-eIF2α (no. 9721; Cell Signaling Technology), pan-actin (no. 8456; Cell Signaling Technology), tubulin (DM1A, no. T9026; Sigma), GFP (to visualize CPL-1 W32A,Y35A ::YFP and hsp-4p –driven GFP; JL-8; BD Bioscience), and anti-rabbit HRP-conjugated (no. 7074; New England Biolabs) or donkey anti-mouse IR-Dye 800 (LI-COR) secondary antibody.

Techniques: Two Tailed Test, Expressing

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo

doi: 10.1073/pnas.1318262111

Figure Lengend Snippet: mdt-15 is not required for mitochondrial protein homeostasis. (A) Micrographs show control(RNAi), mdt-15(RNAi), WT, and mdt-15(tm2182) worms expressing the hsp-6p::GFP transcriptional reporter (Upper row) or the hsp-60p::GFP transcriptional reporter (Lower row). (B) Micrographs show control(RNAi) and mdt-15(RNAi) worms expressing the hsp-6p::GFP transcriptional reporter treated with 10 μg/mL antimycin A for 24 h (Lower row) or not treated (Upper row). For A and B, one of three independent experiments is shown. (C) Bars represent the relative abundance of individual FAs in cardiolipin (Left) and the average levels of cardiolipin relative to TLs or total PLs (Right). Lipids were extracted from control(RNAi) and mdt-15(RNAi) worms. n = 3; error bars represent SEM; *P < 0.05 as determined by two-tailed Student t test.

Article Snippet: Protein concentrations were determined using the RC DC Protein Assay kit (no. 500–0121; Bio-Rad), and SDS/PAGE analysis and immunoblotting were performed as described ( 4 ) with antibodies against Ser51-Phospho-eIF2α (no. 9721; Cell Signaling Technology), pan-actin (no. 8456; Cell Signaling Technology), tubulin (DM1A, no. T9026; Sigma), GFP (to visualize CPL-1 W32A,Y35A ::YFP and hsp-4p –driven GFP; JL-8; BD Bioscience), and anti-rabbit HRP-conjugated (no. 7074; New England Biolabs) or donkey anti-mouse IR-Dye 800 (LI-COR) secondary antibody.

Techniques: Expressing, Two Tailed Test

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: Activation of the endoplasmic reticulum unfolded protein response by lipid disequilibrium without disturbed proteostasis in vivo

doi: 10.1073/pnas.1318262111

Figure Lengend Snippet: mdt-15 inactivation and defects in PL biosynthesis do not enhance misfolded-protein aggregation. (A, Left) Micrographs depict SRP-2H302R::GFP foci in control, mdt-15–, SCD-, sams-1–, ire-1–, enpl-1–, and sca-1–depleted worms. (Right) The dot plot represents the quantification of GFP aggregates; data points were pooled from three independent experiments. *P < 0.0001 compared with control RNAi. The nonsignificant P values are mdt-15, 0.89; SCD, 0.80; and sams-1, 0.48. (B) The immunoblot shows the levels of CPL-1W32A,Y35A::YFP and tubulin (loading control) in lysates from control, mdt-15–, SCD-, sams-1–, ire-1–, sca-1–, enpl-1–, and sel-1–depleted worms. The numbers below the blots represent the intensity of the bands relative to corresponding tubulin bands. One of four independent experiments is shown.

Article Snippet: Protein concentrations were determined using the RC DC Protein Assay kit (no. 500–0121; Bio-Rad), and SDS/PAGE analysis and immunoblotting were performed as described ( 4 ) with antibodies against Ser51-Phospho-eIF2α (no. 9721; Cell Signaling Technology), pan-actin (no. 8456; Cell Signaling Technology), tubulin (DM1A, no. T9026; Sigma), GFP (to visualize CPL-1 W32A,Y35A ::YFP and hsp-4p –driven GFP; JL-8; BD Bioscience), and anti-rabbit HRP-conjugated (no. 7074; New England Biolabs) or donkey anti-mouse IR-Dye 800 (LI-COR) secondary antibody.

Techniques: Western Blot