Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in
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1 Supplementary Information Neuronal GPCR OCTR-1 regulates innate immunity by controlling protein synthesis in Caenorhabditis elegans Yiyong Liu 1, Durai Sellegounder 1, and Jingru Sun 1 * 1 Department of Biomedical Sciences, Elson S. Floyd College of Medicine, Washington State University, Spokane, Washington, USA *Corresponding. jingru.sun@wsu.edu
2 Figure S1: Wild-type animals treated with rps-1 RNAi have a lifespan similar to that of wildtype animals treated with empty vector control RNAi. Wild-type animals grown on doublestranded RNA (dsrna) for vector control or dsrna for rps-1 were scored for survival over time. WT+vector versus WT+rps-1 RNAi, p = The survival graph represents assays of two independent experiments. n = 45 animals per strain.
3 Figure S2: rps-1 RNAi and eif-3.j RNAi in wild-type and octr-1(ok371) animals. (A) qrt-pcr analysis of expression of rps-1 and eif-3.j in wild-type animals. Bars represent mean ± SEM. n = 3 independent experiments. * denotes a significant difference (p < 0.001) between the means of the WT+RNAi animals and the WT+vector animals. (B) qrt-pcr analysis of expression of rps- 1 and eif-3.j in octr-1(ok371) animals. Bars represent mean ± SEM. n = 3 independent experiments. * denotes a significant difference (p < 0.001) between the means of the octr- 1+RNAi animals and the octr-1+vector animals.
4 Figure S3: rps-1 RNAi and eif-3.j RNAi in JRS9 (octr-1(ok371);phsp-4::gfp(zcls4)) animals. qrt-pcr analysis of expression of rps-1 and eif-3.j in JRS9 animals. Bars represent mean ± SEM. n = 3 independent experiments. * denotes a significant difference (p < 0.001) between the means of the JRS9+RNAi animals and the JRS9+vector animals.
5 Table S1. Proteins downregulated in wild-type N2 animals upon P. aeruginosa infection Protein Gene ID Fold Change # p-value GRD-14 t01b10.2 only in the uninfected C05C8.7 c05c8.7 only in the uninfected GPX-5 c11e4.1 only in the uninfected HACD-1 r09b5.6 only in the uninfected Y54G11A.7 y54g11a.7 only in the uninfected Y43F8C.13 y43f8c.13 only in the uninfected Y49E10.18 y49e10.18 only in the uninfected BAF-1 b only in the uninfected CLEC-5 c35d10.14 only in the uninfected SEC-24.2 zc518.2 only in the uninfected ZK zk only in the uninfected NDX-2 w02g9.1 only in the uninfected CYTB-5.2 w02d3.1 only in the uninfected THN-2 f28d1.5 only in the uninfected F28B4.3 f28b C49C3.4 C49c # only in the uninfected: the protein was only detected in the uninfected wild-type N2 animals, not in the infected wild-type N2 animals.
6 Table S2. Proteins upregulated in octr-1(ok371) animals upon P. aeruginosa infection Protein Gene ID Fold Change # p-value METR-1 r03d7.1 only in the infected DOD-24 c32h11.12 only in the infected F20D6.11 f20d6.11 only in the infected M60.2 m60.2 only in the infected F55G11.2 f55g11.2 only in the infected CPG-2 b only in the infected SKPO-1 f49e12.1 only in the infected RUVB-1 c27h6.2 only in the infected F55G11.4 f55g11.4 only in the infected D d only in the infected C32H11.4 c32h11.4 only in the infected C29E4.12 c29e4.12 only in the infected C14C6.5 c14c6.5 only in the infected C33A12.1 c33a12.1 only in the infected Y18D10A.9 y18d10a.9 only in the infected T23D8.3 t23d8.3 only in the infected DOD-17 k10d11.1 only in the infected LYS-2 y22f5a.5 only in the infected C01G10.9 c01g10.9 only in the infected C29G2.6 c29g2.6 only in the infected HMG-11 t05a7.4 only in the infected C17H12.8 c17h12.8 only in the infected CAT-4 f32g8.6 only in the infected CLEC-67 f56d6.2 only in the infected CLEC-66 f35c5.9 only in the infected SRP-6 c03g6.19 only in the infected KIN-10 t01g9.6 only in the infected GST-38 f35e8.8 only in the infected SNA-1 w02f12.6 only in the infected T22C1.6 t22c1.6 only in the infected F29C4.2 f29c4.2 only in the infected CD4.3 cd4.3 only in the infected F35E12.6 f35e12.6 only in the infected IRG-3 f53e10.4 only in the infected GST-5 r03d K07H8.10 k07h E-05 PUD-1.1 f15e ASP-14 k10c HRP-1 f42a E-05 MMCM-1 zk
7 F55B11.2 f55b Y41C4A.11 y41c4a PUD-2.1 f15e CTH-2 zk GST-7 f11g GLB-1 zk CLEC-63 f35c F59B1.2 f59b T13F2.2 t13f DNC-2 c28h SDHA-2 c34b F32A11.3 f32a RPB-2 c26e E-05 F28H7.3 f28h Y47G6A.21 y47g6a GST-4 k08f C36A4.4 c36a GCS-1 f37b KLO-1 c50f F45H10.3 f45h RPB-9 y97e10ar C42D4.1 c42d Y45F10C.4 y45f10c F58H1.3 f58h E-05 MRPL-12 w09d SKR-3 f44g F13H6.3 f13h C14B9.2 c14b CYSL-2 k10h HSP-4 f43e ATP-4 t05h MBF-1 h21p MTSS-1 par RPL-18 y45f10d RPS-10 d C45B2.1 c45b C27H6.8 c27h H28O16.1 h28o Y71F9AL.9 y71f9al CEY-4 y39a1c C39D10.7 c39d DDP-1 y39a3cr TAX-6 c02f
8 DPY-30 zk RPL-13 c32e Y25C1A.13 y25c1a ATP-2 c34e ZK418.9 zk C18H9.3 c18h Y39B6A.33 y39b6a PRX-19 f54f MRPS-28 y43f8c ACDH-7 t25g Y73E7A.1 y73e7a DJR-1.1 b SAMS-4 c06e ZK zk PAM-1 f49e F42A10.5 f42a MAI-2 b TIN-13 dy CTS-1 t20g RPS-11 f40f DPYD-1 c25f PRDX-6 y38c1aa VIG-1 f56d EIF-3.J y40b1b CEY-2 f46f C44E4.4 c44e PDI-1 c14b RPS-1 f56f K07C5.4 k07c RPS-28 y41d4b # only in the uninfected: the protein was only detected in the infected octr-1(ok371) animals, not in the uninfected octr-1(ok371) animals.
9 Table S3. Effects of knockdown of protein synthesis factors by RNAi on C. elegans development RNAi Target* Effect on wild-type worms Effect on octr-1(ok371) worms culture for 48 hrs, the worms were normal in culture for 48 hrs, the worms were normal in F56F3.5 egg laying and development compared to egg laying and development compared to (rps-1) worms treated with vector control RNAi. worms treated with vector control RNAi. F40F11.1 (rps-11) W09D10.3 (mrpl-12) C32E8.2 (rpl-13) Y45F10D.12 (rpl-18) D (rps-10) defective egg laying. 20 Gravid worms synchronized for 1 hr at 25 C only laid 20 eggs per worm in average while worms with empty vector control RNAi laid approximately 110 eggs per worm. The eggs from worms with rps-11 RNAi were hatched very slowly, and development was arrested at L1 stage. Worms displayed immature oocyte. normal in egg laying phenotype. These eggs hatched normally but their development was arrested at L1 stage. defective egg laying phenotype. 20 Gravid worms synchronized for 1 hr at 25 C only laid 12 eggs per worm in average. None of the laid eggs hatched. normal in egg laying phenotype. However, few eggs hatched and development was arrested at L1-L2 stages. Worms displayed dumpy phenotype RNAi at same development stage. normal in egg laying and development control RNAi. defective egg laying. 20 Gravid worms synchronized for 1 hr at 25 C only laid 24 eggs per worm in average while worms with empty vector control RNAi laid approximately 110 eggs per worm. The eggs from worms with rps-11 RNAi were hatched very slowly, and development was arrested at L1 stage. Worms displayed immature oocyte. normal in egg laying phenotype. These eggs hatched normally but their development was arrested at L1 stage. defective egg laying phenotype. 20 Gravid worms synchronized for 1 hr at 25 C only laid 14 eggs per worm in average. None of the laid eggs hatched. normal in egg laying phenotype. However, few eggs hatched and development was arrested at L1-L2 stages. Worms displayed dumpy phenotype RNAi at same development stage. normal in egg laying and development control RNAi.
10 K07C5.4 C27H6.2 (ruvb-1) Y40B1B.5 (eif-3.j) normal in egg laying phenotype. Few eggs were hatched, and development was arrested at L1-L2 stages. normal in egg laying phenotype. These eggs hatched normally but their development was arrested at L1 stage. normal in egg laying and development control RNAi. normal in egg laying phenotype. Few eggs were hatched, and development was arrested at L1-L2 stages. normal in egg laying phenotype. These eggs hatched normally but their development was arrested at L1 stage. normal in egg laying and development control RNAi. * Y41D4B.5 (rps-28) RNAi clone is not available in either Ahringer C. elegans RNAi library or Geneservice C. elegans RNAi library.
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Supplementary Figures
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SUPPLEMENTARY INFORMATION
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Development Supplementary information. Supplementary Figures * * +/+ +/- -/- +/+ +/- -/-
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Supplemental Figure 1
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Supplementary Materials
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