Methods

Strains used in the study

C. elegans strains used in the study were wild-type N2 (Bristol) and TP12 (Pcol-19::GFP).

RNAi interference

Systemic RNA interference was done as described (Fraser et al. 2000; Kamath et al. 2001). HT115(DE3), an E. coli strain, expressing double-stranded RNA for the target gene was grown in LB broth containing carbenicillin (50 g/ml), 8 hours, at 37°C. Bacterial culture was plated onto NGM plates containing 50 g/ml carbenicillin and 5 mM isopropyl D-thiogalactoside (IPTG) and incubated at 25°C for 12 hours before use. Clones were confirmed by sequencing. Gravid adults were allowed to lay eggs on empty vector control or target RNAi plates. Eggs were allowed to grow at 20°C until L4 stage for RT PCR analysis or until the young adult stage for survival and Hoechst staining experiments.

Cuticle permeability assay

Cuticle permeability was assessed using Hoechst 33258 stain animals as described (Moribe et al. 2004). Gravid adults were allowed to lay eggs on empty vector control or target RNAi plates. Eggs were allowed to grow at 20°C until young adult stage. Worms were stained with 10 ug/ml Hoechst 33258 for 30 mins at room temperature (RT). Unbound stain was removed by washing with M9 buffer before visualization and imaging under DAPI filter using Zeiss Apotome.

Quantitative Real-time PCR

Synchronized L4 nematodes grown on control or target RNAi at 20°C were harvested by washing the plates with M9 buffer and frozen in TRizol reagent at -80°C. RNA was extracted using RNeasy Plus Universal Mini Kit according to the manufacturer’s instruction (Qiagen). cDNA was prepared using the iScript cDNA synthesis kit (BIO-RAD). qRT-PCR was conducted using the BIO-RAD TaqMan One-Step Real-time PCR protocol using SYBR Green fluorescence (BIORAD) on an Applied Biosystems QuantStudio 3 real-time PCR machine in 96-well plate format. Fifty nanograms of RNA were used for real-time PCR. 10 ul reactions were set-up in two replicates. Relative -fold changes were calculated using the comparative CT (2 -ΔΔCT) method and normalized to actin-1 (Livak and Schmittgen 2001). Three or more biological replicates were used for qRT-PCR analysis.

Survival assay

Young adult animals were synchronized on control or target RNAi bacterial plates at 20°C. ≥50 animals were exposed to 20 mM paraquat on NGM plates with OP50 at 20°C for oxidative stress induction. Animals were scored for survival every 6-8 hours (Park et al. 2009; Sandhu et al. 2021).

Paralysis assay

For paralysis experiments, animals were synchronized on control or target RNAi plates until young adult stage at 20°C. Levamisole assays were done by scoring the number of paralyzed worms after a 30 mins exposure to 125 μM levamisole on NGM plates with OP50 at RT (Lewis et al. 1980). For ivermectin stress, adult animals were exposed to 50 μM ivermectin in M9 buffer and scored for paralysis at 8 minutes at RT (Kass et al. 1980).

Statistical analysis

Survival assays were plotted using the PRISM 5.01 (Kaplan-Meier method). Survival curves with p values <0.05 for Mental-Cox test were considered significantly different. Statistics for survival assays are presented as extended data in Table 2.

Extended Data

Table 1. List of 286 Transcription factors RNAi clones tested for Hoechst 33258 permeability. https://doi.org/10.22002/D1.2053

Table 2. Statistics for survival assays. https://doi.org/10.22002/D1.2054