T7 ribomaxtm express rnai system

The T7 RiboMAX^TM Express RNAi System (Promega, cat. No. P1700, Madison, WI, USA) was used for in vitro dsRNA synthesis according to the manufacture’s instruction. A cDNA sequence of the ACT gene was acquired from previous research [34 (link)]. A 200 bp DNA fragment, covering nucleotides +579 to +778 of the ACT-coding region, was PCR amplified using specific primers (Supplementary Table S1) containing the T7 promoter sequence. The other DNA fragments with mutations were synthesized (Genecreate, Wuhan, Hubei, China) and used as template for dsRNA synthesis with primers containing the T7 promoter sequence (Supplementary Table S1). The reaction mixture contained 2 μL T7 Express Enzyme Mix, 10 μL RiboMAX^TM Express T7 2 × Buffer, and 1 μg of DNA template. The mixture was incubated at 37 ℃ for 2–6 h and 70 ℃ for 10 min, then slowly cooled to room temperature (~20 min) to form the dsRNA. The DNA template and single-strand RNA (ssRNA) were removed by DNase (removing DNA template) and RNaseA (removing ssRNA) treatments, respectively. The yield of dsRNA was determined by a Nano Photometer (Implen, Munich, BY, Germany) and the integrity of the full length was detected by gel electrophoresis and stored at −80 ℃ until further use.

The T7 Ribo-MAX^TM Express RNAi System (Promega, Madison, MI, United States) was used for the composition of dsRNA. RNAi primers (Supplementary Table S1) were designed based on the full-length DaAqps. The final dsRNA products (diluted to 1000 ng/μl in diethyl pyrocarbonate (DEPC)-treated water) were stored at –80°C and used within 6 months.
Before injection, D. armandi larvae were placed in an ice bath for 10 min. The larvae were immobilized on an agarose plate using manual forceps (Wang et al., 2016b (link)). Afterward, each D. armandi larvae was injected with 0.05 μl DEPC treated water or dsRNA solution (200 ng/μl), using Hamilton Microliter^TM syringes with 32 G sharp-point needles (Hamilton, Bonaduz, Switzerland) (Chen et al., 2010 (link); Tian et al., 2012 (link); Choi et al., 2014 (link)). The untreated larvae were used as the control group for the experiment. Each treatment group contained 80 larvae. Following injections, the larvae were kept at 4°C in a refrigerator. Nine larvae were removed at different time intervals (24, 48, and 72 h) from each treatment group and frozen in liquid nitrogen for storage at –80°C until RT-qPCR analysis.

For RNA interference, the templates of BmAtg4 and BmAtg7 were amplified by PCR from total cDNA obtained from B. mori fat body. Double-stranded RNA (dsRNA) was generated using the T7 RiboMAX^TM Express RNAi system (Promega, WI, USA, P1700), according to the manufacturer’s instructions. dsRNA (50 μg/larva) was injected into the larvae 12 h before IW, with egfp dsRNA injection as control. The fat body was collected 24 h after injection for further analyses. All primers used in this study are listed in Table S1.

A total of 500 orb2 and 495 bp of GFP fragments were amplified using PCR primers and sequenced. Then, T7 promoter sequence was added with PCR primers (Table S1) and the PCR product was amplified from 1μg cDNA, which was used as template for dsRNA synthesis. dsRNA was synthesized from purified PCR products using kit (T7 Ribo MAXTM Express RNAi System, Promega, Madison, WI, USA). Bands of the expected sizes confirmed the integrity of dsRNA and concentrations of dsRNAs were measured by spectrophotometry (Nano Drop 1000, Thermo Scientific, Ann Arbor, MI, USA). dsRNAs were stored at −80 °C. Agomirs and antagomirs were bought from company Gene Pharma (Shanghai, China).

The dsRNA of LsFoxO was produced using the T7 RiboMAXTM Express RNAi System (Promega, Sunnyvale, CA, United States). After synthesis, dsLsFoxO (MF197906, 431 bp) and dsGFP (DQ389577, 495 bp) were quantified by an ultramicro-spectrophotometers (NanoDrop 2000, Thermo Fisher, Scotts Valley, CA, United States) and were maintained at -80°C until use (Table 1). The sequence was verified by sequencing (Sangon Biotech, Shanghai, China). Before injection, the dsRNA and phenol red solution were mixed for observations. Under carbon dioxide anesthesia, nymphs were immobilized on the agarose injection plate with the ventral side upward, under CO₂ anesthesia. The purified dsLsFoxO and dsGFP were slowly injected on one side of the metathorax using the Nanoject II (Drummond, Broomall, PA, United States). The injected individuals were placed in a glass tube (length: 200 mm; diameter: 25 mm) on fresh rice seedlings for further observation. Data on developmental duration were recorded every day until the adult emerged.

The gene expression of tumor protein 53 (p53) and the stress-inducible heat shock protein 70 (hsp70) was, among others (mcm6, pcna, CuZnsod, foxo and smedinx11), selectively knocked down and effects on cell proliferation were studied after 17 days exposure to 50 μM MMS (genotoxic carcinogen), 10 μM MPH (non-genotoxic carcinogen) or culture medium (control). This exposure set-up was chosen on the basis of previous experiments that showed the strongest increases in cell proliferation after 17 days exposure to 50 μM MMS (S5). RNA interference was performed using double stranded RNA (dsRNA) probes, generated by an in vitro transcription system (T7 RibomaxTM Express RNAi System, Promega) as indicated by the manufacturer. The primer and probe details are summarized in Supplemental Fig. S7. The animals were injected with three 32 nl injections of 1 μg/μl dsRNA for 2 consecutive days in the pre-pharyngeal part of the gut using the Nanoject II (Drummond Scientific). The non-RNAi group was injected with water. At day 3, the animals were transversally cut in two and exposed to the different compounds. All RNAi approaches were validated with real time qPCR, with an inhibition between 20–50%).

The dsRNAs were synthesized using the T7 RiboMAX^TM Express RNAi System (Promega, Madison, WI, USA). The DNA templates were obtained by PCR amplification using primers containing T7 RNA polymerase promoter sequences (Table S1). The dsRNAs for enhanced green fluorescent protein (EGFP), dsEGFP, served as negative controls. Subsequently, dsNotch and dsEGFP were prepared according to the instructions in the manual, and the purified dsRNAs were dissolved in nuclease-free water. The purity and integrity of the dsRNAs were analyzed using a spectrophotometer and 1% agarose gel electrophoresis before usage.