Dfc310

Cultured cells were fixed for 10 min with 4% paraformaldehyde with/without permeabilization by 0.2% X-100 and blocked with 3% fetal bovine serum and 1% bovine serum albumin buffer for 1 h at room temperature. After blocking, cells were incubated with anti-GnRH-R (1:100, Cat: NBP2–45300-0.1mg; Novus Biologicals, Littleton, CO, USA), anti- E-cadherin (1:1,000, Cat: 3195s, Cell Signaling Technology), anti-RAD 51 (1:10,000, Cat: ab133534, Abcam), or anti-γH2AX (1:800, Cat: 2577s; Cell Signaling Technology) at 4 °C overnight. After washing with PBS three times, cells were incubated with Alexa 488- or 564-labeled secondary antibodies (1:250 in blocking buffer; Jackson ImmunoResearch Laboratories) as recommended by the manufacturer. Nuclear staining was achieved using Hoechst 33258 (1:10,000; Invitrogen, Darmstadt, Germany). ProLong® Diamond Antifade Mountant (Thermo Fisher Scientific) was then used to mount the stained cells on slides, which were covered with new, clean coverslips. The fluorescence signal was imaged under a Leica DM4000 B LED microscope with a Leica DFC310 digital camera or a laser scanning multiphoton confocal microscope (TCS SP5 MP; Leica Microsystems, Buffalo Grove, IL). All experimental groups were analyzed with the same settings.

For body length measurements, synchronized L1 larvae were placed on NGM agar plates with fath-1 RNAi bacteria or HT115 control and allowed to grow until control group reached young adult stage. Bright-field images of the worms on plates were taken using OLYMPUS SZX16 stereomicroscope fitted with a LEICA DFC310 camera. The body length of each individual was measured from the nose to the tail tip using ImageJ software. At least 40 individuals were examined per treatment group. The life-span assay was performed essentially as previously described [28 (link)]. At least 50 individuals were examined per treatment group.