Microinjector

All zebrafish work was done in the Tg(fli1:GFP) line [42 (link)] using an Animal Study Protocol (G-05–5 to RS) approved by the National Human Genome Research Institute’s Animal Care and Use Committee. Embryos were generated from a breeding stock, maintained at 28 °C in E3 water, and treated with 0.3 mg/mL PTU (N-Phenylthiourea) from 24 h post-fertilization (hpf) to suppress pigmentation. At 48 hpf, embryos were manually dechorionated, and anesthetized with 0.16 mg/ml tricaine (Syndel, Ferndale, WA). Seventy-five freshly thawed passage 4 HPSLFs or NLFs were suspended in 4 nL of 3 mg/mL Matrigel (Corning, Corning, NY) and were injected into the yolk sac of 48 h post-fertilization zebrafish embryos using a microinjector (World Precision Instruments, Sarasota, FL). Zebrafish embryos were screened for CM-Dil signal using a Leica MZ16F microscope (Leica, Wetzlar, Germany) 2 h after injection. Tile images from zebrafish mounted on glass-bottom microwell dishes (MatTek, Ashland, MA) in 0.8% low-melt agarose (Sigma-Aldrich, St. Louis, MO) containing 0.16 mg/mL tricaine were obtained at 24 and 48 h after injection using a Zeiss LSM 810 confocal microscope (Zeiss, Oberkochen, Germany). At least 20 zebrafish embryos were analyzed for experiments performed in triplicate using cells from 2 different patients with HPS-1 pulmonary fibrosis.

We refered to the methods in the literature and apply them in our experiments (20 (link)).After anesthetizing the mice with sodium pentobarbital, mice were placed on the stereotaxic apparatus, drill a 2mm small hole was drilled by a dental drill, and a 26-G stainless steel tube implanted into the right ventricle of the mouse. The guide sleeve was fixed by dental cement, fixed by stainless steel screws fixed on the skull, and sealed with stainless steel wire to prevent blockage. Injections into lateral ventricles were performed using the following coordinates from Bregma: 0.8 mm posterior, 0.2 mm lateral, and 2.4 mm ventral. JWH133 (3μg) or AM630 (3μg) were infused intoventricle at a rate of 1 μl/min using a microinjector (World Precision Instruments) fitted with a 30 gauge needle attached to a 10 μl gas-tight syringe (Hamilton, 7634-01). The needle was left in place for 2 min post injection before slowly withdrawing.

Embryos at one cell stage were injected using a Narishige micro-injector and glass needles prepared by horizontally pulling standard capillaries (filament, 1.0 mm, World Precision Instruments) with aP-97 Flaming/Brown Micropipette Puller (Sutter Instrument Company). A total of 30 pg for DNA and between 50 and 100 pg for mRNA in 1 nl volume were injected in the embryos in the cell or the yolk, respectively. Drug treatments were performed on manually dechorionated embryos at the desired developmental stage in E3 medium. The following compounds were used: blebbistatin (100 μM for 2.5 hr; Calbiochem); paranitroblebbistatin (20 μM; Optopharma), Ableb (5 μM for 15 min before photoactivation; Optopharma), and nocodazole (10 ng/μl for 2.5 hr; Sigma).

Transgenic zebrafish (fli-1-EGFP) embryos were incubated at 28°C in egg water. At 48 h postfertilization (hpf), zebrafish embryos were anesthetized with 0.016% tricaine and transferred onto agar plates for microinjection. Fifty MDA-MB-231 cells suspended in 9.6 nl of serum-free DMEM were injected into the yolk of each embryo using a microinjector (World Precision Instruments, UK). The injected zebrafish embryos were individually transferred to single wells of 12-well plates with 1 ml of egg water. The zebrafish embryos were incubated at 28°C for 70 h. The embryos were fixed in 4% paraformaldehyde overnight at 4°C and imaged using a Perkin Elmer confocal spinning disk. All experiments and maintenance of zebrafish were in compliance with the approved protocols by the NUS Institutional Animal Care and Use Committee.

For virus microinjection, WT or KIF2C cKO mice were randomly allocated to experimental groups and processed. A small craniotomy was performed after anesthetizing the mouse. The flow rate (40 nl/min) was controlled by a micro-injector (World Precision Instruments). Virus was microinjected in both left and right hippocampal CA1 region (from bregma, 3 weeks: ML: 1.45 mm; AP: 1.8 mm; DV: 1.5 mm; 2 months: ML: 1.65 mm; AP: 1.82 mm; DV: 1.62 mm). For electrophysiology, 4- to 6-week-old mice were subjected for experiments at least 3 days after injection. For KIF2C re-expression mice subjected for behavioral tests, mice were injected and bred until 2- to 3-month-old, then took water containing (40 μg/ml) doxycycline for 2.5–3 days to induce KIF2C expression before tests.

Embryos were anaesthetized with 0.112 mg ml⁻¹ Tricaine in E3 medium and mounted in 0.7% (w/v) low-melting agarose (NuSieve GTG Agarose, Lonza) in glass bottom microscopy dishes (MatTek). For the microangiography Texas Red-dextran with a molecular weight of 70 kDa (Thermo Fisher Scientific, Supplementary Table 6) was solubilized in E3 medium to a concentration of 2 mg ml⁻¹. Using a glass microneedle and a microinjector (World Precision Instruments), the dextran was injected into the sinus venosus. Subsequently, images were taken with an SP8 confocal microscope (Leica).

Zebrafish embryos were injected at the one-cell stage with a glass microneedle and a microinjector (World Precision Instruments). For transgenesis tol2 mRNA, transcribed from pCS2FA with the mMESSAGE mMACHINE SP6 Transcription Kit, a kind gift from Koichi Kawakami, was injected at a concentration of 25 ng µl⁻¹ together with Tol2 destination vectors^{69 (link)}. For mutagenesis Cas9 mRNA was in vitro transcribed from the MLM3613 plasmid (Addgene plasmid #42251) using the mMESSAGE mMACHINE T7 ULTRA Transcription Kit (Thermo Fisher Scientific); sgRNA target sequences were cloned into DR274 (Addgene plasmid # 42250) and transcribed with the MAXIscript T7 transcription kit (Thermo Fisher Scientific). Subsequently, 1 nl of a mixture of 600 ng ml⁻¹Cas9 mRNA and 50 ng ml⁻¹ sgRNA were co-injected into one-cell stage embryos^{29 (link)}.