Doxycycline

The generation and genotype of ATP6ap2^flox/y has been already described [59 (link)]. Heterozygous ATP6ap2 ^flox/x females were crossed with males both positive for Pax8-rtTA and Cre [44 (link), 69 (link)]. Expression of Cre-recombinase was induced in male ATP6ap2^{flox/y,Pax8Cre+} and ATP6ap2^+/+,Pax8Cre+ transgenic mice by 1 mg/mL of doxycycline (Sigma) in drinking water containing 2% of Sucrose (Sigma) for 5 days followed by 5 days induction with 0.25 mg/mL doxycycline in 2% Sucrose drinking water and 4 days without doxycycline [44 (link)]. A second group of ATP6ap2^{flox/y,Pax8Cre+} and ATP6ap2^+/+,Pax8Cre+ transgenic mice received 2 mg/mL doxycycline for 5 days followed by 5 days of 1 mg/mL doxycycline in water containing 2% Sucrose [69 (link)]. These groups are accordingly referred to as 1 mg and 2 mg doxycycline. Animals used for experiments were 2- to 3-month-old male mice bred in a C57BL/6 background. All animal studies were performed according to Swiss welfare laws and with approval of the local veterinary authorities.

The control group (ii) was kept untreated (administered 2% Tween80 solution) and was sacrificed at 38 days post-infection. The other groups were administered a daily oral gavage for 21 days, as follows: (iii) antibiotic only, (iv) antibiotic and extract, and (v) extract only. The respective doses for each mouse in these groups were as follows: 2 mg doxycycline in 0.1 ml (80 mg/kg/day), 1 mg doxycycline + 20 mg extract in a total of 0.3 ml, and 20 mg extract in 0.25 ml (800 mg/kg/day) at intervals of 24 h. doxycycline was purchased from Sigma Aldrich for use as an antibacterial treatment.

Human 293-derived Flp-In-293 cells (Invitrogen, Madison, WI, USA) were cultivated in Dulbecco's modified Eagle's medium (DMEM) containing 10% foetal bovine serum (FBS) (Thermo Fisher Scientific). Human neuroblastoma SH-SY5Y-derived Aβ-GFP [23 (link)] and ∆K280 tau_RD-DsRed cells [24 (link)] were kept in DMEM/nutrient mixture F12 (DMEM/F12) containing 10% FBS, 5 μg/ml blasticidin, and 100 μg/ml hygromycin (InvivoGen, San Diego, CA, USA). Doxycycline (Sigma-Aldrich) was applied to induce the expression of Aβ-GFP (5 μg/ml Doxycycline) or ∆K280 tau_RD-DsRed (2 μg/ml Doxycycline).

All cells were cultured at 37°C and 5% constant CO₂ supply. All cell culture media and ingredients were purchased from Gibco unless stated otherwise. HeLa cells and MEFs were grown in Dulbecco’s modified eagle medium (DMEM) including 4.5 g/l glucose supplemented with 2 mM L-glutamine, 2% penicillin (10,000 u/ml)/streptomycin (10,000 µg/ml) and 10% FetalClone III serum (Hyclone, Thermo Fisher Scientific). HEK293T cells were cultured in DMEM including 4.5 g/l glucose supplemented with 2 mM L-glutamine, 1 mM sodium pyruvate, 1% nonessential amino acids, 10% tetracycline-free fetal bovine serum (Biochrom AG), 1.5 mg/ml hygromycin, and 150 µg/ml blasticidin. To induce overexpression, cells were treated with 1 µg/ml tetracycline (Sigma-Aldrich) for 16 hr. Doxycycline inducible CRISPR-Cas9 HeLa WT, SPAG5 and KNSTRN KO cells were grown in DMEM containing 4.5 g/l glucose supplemented with 2 mM L-glutamine, 2% penicillin (10,000 u/ml)/streptomycin (10,000 µg/ml), and 10% tetracycline free fetal bovine serum (Sigma-Aldrich). To induce Cas9 expression, cells were treated with 1 µg/ml Doxycycline (Sigma-Aldrich) for 4 consecutive days adding fresh Doxycycline each day. To obtain stable SPAG5 and KNSTRN KO cell lines, induced cells underwent monoclonal selection after serial dilutions.