C57bl 6 mice

Eight male C57BL6 mice (20–25 g) were purchased from BioLASCO (Taipei, Taiwan), and 16 male Apoe^tm1Unc/J mice (20–25 g) were obtained from Jackson Laboratory (Bar Harbor, ME, USA). The 24 mice were divided into three equal groups (8 per group): C57BL/6 of sham fed normal chow (the control group), ApoE^−/− of 5/6 nephrectomy (5/6 Nx) fed a high-phosphorus diet (the VC group), ApoE^−/− of 5/6 nephrectomy fed both a high-phosphorus diet and empagliflozin (the VCE group; Figure 10). The VCE group received oral empagliflozin 10 mg/kg for 8 weeks after 5/6 nephrectomy. All mice were housed at the Laboratory Animal Center of Tzu Chi University (Hualien, Taiwan). The experimental protocols were approved by the Institutional Animal Care and Use Committee of Hualien Tzu Chi Hospital (approval number 108-48).

C57BL/6 mice (20‐25 g, male, 5‐6 weeks old) were obtained from BioLasco (Taipei, Taiwan). Approval was obtained for all procedures by submitting an Affidavit of Approval of Animal Use Protocol‐Taipei Medical University (Approval No. LAC‐2016‐0081) and the procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (8th edition, 2011).

Adult male C57BL/6 mice (3-month-old) were purchased from BioLASCO Co., Ltd., and group housed under environmentally controlled conditions (lights on at 7:00 a.m. and lights off at 7:00 p.m.; 24 ± 2°C constant room temperature; 50 ± 10% relative humidity). Animals were allowed to habituate for two weeks after their arrival and had free access to autoclaved food and drinking water. Animal experiments were conducted under the animal protocol reviewed and approved by the Animal Research Ethics Board at National Health Research Institutes in Taiwan (Permit Number: NHRI-IACUC-111054-A). The ARRIVE guidelines were used for the animal experiments. Animals were numbered randomly using the RAND function in Mac’s Numbers application for allocation to control and treatment groups. The researchers who conducted the treatment were aware of the group allocation. The animal care staffs and the researchers analyzing the data were unaware of the group allocation.

Eight-week-old male C57BL/6 mice were purchased from BioLASCO Taiwan Co., Ltd. (Taipei, Taiwan). Pulmonary fibrosis was induced by intratracheal administration of 2 U/kg body weight of bleomycin (Sigma-Aldrich, St. Louis. MO) in 50 μl of sterile phosphate-buffered saline (PBS). The control group received the same volume of sterile PBS. Animals were sacrificed 14 days after bleomycin treatment, and lungs were removed for analysis. For administration of ER stress inhibitors and PI3K inhibitor, 4-PBA (200 mg/kg body weight), TUDCA (200 mg/kg body weight) or PI3K inhibitor were dissolved in PBS and administered intraperitoneally from day 0 (prevention group) or day 7 (treatment group) to each animal treated with bleomycin. Animals were sacrificed at 14 days for further studies. All animal experiments were conducted in accordance with the committee guidelines of Taipei Veterans General Hospital and approved by the IACUC (Institutional Animal Care and Use Committee of Taipei Veterans General Hospital, No. IACUC 2014-099).

Adult male Sprague-Dawley rats (250–350 g) and C57BL/6 mice were purchased from the BioLASCO and the National Laboratory Animal Center, Taiwan, respectively. The APP/PS1 mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA) (strain name B6.Cg-Tg(APPswe,PSEN1dE9)85Dbo/Mmjax, stock no. 005864). All animals were bred and maintained on a 12-h light/12-h dark cycle (light on at 8:00 am) at the Animal Facility of the Institute of Biomedical Sciences (IBMS), Academia Sinica with food and water continuously available. Experimental procedures follow the Guide for the Care and Use of Laboratory Animals of the National Institute of Health and were approved by the Animal Committee of IBMS, Academia Sinica.

Adult male C57BL/6 mice aged 6–8 weeks purchased from (BioLasco Taiwan Co., Ltd., Taipei, Taiwan) were used in all experiments. All of the mice were caged at 22 °C with a 12-hour light-dark cycle, and allowed free access to food (Laboratory Autoclavable Rodent Diet 5010) or a high-fat diet (HFD) (D12492; containing 34.9% fat (mostly saturated) by weight, which yields ∼60% calories from fat and has a caloric density of 5.24 kcal/g) and water. This study was approved by the Animal Experiment Committee of Taipei Veterans General Hospital and performed according to the “guides for the care and use of laboratory animals” prepared by the National Academy of Science, USA.

Seven- to eight-week-old female C57BL/6 mice (BioLASCO Taiwan Co., Ltd) or A/J mice (Japan SLC, Inc.) were used to produce the murine intranasal infection model. C. neoformans or C. deuterogattii strains were cultured in YPD broth overnight at 30 °C with shaking at 200 rpm, and washed twice with PBS. Cells were counted with hemocytometer, resuspended in PBS, and diluted to 10⁶ cells (for C57BL/6 mice) or 10⁷ cells (for A/J mice) per ml. Appropriate dilutions of the cells were plated onto YPD agar plate and incubated at 30 °C for 48 h in order to confirm CFU numbers and viability. Groups of 10 (C57BL/6) or 7 (A/J) mice were anesthetised with isoflurane (Panion & BF Biotech Inc.) for C57BL/6 mice or 2% tribromoethanol (T48402, Sigma-Aldrich) for A/J mice, and inoculated with C. neoformans or C. deuterogattii strains via intranasal instillation of inocula of 5 × 10⁴ cells (for C57BL/6 mice) or 5 × 10⁵ cells (for A/J mice) in 50 µl PBS. The survival of mice was monitored twice daily for 49 days, and moribund mice (mice that were unable to eat or drink, their body weight was reduced by 15%, or were hunched) were euthanised with CO₂. The survival curve was created with Prism 8. The significance of differences was determined using Log-rank (Mantel-Cox) test.