Doxorubicin hydrochloride

ADR (doxorubicin hydrochloride; Wako, Osaka, Japan) was diluted in 0.9% saline and was administrated to non-anesthetised wild-type and Rac1 conditional-knockout (cKO) mice without detectable glomerulosclerosis at a dose of 8–9 mg/kg body weight (BW) via tail-vein injection. Rac1^flox/flox mice were used as the control. Urine was collected once every few days. The urinary albumin/creatinine ratio (ACR) was measured using an immunoassay (DCA 2000 Systems; Siemens Medical Solutions Diagnostics, Tarrytown, N.Y., USA) with a Bayer DCA 2000+ chemical analyser (Bayer Diagnostics, Elkhart, Ind., USA). After anesthesia with sodium pentobarbital (100 mg/kg BW; Dainippon Sumitomo Pharma, Osaka, Japan), 3–5 mice per day were euthanised on days 0, 7, 14, 21, and 28 after ADR injection. All mice were housed under specific pathogen-free conditions using standard animal cages with free access to standard chow and drinking water.

Female BALB/c mice were purchased from a commercial vendor (Oriental Yeast Co., Ltd., Tokyo, Japan), and ADR nephropathy was induced as previously described45 (link). In brief, ADR (doxorubicin hydrochloride; Wako, Osaka, Japan) diluted with 0.9% saline was injected into eight-week-old BALB/c mice via the tail vein at a dose of 11 mg/kg. Age-matched control mice were injected with an equal volume of PBS only. After anaesthesia with sodium pentobarbital (100 mg/kg BW; Dainippon Sumitomo Pharma, Osaka, Japan), mice were euthanized on days seven and 14 after the injection of ADR. All mice were housed under specific pathogen-free conditions in standard animal cages with free access to standard chow and drinking water. All animal handling and experiments were performed strictly in accordance with the recommendations of the guideline for the Care and Use of Laboratory Animals of the Juntendo University Faculty of Medicine. The experimental protocol was approved by the Animal Care and Use Committee of Juntendo University, Tokyo, Japan.

Conditionally immortalized mouse podocytes were cultured as previously described^{29 (link)}. Briefly, undifferentiated podocytes were cultured in RPMI1640 medium (Sigma-Aldrich, Tokyo, Japan) with 10% fetal bovine serum (FBS), 100-units/ml penicillin and streptomycin (Pen/Strep; Life Technologies, Carlsbad, CA, USA), and 10-U/ml γ-interferon (IFN) at 33 °C. For differentiation, the podocytes were transferred to non-permissive conditions at 37 °C in the absence of γ-IFN and grown for 7–14 days. To detect the reaction of ADR (doxorubicin hydrochloride; Wako, Osaka, Japan), 0.25-μg/ml ADR was added to the differentiated podocytes in a regular medium for the indicated periods.
HEK293T cells were cultured in Dulbecco’s Modified Eagle Medium (Sigma) containing 10% FBS and 100-units/ml Pen/Strep. Transient transfection of HEK293T cells was performed using Fugene 6 reagent (Roche Diagnostics, Basel, Switzerland) at a DNA-to-Fugene ratio of 1:3, according to the manufacturer’s protocol.

Human normal cells (TIG-3 and MRC5) and cancer cell lines, breast cancer (MDA-MB 231, MCF-7), osteosarcoma (U2OS, Saos-2), cervical carcinoma (HeLa), hepatocellular carcinoma (HUH-6, HUH-7), ovarian carcinoma (SKOV3), adenocarcinoma (A549) and colorectal adenocarcinoma (DLD-1, COLO 320 and HCT116), procured from JCRB or DS Pharma, Japan, were cultured in DMEM (Life Technologies). Human melanoma (G361) (JCRB, Japan) was cultured in McCoy’s 5A (Life Technologies). Mortalin-overexpressing cells were generated by retroviral vector as described previously15 (link)22 (link). Mortalin-targeting adenovirus expressing mortalin shRNA (#009-GAATGA GGCTAGACCTTTA) was generated and used as described earlier32 (link). Plasmid based mot-shRNA 2166 (5′-ACCATCTCGCACACAGCAATTCAAGAGATTGCTGTGTGCGAGATGGTT-3′) was constructed and used as described earlier18 (link). Chemotherapeutic drugs were purchased from Sigma (Nocodazole, Paclitaxel, Doxorubicin hydrochloride, Cyclophamide monohydrate) or Wako (Methotrexate, Epirubicin hydrochloride and Docetaxel). Anti-mortalin antibodies (polyclonal and monoclonal) were raised in our laboratory. Mortalin targeting shRNA-expressing adenovirus were generated as described earlier32 (link).

Dipalmitoylphosphatidylcholine (DPPC), 1,2-distearoyl-sn-glycerol-3 phosphoethanolamine-N-[methoxy (polyethylene glycol)-2000] (mPEG–DSPE) and 1,2-distearoyl-sn-glycerol-3-phosphoethanolamine-N-[maleimide (polyethylene glycol)-2000] (Mal–PEG–DSPE) were obtained from NOF Co. (Tokyo, Japan). Cholesterol (Chol) was purchased from Kanto Chemical Co., Inc. (Tokyo, Japan). Thiazolyl blue tetrazolium bromide (MTT), 2-iminothiolane hydrochloride, human IgG, RPMI 1640 medium, and DMEM were from Sigma-Aldrich (St Louis, MO, USA). Doxorubicin hydrochloride was purchased from Wako Pure Chemical (Osaka, Japan). Amicon Ultra filters were from Merck Millipore Ltd. (Billerica, MA, USA). PD-10 columns were from GE Healthcare (Carlsbad, CA, USA).

Dimethylformamide (DMF, Sigma-Aldrich, USA), permeable membrane tube (MWCO 3.5 kDa, Spectrum Laboratories Inc., Japan), tetraethyl orthosilicate (TEOS, Sigma-Aldrich, USA), ammonia (NH₃, China), Doxorubicin hydrochloride (DOX, Wako, Japan), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT, Roche Diagnostics, Japan), dimethyl sulfoxide (DMSO, China), Dulbecco’s Modified Eagle’s Medium (DMEM, Gibco, USA), fetal bovine serum (FBS, Sigma-Aldrich, USA), and antibiotics (a mixture of penicillin, streptomycin, and neomycin, Sigma-Aldrich, USA) were purchased.