Ru360

N. crassa was handled according to standard procedures. Vogel's minimal medium plus 1.5% (w/v) sucrose was used in all experiments (Davis and de Serres, 1970 (link)). Wild-type and deletion strains used in this study are listed in supplementary material Table S1. The following chemicals were used: staurosporine (LC Laboratories, Woburn, MA); DMSO, ML204, flufenamic acid and xestospongin C (Sigma-Aldrich, St Louis, MO); 4-methyl-4′-[3,5-bis(trifluoromethyl)-1H-pyrazol-1-yl]-1,2,3-thiadiazole-5-carboxanilide (YM-58483/BTP2) and 2-aminoethoxydiphenyl borate (2-APB) (Tocris Bioscience, Bristol, UK); 1,2-bis(ortho-aminophenoxy)ethane-N,N,N′,N′-tetrasodium (BAPTA), thapsigargin, Ru360, LiCl₂ and 7-hydroxystaurosporine (UCN-01) (Merck Millipore, Darmstadt, Germany); 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-1H-pyrrole-2,5-dione (U-73122) (Alexis Biochemicals, San Diego, CA) and bafilomycin A1 (Wako Chemicals, Richmond, VA).
Mycelial extension rates at 26°C were measured after inoculating 5 µl containing 1×10³ conidia onto the centre of large Petri dishes (14.2-cm diameter) containing solid minimal medium. For growth measurements in liquid minimal medium, 1×10⁴ conidia/ml were incubated at 26°C, 100 rpm, under constant light in 96-well plates (200 µl total volume per well) and absorbance was monitored at 450 nm over 24 hours.

BAECs were plated at density of 5 × 10⁴ for each well in 24-well plates. After incubation with PTH 0.1 nM for both 1 and 3 h, the cells were incubated with either 5 μM H₂DCFDA (Invitrogen) for 30 min at 37°C for total ROS (tROS) detection or with 5 μM Mitosox (Invitrogen) for 10 min at 37°C for mitochondrial ROS (mROS) detection, in a humidified atmosphere (5% CO₂, 95% air). After incubation, cells were washed twice with PBS, and fresh medium was added. The fluorescence was immediately measured by a plate reader (Tecan Infinite 200 Pro) using excitation/emission wavelengths of 492/520 nm for H₂DCFDA and 510/580 nm for Mitosox. Then, the cells were trypsinized and collected for cytofluorimetric analysis. In some experiments, cells were also treated with either 5 μM MitoTEMPO (Sigma-Aldrich), 100 nM calcipotriol hydrate, a vitamin D analogue (Sigma-Aldrich), or 10 μM Ru360 (Merck-Millipore). MitoTEMPO was administered 30 min before PTH, while both calcipotriol hydrate and Ru360 were coadministered with PTH.

The IP₃R inhibitor Xestospongin C (XeC, ×2628, 10 μM, Sigma) [14 (link)] and the MCU inhibitor Ru360 (557,440, 10 μM, Merck, Kenilworth, New Jersey, USA) [15 (link)] were used to block ER calcium release and mitochondrial Ca²⁺ uptake, respectively. Podocytes were pre-treated with the above inhibitors for 60 min before treatment with ADR or Ang II, respectively. Specific siRNA targeting the bridging protein Grp75 and a non-targeted negative control siRNA were synthesized by Invitrogen. Podocytes were plated in six-well plates and treated with 100 pmol/well siRNA duplexes using 10 μl RNAiMAX reagent (Invitrogen) according to the manufacturer’s protocol. After 8–12 h, the media were changed according to the status of cell growth at 40–50% confluence. The podocytes were collected for further experiments 24 h after transfection.

Metformin and phenformin were suspended in culture medium (without FBS and penicillin) and used at the respective concentrations of 5 mM and 100 µM, unless stated otherwise. Metformin, phenformin, 2-APB, KB-R7943 and cyclosporin A were purchased from Sigma-Aldrich. RU-360 was purchased from Merck Millipore.

NCI-H929 and U266 multiple myeloma (MM) cells were obtained from American Type Culture Collection (Manassas, VA) and maintained in RPMI-1640 medium supplemented with 10% FBS and 1% penicillin/streptomycin/glutamine. NCI-H929 cells were also supplemented with 0.05 mM beta-mercaptoethanol. The HYD1 drug-resistant NCI-H929 cell line H929-60 was developed as previously described^{15 (link)}. The parental and drug resistant line was validated by short tandem repeat (STR) analysis. 5TGM1 myeloma cells were derived from murine myeloma 5T33 and kept in similar media as U266 cells. All cell lines were tested for mycoplasma every six months. HYD1 (kikmviswkg) and MTI-101 were synthesized by Bachem (San Diego, CA). Bortezomib was purchased from Selleck Chem. The compounds U73122 (Sigma), 2-APB (Sigma) and Ru360 (Merck Millipore) were all used to inhibit Ca²⁺ signaling.

The following inhibitors were used: 5 μM or 10 μM Fbp inhibitor (5-chloro-2-(N-(2,5-dichlorobenzenesulfonamido))-benzoxazole; Cayman Chemicals, Ann Arbor, MI, USA, 18860), 25 μM MCU inhibitor (Ru360; Merck KGaA, Darmstadt, Germany, 557440), 5 μM SERCA inhibitor (thapsigargin; Merck KGaA, Darmstadt, Germany, T9033). Each of the inhibitors was added to neuronal culture 30 min prior to the LTP induction.