Multisizer 4e coulter counter

MBs were prepared by the lyophilization method. 1,2-diacyl-sn-glycero-3-phosphocholine (Advanced Vehicle Technology Pharmaceutical Ltd, Shanghai, China), 1,2-dioctadecanoyl-sn-glycero-3-phosphocholine (Advanced Vehicle Technology Pharmaceutical Ltd, Shanghai, China), Tween-80 (Solarbio, Beijing, China), and poloxamer 188 (Sigma-Aldrich, MO, United States) were dissolved in tert-butanol and stored at 4°C overnight. The coagulated solution was lyophilized at 5 × 10⁻⁴ Pa pressure for 24 h (primary drying at −48°C for 20 h and the temperature was gradually raised to 5°C for 4 h). A certain amount of freeze-dried powder was placed in a vial under vacuum. NO was deoxygenated with saturated NaOH solution. About 10 ml of sulfur hexafluoride (SF₆) or a gaseous mixture of NO and SF₆ (volume ratio, 1:9) was injected into the vials using a 10-ml syringe to obtain MBs and NO-MBs. The MBs and NO-MBs samples were assessed under an optical microscope (Nikon, Tokyo, Japan). The mean diameter and concentration of the MBs and NO-MBs were measured using a Multisizer 4e Coulter counter (Beckman Coulter Inc., Brea, CA, United States).

Cr CC1690 cells were grown photoautotrophically in TP^{62 (link)}, TP10, or B&D medium^{63 (link)} at 25 °C, and the illumination of 125 μmol m⁻² s⁻¹ under continuous light conditions. Cultures were kept in a rotatory shaker at 70 RPM. Cells in the mid-logarithmic phase were used as inocula for the different experiments. Cell growth was determined either by measuring samples in a Multisizer 4e Coulter counter (Beckman Coulter Inc., California, USA) particle counter with the Beckman Coulter Multisizer software (v4.03) or using an Infinite M200Pro (TECAN Austria GmbH, Grödig, Austria) plate reader with the TECAN i-control software (v2.0.10.0), to determine either absorbance at 750 nm or chlorophyll fluorescence (excitation 440/9 nm, emission 680/20 nm).

Starch purification, scanning electron microscopy and polarised light microscopy were performed as described in Hawkins et al. (2021 (link)). Briefly, starch granules were purified from homogenates using filtration and a Percoll cushion. Granule size distribution was analysed and plotted in relative volume/diameter using the Multisizer 4e Coulter Counter (Beckman Coulter, High Wycombe, UK). Morphology of starch granules was examined using a Nova NanoSEM 450 (FEI, Hillsboro, OR, USA) scanning electron microscope and a DM6000 microscope (Leica Microsystems, Milton Keynes, UK) for polarised light microscopy. Full methods are provided in Methods S5.

TSC2-deficient kidney tumor cell lines, LAM 621-101 (human, Research Resource Identifier [RRID]: CBCL_S897) (28 (link)), 105K (mouse) (65 (link)), and TMKOC (mouse) (66 (link)), were provided by Drs Jane Yu and Elisabeth Henske. TMKOC was originally generated by Dr Vera Krymskaya (67 (link)). HEK293E cell line (RRID: CVCL_6974), MCF7 (RRID: CVCL_0031), BT549 (RRID: CVCL_1092), and UMB1949 (RRID: CVCL_C471) were obtained from American Type Culture Collection. LAM 621-101, UMB1949, MCF7, BT549, TMKOC, 105K, and HEK293E cells were grown in Dulbecco's modified Eagle's medium (GIBCO) with 10% fetal bovine serum (FBS) (Sigma–Aldrich) at 37 °C with 5% CO₂. About 5 × 10⁶ cells counted by Multisizer 4e Coulter Counter (Beckman) were plated on a 60 mm plate and serum starved for 24 h unless otherwise indicated. Rapamycin (Calbiochem) dissolved in dimethyl sulfoxide (DMSO) was treated at the final concentration of 20 nM (LAM 621-101, UMB1949, MCF7, and BT549) or 100 nM (TMKOC and 105K). MAPK13-IN-1 (MAPK13 inhibitor; MedChemExpress) dissolved in DMSO was treated at the final concentration of 5 μM unless otherwise indicated.

For analysis of spore size distributions, triplicate spore preparations were made in 3 ml of 20% glycerol and suspended in Isoton II (Beckman Coulter, Brea, CA, USA). The particle volume was then measured for a minimum of 100,000 particles per sample using a Multisizer 4e Coulter counter (Beckman Coulter) equipped with a 30 µm aperture tube (size range 0.6–18 µm, aperture current 600 μA). All measurements were conducted with logarithmic spacing into 400 bins using the Multiszier 4 software (Version 4.01) but are presented on a linear x-axis for clarity. For analysis of spore efficiency, triplicate spore preparations were measured in the same way but instead counting the total number of particles in 50 µl. The dilution factor (1:500 for all samples) was then used to calculate the number of particles per µl as presented.