Mm300

Chlorella cultivated in N-deficient media were harvested by centrifugation at 8,000 rpm for 20 min, and then, the spent medium and Chlorella were sampled. For extraction of total protein from Chlorella cells, 50 mg of cells frozen in liquid N were homogenized in a mixer mill MM300 (Qiagen, Germany) and then resuspended in 1 M PBS buffer (pH 7.4) including 1 × cOmplete™ (Roche, Germany) at 4 °C. The solution was centrifuged at 14,000 rpm for 10 min, and then, the supernatant was transferred into a new tube. To determine the concentration of total proteins in the medium, the spent medium was filtered through a 0.2 μm membrane and was concentrated up to 250 times via 5 kDa size cut-off Viva Flow 200 and Vivaspin 20 in turn (Sartorius, USA). After the cell lysates and total proteins from the medium were separated in 12% NuPAGE gel (Invitrogen, USA), they were transferred onto nitrocellulose membranes using the TurboTransfer system (Bio-Rad, USA). After the membrane was incubated with the polyclonal antibody of hG-CSF (Abcam, USA) for 1 h (1:2000 dilution), it was exposed to anti-rabbit IgG-HRP, which was diluted to 1:5,000 (Abcam, USA). Recombinant hG-CSF synthesized in CHO cells (Abcam, USA) was used as the positive control.

To extract nucleic acids, Chlorella cells were harvested by centrifugation, frozen quickly using liquid N, and then ground using a mixer mill MM 300 (Qiagen, Germany) as described by Kumar et al.¹⁵ . Genomic DNA was extracted from the powder using the cetyl trimethylammonium bromide method^{70 (link)}. To investigate the presence of transgenes in the hygromycin-resistant cell lines, PCR was performed on genomic DNA using hG-CSF primers, which were designed to propagate 351 bp-long transcripts, from 31 to 381 nt of the codon-optimized hG-CSF gene. Total RNA was isolated using Trizol (Invitrogen, USA). After DNase I treatment, the RNA was reverse transcribed at 50 °C for 1 h using TOPscript cDNA Synthesis Kits (Enzynomics, Korea) and subjected to RT-PCR and qPCR using primers for detecting hG-CSF transcripts. For RT-PCR, the normalization for quantification was performed by PCR using Chlorella ubiquitin (CvUbi) and/or Actin1 (CvAct1) gene primers. The number of cycles for RT-PCR was 25 cycles for CvUBI and 32 cycles for hG-CSF. For qPCR, the comparative threshold cycle method (ΔΔCt) was used (LightCycler 96; Roche, USA). The CvUbi gene was used as an internal reference. The primers used in this study are summarized in Supplementary Table 2.