Ibright software

Cells were trypsinized and suspended in DMEM with either DMSO, Iono, or Tg treated for 5 min in a 6-well low-attached plate (Corning). Cells were centrifuged at 1000 rpm for 5 min. Lysates were prepared and immunoblot analysis was conducted as previously described [28 (link)] using the following antibodies: Anti-Acetyl-α-tubulin (Lys40) (D20G3) XP (1:1000, Cell Signaling Technologies, CST5335, Danvers, MA, USA), Anti-α-tubulin (1:1000, Sigma T6199), Anti-Detyrosinated alpha tubulin (1:1000, Abcam, ab48389, Cambridge, UK), Anti-GAPDH (1:1000, Santa Cruz sc32233, Santa Cruz, CA, USA), Anti-Myosin Light Chain 2 (D18E2) (1:1000, CST8505), Anti-phospho-myosin light chain 2 (Ser19) (1:1000, CST3675), Anti-myosin phosphatase 1 (D6C1) (1:1000, CST8574), Anti-phospho-myosin phosphatase (Thr853) (1:1000, CST4563), Anti-phospho-cofilin (Ser3) (77G2) (1:1000, CST3313), Anti-cofilin (D3F9) (1:1000, CST5175). Densitometry analysis was conducted across 3 independent experiments using ImageJ software. Original immunoblot images and additional detailed densitometry method and analysis using the iBright Software (ThermoFisher, Waltham, MA, USA) can be found in the supplementary material method S1, Supplementary Figures and File S1.

To examine the induction of recombinant VHSV glycoprotein expression, various concentrations of NaCl were added in the early log phase (long-term induction) or late log phase (short-term induction) according to the Chlorella growth curve. Transformed C. vulgaris was inoculated into BGPK broth with 200 mM KClO₃ at an initial concentration of 1.5 × 10⁶ cells/mL. For long-term induction, cells were cultured for 2 days and then treated at the same concentrations of NaCl mentioned above. After incubation for an additional 5 days, protein was extracted and Western blotting analysis was performed. For short-term induction, cells were cultured for 7 days and NaCl was added at those same concentrations. After 30, 60, or 120 min, total protein was extracted and Western blotting was performed as described above. The expression level was compared via density analysis using iBright software (Thermo Fisher). The effects of two induction variables, i.e., NaCl concentration and treatment time, on the expression of VHSV glycoprotein in transformed Chlorella were evaluated using Design Expert 13 software (Stat-Ease Inc., Minneapolis, MN, USA).

The supernatant (4 μg/μL) mixed with a certain proportion of 5 ×  and 1 ×  SDS-PAGE loading buffer (Solarbio, China) was boiled at 95°C for 10 min. Equal amounts of protein (50 μg/20 μL) were separated by 10% SDS-PAGE (SDS-PAGE Gel Kit, Solarbio Life Sciences, China). Proteins were electrophoretically transferred to polyvinylidene difluoride membranes (Bio-Rad, Richmond, USA), which were then blotted with an antibody against ATP synthase subunit epsilon (ATP5E) (ThermoFisher, USA) diluted 1 : 500, voltage-dependent calcium channel subunit alpha-2/delta-1 (CACNA2D1) (Abcam, Hong Kong) diluted 1 : 10000, glutathione peroxidase 3 (Gpx-3) (Santa, USA) diluted 1 : 2000, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (Immunoway, USA) diluted 1 : 20000. A secondary antibody (Abcam, Hong Kong) was used to detect the primary antibody. Anti-beta actin antibody (Immunoway, USA) and secondary antibody (Abcam, Hong Kong) were used as references. Finally, chemiluminescence analysis, development, and fixation were carried out. Band signals were detected using an iBright CL750 western blot imaging system (ThermoFisher, USA) and analyzed using iBright Software (ThermoFisher, USA).