Microreader

For the CCK-8 assay, ICP-1 cells were inoculated in 96-well plates and transfected with plasmids or siRNA. Cell proliferation and viability were then monitored at 12, 24, 36, and 48 h using the CCK-8 kit (TransGen Biotech, Beijing, China) following the manufacturer’s instructions. This analysis was performed using a microreader (Bio-Rad, Hercules, CA, United States) to measure the absorption value at 450 nm.

Cell viability was measured by a 3- (4,5-dimethyl-2-thiazolyl)- 2,5-diphenyl -2 -H-tetrazolium bromide (MTT) assay. eIF4E or negative control shRNA-transfected EC9706 cells were seeded into 96-well plates (4 × 10⁵/well) in culture medium for 24 h. The medium was then changed to a medium containing the following concentration of DDP (cisplatin) at 0, 6.0, 12.0, 24.0, and 48.0 μg/ml. The cell viability was assessed at 24 h and 48 h incubation. Every well was stained with 5 mg/ml MTT for 4 h and lysed for 15 min. Then the optical density (OD) of culture plates was determined on a microreader (Bio-Rad) at 570 nm. Inhibitory rate was calculated as (1- OD 570_{treated group}/OD 570_{untreated group}) *100%. Each experiment was repeated three times.

OS cell viability was measured with A CCK-8 assay (Dojindo Molecular Technologies, Japan). Specifically, cells with a density of 7,000 cells/well were firstly seeded in 96-well plates. After 6 h of culture, cells were transfected with MG-63-NC, MG-63-FAM64, U-2 OS-NC, and U-2 OS-FAM64A, respectively, and then incubated at 37°C with 5% CO₂ for 0, 24, 48, and 72 h, respectively. At each time-point, a CCK-8 reagent of 10 μl was added into each well, and the incubation was subsequently extended for an additional 2 h at 37°C. The absorbance of each well was measured with a microreader (Bio-Rad, USA) at 450 nm.

The impact of CUL4A expression on the proliferation of HCC cells was determined by cell proliferation assay as described previously38 (link). Briefly, BEL7402 or HepG2 cells at 10⁴ cells/well were cultured in 96-well plates overnight and transfected with CUL4A-siRNA or NC-siRNA using LipofectamineTM 2000 (Invitrogen), followed by incubation for four days. The proliferation of HCC was determined longitudinally using the CCK-8 kit (Beyotime, Shanghai, China) and measuring the absorbance at A450 in a microreader (Bio-Rad, Tokyo, Japan). CUL4A expression was quantified by Western blot.

To assess autoantibody against dsDNA, serum was harvested from aged mice (~6 months) and mouse anti-dsDNA ELISA Kits (Shibayagi) were used to perform ELISA according to the manufacturer’s instructions. To measure SRBC-specific IgG production, serum was harvested from mice on D14 after SRBC immunization and assessed by using SRBC IgG ELISA kits (Abnova) according to the manufacturer’s instructions. For NP immunization experiments, mice were immunized with NP-KLH and NP-specific antibodies that bound to NP₂₀-BSA or NP₇-BSA (Biosearch Technologies) to be determined by ELISA. In brief, 96-well plates were coated overnight at 4 °C with NP₂₀-BSA or NP₇-BSA, followed by blockade of non-specific binding by incubation with blocking buffer (1% of BSA in PBS) for 1 h at room temperature. Mouse serum was diluted to 10⁻⁵ of the original concentration in blocking buffer and then added to the plates, followed by incubation for 1 h at room temperature. Plates were washed with washing buffer (0.05% Tween-20 in PBS) three times. Bound antibodies were detected by HRP-conjugated anti-mouse IgG1 antibodies (Southern Biotech 5300-05, 1:1000). The reactions were developed by incubation for 15 min at room temperature with TMB substrate (Biolegend) and were stopped by the addition of 2 N H₂SO₄. Absorbance was measured by a microreader (Bio-Rad) at 450 nm.

The transfected cells were induced to differentiate into adipocytes by the culture medium with 15% fetal serum and 0.2% oleic acid (Sigma, CA, United States). The cells were washed with PBS and then, 4% formaldehyde was added to fix the cells for 30 min. Next, the cells were washed with PBS, followed by wash with 60% isopropanol, and finally stained with oil red O reagent for 30 min. The cells were then treated with 60% isopropanol for 15 s and rinsed with PBS 3 times for 3 min each time. The stained cells were observed, and images were captured under an electron microscope (Nikon, Tokyo, Japan). Oil red O dye was extracted from the cells in isopropanol solution and quantified in a microreader (Bio-Rad, CA, United States) at 510 nm. Cells were collected and resuspended in a 1.5 mL tube with 1 mL PBS. Finally, cells were counted in a cell counter (CountStar, Shanghai, China).

Cell proliferation was evaluated by MTT (Sigma, St Louis, MO, USA) assay at indicated time points [36 (link)]. HLE-B3 cells (2 × 10⁵ cells/well) were seeded in 6-well plates. The next day, cells were transfected with pCDNA3.1-c-Src^Y530F, pSlience4.1-ShSrc and control vectors. After transfection for 24 h, cells in activation of c-Src kinase group, inhibition of c-Src kinase group and control group were trypsinized and seeded in 96-well plates (6 × 10³ cells/well). At different time points (after sticking for 0, 12, 24, 48 and 72 h), the medium was replaced with 100 μl MTT (5 mg/ml), and the plate was incubated at 37 °C for another 4 h. After incubation, the culture medium was removed gently, and 100 μl DMSO was added. Finally, the absorbance was determined on a microreader (Bio-Rad, Hercules, CA, USA) at 570 nm. All experiments were performed three times independently. The cell proliferation diagram was plotted using the absorbance at each time point.