Fig 1: Increase in sorafenib-induced apoptosis of HCC cells by silencing ORMDL3. A Western Blot was used to analyze the expression levels of Caspase 3, Caspase 9, PARP, Bax, and Bcl-2 in SMMC-7721 cells after ORMDL3 was silenced or overexpressed under the action of sorafenib. B, C Quantitative analysis of Western Blot gray value. Statistical analysis was performed using one-way ANOVA. **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 3). D, E Silenced and overexpressed ORMDL3 hepatoma cells SMMC-7721 were treated with sorafenib (4 µM) for 24 h, and apoptosis and necrosis were detected. Representative images under ×100 inverted microscope after PI/Hoechst kit staining. Scale bar, 100 µM. Differences between the two groups were analyzed using Student’s t test. *P < 0.05. The data are presented as the means ± SD (n = 3)
Fig 2: In vivo study—establishment of subcutaneous tumor transplantation model in mice. A Nude mice were divided into groups, and subcutaneously transplanted tumor models were constructed. The mice were given drugs every 2 days, and changes in body weight and tumor volume were recorded. On the 28th day, the mice were sacrificed and subcutaneous tumor masses were removed for analysis. B Mice were weighed every two days to make 28-day weight changes after drug injection. C The weight data of mice before death were obtained. D Tumor weight was detected after the mice were sacrificed. E Tumor growth trend analysis. Statistical analysis was performed using one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 5). F H&E staining was performed to observe the tumor tissue sections. Immunohistochemical evaluation of Ki67, LC3B, Beclin1, ATF4, and PERK expressions in xenograft tumors of nude mice with different treatments. The brown expression was positive. Scale bar, 200 µM. G Image ProPlus (IPP) was used to analyze the expressions of Ki67, LC3B, Beclin1, ATF4, and PERK in immunohistochemical images. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01,***P < 0.001. The data are presented as the means ± SD (n = 5). H Schematic diagram of the mechanism by which silencing ORMDL3 increases the sensitivity of HCC cells to sorafenib. Sorafenib acts on tyrosine kinase receptors of HCC cells, induces endoplasmic reticulum stress, and leads to increased levels of autophagy and ROS. Under the action of sorafenib, silencing of ORMDL3 can inhibit the PERK-ATF4-Beclin1 pathway, thereby inhibiting autophagy, increasing ROS levels, and increasing ROS-mediated apoptosis of liver cancer cells
Fig 3: Inhibition of autophagy through the PERK-ATF4-Beclin1 pathway by silencing ORMDL3. A, B Western Blot assay was performed to detect the protein expression levels of PERK, p-PERK, ATF4, Beclin1 in ORMDL3-silenced human HCC cells and grayscale analysis. C, D Western Blot assay was performed to detect the protein expression levels of PERK, p-PERK, ATF4, Beclin1, P62, and LC3 after overexpression of ATF4 in ORMDL3-silenced human HCC cells SMMC-7721 and grayscale analysis. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 3)
Fig 4: Increase in the apoptosis of sorafenib-induced HCC cells by silencing ORMDL3, by inhibiting autophagy. A, B Western Blot assay was performed to detect the expression levels of autophagy-related proteins LC3, P62, and Beclin1 in human HCC cells SMMC-7721 silenced by ORMDL3, and gray value analysis. C, D The expression of LC3 in HCC cells was detected by immunofluorescence. Scale bar, 25 µM. Five regions were randomly selected from each sample for evaluation. Statistical analysis was performed using one-way ANOVA. *P < 0.05, **P < 0.01. The data are presented as the means ± SD (n = 3). E, F Western Blot assay was used to detect the expression levels of autophagy-related proteins LC3, P62, and Beclin1 in ORMDL3-silenced human HCC cells SMMC-7721 combined with autophagy inhibitor CQ (50 µM), and gray value analysis. G Annexin V-FITC/PI Apoptosis Kit was used to analyze apoptosis levels in the ORMDL3-silenced group by flow cytometry. H Histogram of streaming data analysis. Statistical analysis was performed using one-way ANOVA. **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 3). I The apoptosis and necrosis of ORMDL3-silenced HCC cells SMMC-7721 were detected in the presence of sorafenib (4 µM) combined with Rap (100 nM), an autophagy agonist. Representative images under ×100 inverted microscope after PI/Hoechst kit staining. Scale bar, 100 µM. Differences between the two groups were analyzed using Student’s t test. **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 3)
Fig 5: Increase in the sensitivity of liver cancer cells to sorafenib by silencing ORMDL3. A, B Western Blot and RT-qPCR were used to detect the expression of ORMDL3 at protein and mRNA levels in SMMC-7721 cells after ORMDL3 deletion or overexpression of ORMDL3. C, D MTT assay was used to detect the cell viability and inhibition rate of different groups of human hepatoma cell line SMMC-7721 with ORMDL3 silenced and ORMDL3 overexpression. E, F Clonal formation survival and statistical quantification of ORMDL3 silenced or overexpressing of ORMDL3 HCC cells with or without sorafenib. Statistical analysis was performed using one-way ANOVA. nsP > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001. The data are presented as the means ± SD (n = 3)
Supplier Page from Abcam for Anti-ORMDL3 antibody