Animal ethics.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.</div

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Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.</div

Effects of TGM2 overexpression on proliferation, migration, and invasion of MDCK cells.

(A). mRNA levels of TGM2-overexpressed cells were determined with real-time PCR. (B). Protein levels of TGM2-overexpressed cells determined by Western blotting. (C). Effects of TGM2 overexpression on proliferation ability of MDCK cells determined by cell counting; (D,E) Effects of TGM2 overexpression on plate clone rate of MDCK cells determined by plate clone formation assays. (F,G) The effects of TGM2 overexpression on the invasion ability of MDCK cells were determined using Transwell assays. (H) The effects of TGM2 overexpression on the migration ability of MDCK cells were determined by scratch tests. TGM2-overexpressed cells (TGM2-OE); Vector control cell (Con). The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p

Raw data of the gels and blot.

Madin-Darby canine kidney (MDCK) cells are one of the main cell lines used for influenza vaccine production due to their high virus yield and low mutation resistance. Due to their high tumorigenicity, the safety of vaccines produced from these cells is controversial. TGM2 is a multifunctional protein that plays an important role in the adhesion and migration of cells and is associated with tumor formation. We found that the expression level of TGM2 was significantly up-regulated in low tumorigenic MDCK cells. We first analyzed TGM2-overexpressed and knockout MDCK cells in vitro. Scratch-wound assay and Transwell chamber experiments showed that TGM2 overexpression significantly inhibited the migration and invasion of MDCK cells and significantly reduced their proliferation. TGM2 knockout significantly enhanced cell migration, invasion, and proliferation. The tumorigenesis results in nude mice were consistent with those in vitro. TGM2 knockout significantly enhanced the tumorigenesis rate of MDCK cells in nude mice. We also investigated the effects of TGM2 gene expression on the replication of the H1N1 influenza A virus in MDCK cells. The results showed that TGM2 induced the negative regulation of H1N1 replication. These findings contribute to a comprehensive understanding of the tumor regulation mechanism and biological functions of TGM2.</div

Effects of TGM2 on proliferation of influenza A (H1N1) virus.

(A). mRNA levels of viral NP and NS1 genes overexpressed by TGM2 were determined by real-time PCR. (B, C). The effect of TGM2 overexpression on viral NP protein was determined by Western blotting. (D). Effects of TGM2 overexpression on titer of influenza A H1N1 virus determined by TCID50; (E). mRNA levels of NP and NS1 genes of TGM2 knockout virus were determined by real-time PCR. (F, G). Effect of TGM2 knockout on NP protein of the virus was determined by Western blotting. (H). Effect of TGM2 knockout on titer of influenza A (H1N1) virus measured by TCID50. TGM2-overexpressed cells (TGM2-OE); Vector control cells (Con); TGM2-knockout cells (TGM2-KO); Wild-type cell (WT). The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p

Expression of TGM2 in high and low tumor-forming MDCK cells detected by real-time PCR and Western blotting.

(A). TGM2 mRNA levels in high and low tumorigenesis MDCK cells determined by real-time PCR. (B). TGM2 protein levels in high and low tumor-forming MDCK cells were determined by Western blotting and densitometrically quantified and normalized to β‐actin (C).</p

Effect of TGM2 on MDCK tumorigenicity.

(A). Subcutaneous tumor display in nude mice. (B) Statistical results of subcutaneous tumor number in nude mice. (C). Effect of TGM2 overexpression on tumor volume. (D). Effect of TGM2 knockout on tumor volume. (E). Histological observation of liver, lung and subcutaneous tumors in nude mice 30 days after Con, TGM2-OE, WT, and TGM2-KO cells were injected subcutaneously (H&E staining). A large number of hepatic cells were seen in the tissues with mild watery degeneration, cell swelling, loose cytoplasm, and light staining (black arrow). Focal necrosis of the liver cells was observed with nuclear fragmentation or dissolution (yellow arrows), with a small amount of connective tissue hyperplasia (red arrows), and a small amount of lymphocyte and neutrophil infiltration (blue arrows). Positive control (HeLa cells), Negative control (MRC-5 cells), TGM2 overexpression (TGM2-OE), Carrier control (Con), TGM2 knockout (TGM2-KO), Wild type (WT).</p

Screening of tumorigenesis-related target genes regulated by TGM2.

A: Real-time PCR showing the C-myc, Caspase 9, BAK, BAX, TWIST1, CDH2, P27, P38, Snail1, SOCS3, Snail2, and CCND1 mRNA levels. B: TWIST1 protein levels in high and low tumor-forming MDCK cells and TGM2-KO cells were determined by Western blotting and densitometrically quantified and normalized to β‐actin. The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p

Effects of TGM2 gene knockout on proliferation, migration, and invasion of MDCK cells.

(A). mRNA level of TGM2-KO cells determined by real-time PCR; (B). Protein level of TGM2-KO cells determined by Western blotting; (C). Effect of TGM2 knockout on proliferation ability of MDCK cells determined by cell counting; (D,E). Effects of TGM2 knockout on the plate clone rate of MDCK cells were determined by plate cloning experiments. (F,G). Effects of TGM2 knockout on the invasion ability of MDCK cells determined by Transwell assay; (H). Effects of TGM2 knockout on migration capacity of MDCK cells determined by scratch test. TGM2-knockout cells (TGM2-KO); Wild-type cell (WT). The experimental data are expressed as "mean ± standard deviation". *P < 0.05 and **P < 0.01 indicate significant differences, and ***P < 0.001 indicates extremely significant differences.</p