Fig 1: COL4A1 interacts with NID1 in oral squamous cell carcinoma cells. (A) mRNA expression of NID1 in SCC-4 cells measured via reverse transcription-quantitative PCR. (B) NID1 protein level in SCC-4 cells semi-quantified using western blot assay. (C) Association between COL4A1 and NID1, analyzed using the TNMplot database. (D) Binding of COL4A1 and NID1 analyzed using the STRING database. (E) Experimental verification of the binding between COL4A1 and NID1 using co-immunoprecipitation assay. Results are presented as the mean ± standard deviation. ***P<0.001. NID, nidogen-1; COL4A1, collagen type IV α1 chain.
Fig 2: COL4A1 is upregulated in OSCC cells. (A) mRNA expression of COL4A1 in OSCC cell lines measured via reverse transcription-quantitative PCR. (B) COL4A1 protein level in OSCC cell lines semi-quantified using western blot assay. Results are presented as the mean ± standard deviation. *P<0.05 and ***P<0.001. COL4A1, collagen type IV α1 chain; OSCC, oral squamous cell carcinoma.
Fig 3: Knockdown of COL4A1 suppresses proliferation of SCC-4 cells. (A) mRNA expression of COL4A1 in SCC-4 cells measured via reverse transcription-quantitative PCR. ***P<0.001. (B) COL4A1 protein level in SCC-4 cells semi-quantified using western blot assay. ***P<0.001. Cell proliferation in SCC-4 cells was evaluated using (C) CCK-8 (**P<0.01) and (D) EdU incorporation (magnification, x100). (E) Colony formation assay (magnification, x1) was used to measure the colony-forming ability of SCC-4 cells. Results are presented as the mean ± standard deviation. COL4A1, collagen type IV α1 chain; NC, negative control; si, small interfering; OD, optical density.
Fig 4: COL4A1 silencing inhibits migration, invasion and expression of epithelial-mesenchymal transition-associated protein of SCC-4 cells. (A) Representative wound healing assay of SCC-4 cells transfected with si-COL4A1 (magnification, x100). (B) Cell migration rate measured using wound healing assay. (C) Representative Transwell invasion assay of SCC-4 cells transfected with si-COL4A1 (magnification, x100). (D) Cell invasion rate measured via Transwell invasion assay. (E) Protein levels of N-cadherin, vimentin and E-cadherin in SCC-4 cells transfected with si-COL4A1 semi-quantified using western blot assay. Results are presented as the mean ± standard deviation. ***P<0.001. NC, negative control; COL4A1, collagen type IV α1 chain; si, small interfering.
Fig 5: Upregulation of NID1 reverses the inhibitory effect of COL4A1 silencing on SCC-4 cells. (A) mRNA expression of NID1 in SCC-4 cells transfected with Oe-NID1 was measured via reverse transcription-quantitative PCR. ***P<0.001. (B) NID1 protein levels in SCC-4 cells transfected with Oe-NID1 measured using western blot assay. ***P<0.001. Cell proliferation in SCC-4 cells was evaluated using (C) Cell Counting Kit-8 (**P<0.01, ***P<0.001 vs. si-NC; ##P<0.01, ###P<0.001 vs. si-COL4A1+ Oe-NC) and (D) EdU incorporation (magnification, x100). (E) Colony formation assay (magnification, x1) was used to measure the colony-forming ability of SCC-4 cells. (F) Representative image of the wound healing assay of SCC-4 cells transfected with Oe-NID1 (magnification, x100). (G) Migration rate of SCC-4 cells transfected with Oe-NID1 measured using a wound healing assay. ***P<0.001. (H) Representative image of Transwell invasion assay of SCC-4 cells transfected with Oe-NID1 (magnification, x100). (I) Invasion rate of SCC-4 cells transfected with Oe-NID1 measured via Transwell invasion assay. ***P<0.001. (J) Protein levels of N-cadherin, vimentin and E-cadherin in SCC-4 cells transfected with Oe-NID1 semi-quantified using western blot assay. Results are presented as the mean ± standard deviation. **P<0.01 and ***P<0.001. NID1, nidogen-1; Oe, overexpression; NC, negative control; COL4A1, collagen type IV α1 chain; si, small interfering; OD, optical density.
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