Journal: Clinical and Translational Medicine

Article Title: Hypoxia‐induced secretory autophagy in cancer‐associated fibroblasts promotes ECM remodelling through serglycin secretion in oral squamous cell carcinoma

doi: 10.1002/ctm2.70556

Figure Lengend Snippet: CAFs secrete SRGN via autophagy to promote OSCC cell invasion and migration by facilitating ECM remodelling through interaction with MMP2/9. (A, B) WB analysis of SRGN protein expression levels and quantification in WT CAFs and SRGN KO CAFs. (C) qPCR analysis of SRGN gene expression in WT CAFs and SRGN KO CAFs. (D) UV image of the agarose gel. (E) The supernatant from normoxic and hypoxic WT CAFs, WT CAFs + 3‐MA, and SRGN KO CAFs was collected and co‐cultured with OSCC cells. Invasion ability was assessed by transwell assays. (F) Invasion cell numbers were quantified using ImageJ software. (* p < .05; ** p < .01; *** p < .001). (G) The supernatant from normoxic and hypoxic WT CAFs, WT CAFs + 3‐MA, and SRGN KO CAFs was collected and co‐cultured with SCC9 cells. Migration ability was assessed by scratch assays. (H) Prediction of SRGN‐binding proteins using the STRING database. (I) HEK293T cells were transfected with SRGN‐Flag and incubated for 48 h. Cell lysates were incubated with anti‐Flag beads, and immunoblotting (IB) was performed using anti‐Flag, anti‐MMP11, anti‐MMP9, and anti‐MMP2 antibodies. (J) WB analysis of changes in MMP9, MMP11, MMP2, and SRGN protein expression levels in WT CAFs and SRGN KO CAFs. (K) Gelatin degradation assays were performed to evaluate gelatin degradation after 24 h of co‐culture of CAL27 cells with the supernatants from normoxic and hypoxic WT CAFs, WT CAFs + 3‐MA, and SRGN KO CAFs. Scale bar = 20 µm.

Article Snippet: Primary antibodies used in this assay included: β‐actin (1:4000, 20536‐1, Proteintech), MMP9 (1:1000, bs‐4593R, Bioss), MMP2 (1:1000, CY5189, Abways), MMP11 (1:1000, CY5778, Abways), Flag (1:5000, 80801‐2‐RR, Proteintech).

Techniques: Migration, Expressing, Gene Expression, Agarose Gel Electrophoresis, Cell Culture, Software, Binding Assay, Transfection, Incubation, Western Blot, Co-Culture Assay

Journal: Clinical and Translational Medicine

Article Title: Hypoxia‐induced secretory autophagy in cancer‐associated fibroblasts promotes ECM remodelling through serglycin secretion in oral squamous cell carcinoma

doi: 10.1002/ctm2.70556

Figure Lengend Snippet: CAF‐derived SRGN promotes tumour invasion and ECM degradation via autophagy secretion. (A) In vivo xenograft models were established in nude mice and divided into four groups: (a) CAL27, (b) CAL27 + WT CAFs, (c) CAL27 + WT CAFs (3‐MA), and (d) CAL27 + SRGN KO CAFs. (B) Tumour volume and tumour weight were monitored ( n = 7). (C, D) H&E staining and IHC analysis of COL1, E‐cadherin, MMP2 and MMP9 were performed in orthotopic xenograft tumour tissues. The expression levels of COL1, E‐cadherin, MMP2 and MMP9 were quantitatively analyzed using Fiji software. Scale bar = 100 µm. (* p < .05; ** p < .01; *** p < .001).

Article Snippet: Primary antibodies used in this assay included: β‐actin (1:4000, 20536‐1, Proteintech), MMP9 (1:1000, bs‐4593R, Bioss), MMP2 (1:1000, CY5189, Abways), MMP11 (1:1000, CY5778, Abways), Flag (1:5000, 80801‐2‐RR, Proteintech).

Techniques: Derivative Assay, In Vivo, Staining, Expressing, Software

Journal: Clinical and Translational Medicine

Article Title: Hypoxia‐induced secretory autophagy in cancer‐associated fibroblasts promotes ECM remodelling through serglycin secretion in oral squamous cell carcinoma

doi: 10.1002/ctm2.70556

Figure Lengend Snippet: Mechanism diagram of hypoxic CAFs‐derived SRGN secretion and tumour progression promotion. Under normal conditions, SRGN is translocated into the ER and subsequently transported via the Golgi apparatus for secretion into the extracellular space. Under hypoxic conditions, elevated autophagy levels in CAFs facilitate the release of SRGN into the ECM through secretory autophagy‐mediated plasma membrane fusion. Within the ECM, SRGN interacts with MMP2 and MMP9, enhancing ECM remodelling and ultimately promoting the invasive capacity of OSCC cells.

Article Snippet: Primary antibodies used in this assay included: β‐actin (1:4000, 20536‐1, Proteintech), MMP9 (1:1000, bs‐4593R, Bioss), MMP2 (1:1000, CY5189, Abways), MMP11 (1:1000, CY5778, Abways), Flag (1:5000, 80801‐2‐RR, Proteintech).

Techniques: Derivative Assay, Clinical Proteomics, Membrane

Journal: Molecular medicine reports

Article Title: lncRNA FEZF1‑AS1 promotes migration, invasion and epithelial‑mesenchymal transition of retinoblastoma cells by targeting miR‑1236‑3p.

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Figure 3. Epithelial‑mesenchymal transition of retinoblastoma cells is suppressed by shRNA‑FEZF1‑AS1. Western blotting was used to analyze the protein expression levels of (A) Vimentin, Snail, Slug and β‑catenin, (B) N‑cadherin, E‑cadherin, Claudin‑1 and (C) MMP2 and MMP9 in Y79 cells transfected with shRNA‑NC or shRNA‑FEZF1‑AS1. All data are expressed as the mean ± SEM. **P<0.01, ***P<0.001 vs. shRNA‑NC group. FEZF1‑AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; MMP, matrix metalloproteinase.

Article Snippet: The membranes were then incubated with the following primary antibodies at 4 ̊C overnight: Anti‐Vimentin (1:1,000; cat. no. 3932; Cell Signaling Technology, Inc.), anti‐Snail (1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.), anti‐Slug (1:1,000; cat. no. 9585; Cell Signaling Technology, Inc.), anti‐Claudin‐1 (1:1,000; cat. no. 4933; Cell Signaling Technology, Inc.), anti‐β-catenin (1:500; cat. no. sc‐59737; Santa Cruz Biotechnology, Inc.), anti‐N‐cadherin (1:500; cat. no. sc‐59987; Santa Cruz Biotechnology, Inc.), anti‐E‐cadherin (1:500; cat. no. sc‐8426; Santa Cruz Biotechnology, Inc.), anti‐matrix metalloproteinase (MMP) 2 (1:1,000; cat. no. 10373‐2‐AP; ProteinTech Group, Inc.), anti‐MMP9 (1:1,000; cat. no. 10375‐2‐AP; ProteinTech Group, Inc.) and anti‐GAPDH (1:1,000; cat. no. SAB5600208; Sigma-aldrich; Merck KGaa).

Techniques: Western Blot, Expressing, Transfection, shRNA, Negative Control

Journal: Molecular medicine reports

Article Title: lncRNA FEZF1‑AS1 promotes migration, invasion and epithelial‑mesenchymal transition of retinoblastoma cells by targeting miR‑1236‑3p.

doi: 10.3892/mmr.2020.11478

Figure Lengend Snippet: Figure 6. Continued. Inhibition of miR‑1236‑3p reverses the effects of shRNA‑FEZF1‑AS1 on epithelial‑mesenchymal transition. (A) Western blotting was used to analyze the protein expression levels of (A) Vimentin, Snail, Slug and β‑catenin, (B) N‑cadherin, E‑cadherin and Claudin‑1, and (C) MMP2 and MMP9 in Y79 cells transfected with shRNA‑NC or shRNA‑FEZF1‑AS1 with or without miR‑1236‑3p inhibitor or miR‑NC inhibitor. All data are expressed as the mean ± SEM. ***P<0.001 vs. shRNA‑NC group; #P<0.05, ##P<0.01, ###P<0.001 vs. shRNA‑FEZF1‑AS1 + miR‑NC inhibitor group. FEZF1‑AS1, FEZ family zinc finger 1 antisense RNA 1; shRNA, short hairpin RNA; NC, negative control; miR, microRNA; MMP, matrix metalloproteinase.

Article Snippet: The membranes were then incubated with the following primary antibodies at 4 ̊C overnight: Anti‐Vimentin (1:1,000; cat. no. 3932; Cell Signaling Technology, Inc.), anti‐Snail (1:1,000; cat. no. 3879; Cell Signaling Technology, Inc.), anti‐Slug (1:1,000; cat. no. 9585; Cell Signaling Technology, Inc.), anti‐Claudin‐1 (1:1,000; cat. no. 4933; Cell Signaling Technology, Inc.), anti‐β-catenin (1:500; cat. no. sc‐59737; Santa Cruz Biotechnology, Inc.), anti‐N‐cadherin (1:500; cat. no. sc‐59987; Santa Cruz Biotechnology, Inc.), anti‐E‐cadherin (1:500; cat. no. sc‐8426; Santa Cruz Biotechnology, Inc.), anti‐matrix metalloproteinase (MMP) 2 (1:1,000; cat. no. 10373‐2‐AP; ProteinTech Group, Inc.), anti‐MMP9 (1:1,000; cat. no. 10375‐2‐AP; ProteinTech Group, Inc.) and anti‐GAPDH (1:1,000; cat. no. SAB5600208; Sigma-aldrich; Merck KGaa).

Techniques: Inhibition, Western Blot, Expressing, Transfection, shRNA, Negative Control