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: Experimental and Therapeutic Medicine

Article Title: IL-32γ promotes integrin αvβ6 expression through the activation of NF-κB in HSCs

doi: 10.3892/etm.2017.4956

Figure Lengend Snippet: Effect of different concentrations (0, 5, 10 and 20 ng/ml) of IL-32γ on LX-2 activation phenotypes. (A) Growth curves of LX-2 demonstrated that upregulation of IL-32γ promoted proliferation of LX-2. (B) Reverse transcription-quantitative polymerase chain reaction assessing mRNA levels of α-SMA, (C) collagen I, (D) TIMP1, (E) MMP2 and (F) MMP9, representing the activation level of LX-2. (G) Western blot analysis was used to measure collagen I, MMP9, MMP2, α-SMA, TIMP1 and GAPDH expression in whole-cell extracts. Data are presented as the mean ± standard deviation of three experiments. *P<0.01 and **P<0.05 vs. 0 ng/ml IL-32γ. IL-32γ, interleukin-32γ; α-SMA, α-smooth muscle actin; TIMP1, tissue inhibitor of metalloproteinase 1; MMP, matrix metalloproteinases; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; RT-qPCR, reverse transcription-quantitative polymerase chain reaction.

Article Snippet: Primary antibodies were as follows: Anti-integrin αVβ6 (cat. no. ab97588; Abcam, Cambridge, MA, USA), anti-GAPDH (cat. no. KGAA002-2; Nanjing KeyGen Biotech Co., Ltd.), anti-α-SMA (cat. no. G6669; Sigma-Aldrich; Merck KGaA), collagen type I antibody (cat. no. 600-402-103; Rockland, Limerick, PA, USA), TIMP1 (cat. no. 8946), MMP2 (cat. no. 87809), MMP9 (cat. no. 13667), NF-κB: p65 (cat. no. 8242), p50 (cat. no. 3035) (all Cell Signaling Technology, Inc., Danvers, MA, USA) and IL-32γ (cat. no. 513501; Biolegend, Inc.).

Techniques: Activation Assay, Real-time Polymerase Chain Reaction, Western Blot, Expressing, Standard Deviation, Quantitative RT-PCR