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: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: a , Design of 5 Gram-negative bacteria gavage in germ-free mice without DEN (PBS group n = 6; BT group n = 6; PM group n = 6; EC group n = 6; EH group n = 6; KP group n = 6) and related results ( b , c ). b , Gut permeability assays using 500 kDa FITC-dextran (top), live bacteria translocated into liver cultured in blood agar plates (middle) and Cy3-conjugated EUB338 probe FISH assay of liver tissues (bottom). n = 6 biologically independent samples. c , Quantitative analysis of TEM, Alcian blue staining, E-cad, CLDN3, CLDN1 IHC staining, and Cy3-conjugated EUB338 probe FISH of colon tissues. n = 10 biologically independent samples. d , THP-1-induced macrophage using phorbol ester (top row) and MMP-2/-9 activity in K. pneumoniae and THP-induced macrophage co-culture conditional medium using gelatin hydrolysis assay and gelatin zymography (bottom row). n = 3 independent experiments with similar results. Ctrl: BHI and macrophage co-culture conditional medium; EC: E. coli and macrophage co-culture conditional medium; KP: K. pneumoniae and macrophage co-culture conditional medium. e , Design of colon macrophage depletion using Clodrosome and its effect on K. pneumoniae -mediated gut barrier dysfunction in germ-free mice without DEN treatment (PBS group n = 6; KP group n = 6; KPC group n = 6) and related results ( f – h ). f , In vivo imaging of body and liver after Cy5.5- d -Lys-labelled K. pneumoniae gavage (top), and live bacterial culture of liver tissues on blood agar plates and quantification (bottom). n = 6 biologically independent samples. g , Gut permeability assays using 500 kDa FITC-dextran (top left) ( n = 6 biologically independent samples) and MMP-2/-9 activity in mouse faecal supernatant detected using gelatin hydrolysis assay (top middle), gelatin zymography (top right) ( n = 3 independent experiments with similar results) and dynamic activity assay (bottom) ( n = 6 biologically independent samples). h , Quantitative analysis of TEM, Alcian blue staining, E-cad and CLDN1 IHC staining, bacteria number per slide using Cy3-conjugated EUB338 probe (PBS group) and K. pneumoniae probe (KP and KPC groups) FISH of colon tissues. n = 10 biologically independent samples. In b , c (excluding tight junction disappear rate) and f – h (excluding liver with live bacteria and tight junction disappear rate) data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Liver with living bacteria and tight junction disappearance rate were analysed using Fisher’s exact test. MMP activity was analysed using two-way ANOVA. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: MMP-2 (HY-P70268, MCE) and MMP-9 (HY-P70145, MCE) were used at 0.01 μg ml −1 and co-cultured with NCM460 cells for 3 days.

Techniques: Bacteria, Permeability, Cell Culture, Staining, Immunohistochemistry, Activity Assay, Co-Culture Assay, Hydrolysis Assay, Zymography, In Vivo Imaging

Journal: Nature Microbiology

Article Title: Gut–liver translocation of pathogen Klebsiella pneumoniae promotes hepatocellular carcinoma in mice

doi: 10.1038/s41564-024-01890-9

Figure Lengend Snippet: a , Design of K. pneumoniae gavage for promoting precancerous lesions in germ-free mice without DEN treatment (GF/PBS group n = 10; GF/EC group n = 8; GF/KP group n = 10) and related results ( b – h ). b , In vivo imaging of body and liver after Cy5.5- d -Lys-labelled K. pneumoniae gavage (left), live bacteria culture of liver tissues on blood agar plate (top right) and Cy3-conjugated EUB338 probe (GF/PBS group), E. coli probe (GF/EC group) or K. pneumoniae probe (GF/KP group) FISH detection in liver tissues (bottom right). c , Gut permeability assays using 500 kDa FITC-dextran (left) ( n = 10 (GF/PBS), 8 (GF/EC), 10 (GF/KP)) and quantitative analysis of TEM (middle) ( n = 6 biologically independent samples) and E-cad IHC staining (right) ( n = 6 biologically independent samples). d , MMP-2/-9 activity in mouse faecal supernatant using gelatin hydrolysis assay (bottom left), gelatin zymography (top left) and dynamic activity assay (right). n = 3 biologically independent samples. e , Masson’s trichrome staining (top left) and COLIV IHC staining (top right) of colon tissues and COLIV protein level by western blot (bottom). n = 6 biologically independent samples. f , Representative images of liver gross morphology (top left) (dashed yellow circles indicate nodules), H&E staining of mice liver sections (bottom left), and nodule number and incidence of dysplasia (right). n = 10 (GF/PBS), 8 (GF/EC), 10 (GF/KP). g , IHC staining for PCNA and Ki-67 (left) ( n = 6 biologically independent samples), and western blot assay of PCNA (right) ( n = 3 biologically independent samples). h , Desmin (left) and α-SMA (middle) IHC staining, and Sirius red staining (right) of liver sections. n = 6 biologically independent samples. i , Design of K. pneumoniae gavage on precancerous lesions in SPF mice without DEN treatment (PBS group n = 6; EC group n = 6; KP group n = 6) and relevant results ( j – n ). j , Gut permeability assays using 500 kDa FITC-dextran. n = 6 biologically independent samples. k , Live bacterial culture of liver tissues on blood agar plate (top), and Cy3-conjugated EUB338 probe (PBS group), E. coli probe (EC group) or K. pneumoniae probe (KP group) FISH detection in liver tissues (bottom). n = 6 biologically independent samples. l , TEM (left) and quantitative analysis (right) of colon tissues. n = 6 biologically independent samples. m , Representative images of liver gross morphology (top) (dashed yellow circles indicate nodules), H&E staining of mice liver sections (middle), and nodule number and incidence of dysplasia (bottom). n = 6 biologically independent samples. n , Representative images of IHC staining for PCNA and Sirius red staining of liver sections (left) with quantitative analysis (right). n = 6 biologically independent samples. o , Design of K. pneumoniae gavage on HCC in SPF mice with DEN treatment (DPBS group n = 7; DEC group n = 6; DKP group n = 7) and related results ( p ). p , Representative images of liver gross morphology (left) (dashed yellow circles indicate tumour) and H&E staining of mice liver sections (middle) with tumour incidence and tumour number quantitative analysis (right). n = 7 (DPBS), 6 (DEC), 7 (DKP). In c (excluding tight junction disappear rate), d , e – h (excluding incidence of dysplasia), j,k , m (excluding incidence of dysplasia), n and p (excluding tumour incidence), data are presented as mean ± s.e.m. Each data point in bar plots represents one mouse. Tight junction disappearance rate, incidence of dysplasia and tumour incidence were analysed using Fisher’s exact test. MMP activity was analysed using two-way ANOVA. Unless otherwise stated, statistical significance was calculated using one-way ANOVA. Adjustments were made for multiple comparisons.

Article Snippet: MMP-2 (HY-P70268, MCE) and MMP-9 (HY-P70145, MCE) were used at 0.01 μg ml −1 and co-cultured with NCM460 cells for 3 days.

Techniques: In Vivo Imaging, Bacteria, Permeability, Immunohistochemistry, Activity Assay, Hydrolysis Assay, Zymography, Staining, Western Blot

Journal: Biomedicines

Article Title: Prolonged Cardiopulmonary Bypass Time-Induced Endothelial Dysfunction via Glypican-1 Shedding, Inflammation, and Matrix Metalloproteinase 9 in Patients Undergoing Cardiac Surgery.

doi: 10.3390/biomedicines13010033

Figure Lengend Snippet: Figure 1. Changes in the circulating glypican-1, MMP9, and IL-1β levels in cardiac surgery patients with a normal CPB and prolonged CPB duration. (A–C) Box-and-whisker plots showing the preop- erative (baseline) levels of (A) glypican-1, (B) MMP9, and (C) IL-1β between the normal CPB and prolonged CPB group patients. The lines across each box represent the median value. The lines that extend from the top and bottom of each box represent the lowest and highest observations still inside the lower and upper limits of confidence. (D–F) Line arts showing the change in the dynamic pre- and post-cardiac surgery timepoints of (D) glypican-1, (E) MMP9, and (F) IL-1β plasma level between the groups. * p < 0.05 is considered significant.

Article Snippet: Specifically, the kits used were ELH-GPC1-Glypican1 for glypican-1 (Ray Biotech, Peachtree Corners, GA, USA), KE00164-MMP9 for MMP9 (ProteinTech, Rosemont, IL, USA), and KE00021-IL-1β for IL-1β (ProteinTech, Rosemont, IL, USA).

Techniques: Whisker Assay, Clinical Proteomics

Journal: Biomedicines

Article Title: Prolonged Cardiopulmonary Bypass Time-Induced Endothelial Dysfunction via Glypican-1 Shedding, Inflammation, and Matrix Metalloproteinase 9 in Patients Undergoing Cardiac Surgery.

doi: 10.3390/biomedicines13010033

Figure Lengend Snippet: Figure 2. Spearman’s rank correlation analysis for the Intra-OP and Post-OP circulating glypican-1, MMP9, and IL-1β with each other. (A–C) Scatter plots showing the correlations between the intra- operative (A) glypican-1 and MMP9; (B) IL-1β and glypican-1, and (C) MMP9 and IL-1β; (D–F) Scatter plots showing the correlations between the post-operative (D) glypican-1 and MMP9, (E) IL-1β and glypican-1, and (F) MMP9 and IL-1β. Note—CI: confidence intervals; r: Spearman’s Rho. * p < 0.05 is considered significant.

Article Snippet: Specifically, the kits used were ELH-GPC1-Glypican1 for glypican-1 (Ray Biotech, Peachtree Corners, GA, USA), KE00164-MMP9 for MMP9 (ProteinTech, Rosemont, IL, USA), and KE00021-IL-1β for IL-1β (ProteinTech, Rosemont, IL, USA).

Techniques:

Journal: Biomedicines

Article Title: Prolonged Cardiopulmonary Bypass Time-Induced Endothelial Dysfunction via Glypican-1 Shedding, Inflammation, and Matrix Metalloproteinase 9 in Patients Undergoing Cardiac Surgery.

doi: 10.3390/biomedicines13010033

Figure Lengend Snippet: Figure 3. Spearman’s rank correlation analysis for the Intra-OP and Post-OP circulating glypican-1, MMP9, and IL-1β and the CPB duration. (A–C) Scatter plots showing the correlation between the CPB duration and the intraoperative (A) glypican-1, (B) MMP9, and (C) IL-1β; (D–F) Scatter plots showing the correlation between the CPB duration and the postoperative (D) glypican-1, (E) MMP9, and (F) IL-1β. Note–CI: confidence intervals; r: Spearman’s Rho. * p < 0.05 is considered significant.

Article Snippet: Specifically, the kits used were ELH-GPC1-Glypican1 for glypican-1 (Ray Biotech, Peachtree Corners, GA, USA), KE00164-MMP9 for MMP9 (ProteinTech, Rosemont, IL, USA), and KE00021-IL-1β for IL-1β (ProteinTech, Rosemont, IL, USA).

Techniques: