Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Col6a1 is regulated by glycemic variability to promote metastatic colonization. ( A ) Anoikis assay shows cell viability under anchorage-independent state; H , hyperglycemic cells; L , hypoglycemic cells. ( B and C ) PCR array ( left panel ) and qRT-PCT results ( right panel ) demonstrate altered expression in ECM genes between hyperglycemic and hypoglycemic PDAC cells; H , high glucose; L , low glucose. ( D ) Western blot analyses show expressions of Col6a1, Spp1, Fn1 under anchorage-dependent and -independent states in hyperglycemic and hypoglycemic PDAC cells; Gapdh is used as a housekeeping gene. ( E ) Densitometry analysis reveals reduced expression of Col6a1, but not Spp1 and Fn1. ( F ) Representative pictures of H&E staining ( left panel ) and metastatic area quantification ( right panel ) results demonstrate that Col6a1 overexpression restores the ability of metastatic colonization of hypoglycemic PDAC cells. ( G ) Representative pictures of H&E staining ( left panel ) and metastatic area quantification ( right panel ) show that Col6a1 down-regulation impairs the capacity of metastatic colonization in hyperglycemic PDAC cells. ( I ) Western blot analysis shows Col6a1 overexpression in hypoglycemic PDAC cells and Col6a1 down-regulation in hyperglycemic PDAC cells. ( H ) Representative pictures of H&E staining ( left panel ) and metastatic area quantification ( right panel ) show that Col6a1 down-regulation impairs the capacity of metastatic colonization in hyperglycemic PDAC cells. ( I ) Colony formation ( left panel ) and anoikis assay ( right panel ) determine the cell proliferation rate under anchorage-dependent and -independent state in control and Col6a1-overexpressing hypoglycemic PDAC cells. ( J ) Colony formation ( left panel ) and anoikis assay ( right panel ) show cell proliferation rate under anchorage-dependent and -independent state in control and Col6a1 down-regulated hyperglycemic PDAC cells. ( K ) 2DG assay determines glucose uptake rate in Col6a1-overexpressing hypoglycemic cells (L-Col6a1), hypoglycemic controls (L-C), Col6a1-downregulating hyperglycemic cells (shCol6a1), and hyperglycemic control cells (H-C). All data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is used to examine statistical significance, * P < .05.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Expressing, Western Blot, Staining, Over Expression, Control, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Col6a1 is controlled by the Rarb/Runx3 signal axis. ( A ) Quantitative RT-PCR ( left panel ) and Western blot ( right panel ) analysis show expression of Runx3; H , hyperglycemic cells; L , hypoglycemic cells. ( B ) Quantitative RT-PCR analysis shows expression of Runx1 and Runx2 in hyperglycemic and hypoglycemic PDAC cells. ( C ) Western blot analysis shows expression of Runx/Col6a1 in another (634 cells) hyperglycemic ( H ) and hypoglycemic ( L ) PDAC cells. ( D ) Western blot analysis shows expression of Smad4 and p53 in hyperglycemic ( H ) and hypoglycemic ( L ) PDAC cells. ( E ) Quantitative RT-PCR analysis shows expression of Rarb ( left panel ) and its target gene Egr2 ( right panel ) in hyperglycemic and hypoglycemic cells. ( F ) Real-time PCR analysis shows expression of Rarg and Rara in hyperglycemic ( H ) and hypoglycemic ( L ) PDAC cells. ( G ) Mass spectrometry analysis shows intracellular levels of RA in hyperglycemic and hypoglycemic PDAC cells. ( H ) Quantitative PCR analysis shows expression of Rarb, Runx3, and Col6a1 after Rar inhibition (by pan-Rar antagonist AGN193109, 50 μmol/L) for 24 hours. ( I ) Western blot analysis shows expression of Rarb, Runx3, and Col6a1 after Rar inhibition in hyperglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, * P < .05.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Real-time Polymerase Chain Reaction, Mass Spectrometry, Inhibition, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Rarb relays the signal of glycemic variability to Runx3/Col6a1 via histone modification. ( A ) Sketch view of chromatin immunoprecipitation (ChIP) assay. ( B ) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H2AK119ub, H3K27me3, H3K4me3, and H3K27ac in hyperglycemic and hypoglycemic PDAC cells. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown; H , hyperglycemic cells; L , hypoglycemic cells. ( C ) Quantitative RT-PCR analysis shows expression of Rarb, Runx3, and Col6a1 after 10 μmol/L, 20 μmol/L SAHA treatment for 24 hours. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, * P < .05. ( D ) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and SAHA treated hypoglyemic ( L ) PDAC cells. Data are presented as mean ± standard deviation, and data of 2 independent experiments are shown.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Modification, Chromatin Immunoprecipitation, Standard Deviation, Quantitative RT-PCR, Expressing, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Rarb relays the signal of glycemic variability to Runx3/Col6a1 via promoter methylation. ( A ) Methylation-specific PCR analysis shows the methylation status of Rarb gene in hypoglycemic PDAC cells, but not in hyperglycemic PDAC cells; M.SssI, CpG methyltransferase. ( B ) Quantitative RT-PCR analysis shows the expression of Rarb, Runx3, Col6a1, and Egr2 after decitabine treatment in hypoglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, * P < .05. ( C ) Western blot analysis shows expression of Runx3 and Col6a1 in hypoglycemic 399 ( left panel ) and 634 ( right panel ) cells after restoration of hyperglycemic culturing conditions.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Methylation, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: PDAC tissues of diabetic patients show increased expression of COL6A1. ( A and B ) Representative IHC pictures and statistical analysis show positive rate of RUNX3 and RARB in diabetic and non-diabetic PDAC slides; scale bar , 50 μm. ( C ) Representative COL6A1 IHC pictures and statistical analysis show positivity rate of COL6A1 in diabetic and non-diabetic PDAC slides, scale bar , 50 μm. ( D ) Representative H&E pictures ( left panel ) and metastatic area quantification ( right panel ) show metastatic colonization of PDAC cells in normal (Con) and diabetic samples (STZ). Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, * P < .05.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Expressing, Standard Deviation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Activity of the Rarb/Runx3/Col6a1 signal axis is inhibited by metformin. Quantitative RT-PCR results show expression of Rarb, Runx3, and Col6a1 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hyperglycemic PDAC cells. ( B ) Western blot analysis shows expression of Rarb, Runx3, Col6a1, and phospho-AMPKα Thr172 after metformin and 2DG treatment for 48 hours in hyperglycemic PDAC cells. ( C ) Quantitative RT-PCR analysis shows expression of Runx1, Runx2, Rara, Rarg, and Egr2 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hyperglycemic PDAC cells. ( D ) Glucose uptake assay shows reduced cellular uptake after metformin treatment for 24 hours. ( E ) Representative H&E pictures ( left panel ) and metastatic area quantification ( right panel ) show metastatic colonization in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation; data of 3 independent experiments are shown. Unpaired t test is applied, * P < .05. ( F ) Chromatin immunoprecipitation (ChIP) analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 2 independent experiments are shown. H , hyperglycemic cells; Met , metformin.

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Chromatin Immunoprecipitation

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Antibody List

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques:

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Sequences of Primers for qRT-PCR Analysis

Article Snippet: For Col6a1 stable knock-down transfection, hyperglycemic PDAC cells were transfected with 1 μg of either COL6A1 shRNA plasmid (sc-35086-SH; Santa Cruz Biotechnology, Heidelberg, Germany) or control shRNA plasmid (sc-108060; Santa Cruz Biotechnology).

Techniques:

Journal: Nature medicine

Article Title: Altered perivascular fibroblast activity precedes ALS disease onset.

doi: 10.1038/s41591-021-01295-9

Figure Lengend Snippet: Fig. 2 | Perivascular fibroblast marker proteins COL6A1 and SPP1 accumulate in enlarged perivascular spaces during ALS progression. a, Col6a1 and Spp1 mRNA specificity within central nervous system cell types. Bars represent relative count of RNA per cell ± s.e.m. b, COL6A1 and SPP1 histochemistry in sALS and control spinal cords. Scale bar, 10 μm. c, Quantifications of human tissue histochemistry from full-frame ×4 photos. sALS and Ctrl n = 4 individuals (two-tailed Student’s t-test P value). All box plots show median and second to third quartiles, and whiskers show ±1.5 × IQR. d, Col6a1 and Spp1 accumulate around blood vessels (outlined with podocalyxin; cyan) in 14-week SOD1G93A mouse spinal cords. Immunofluorescence z stack renderings of 16-μm thick sections. Scale bars, 100 μm (overview) and 10 μm (inset). DAPI, 4,6-diamidino-2-phenylindole. e, Quantifications of immunofluorescence staining in mice from full-frame ×20 photos. SOD1G93A and BL/6 n = 4, SOD1wt n = 3 mice (two-tailed Student’s t-test P value). f, Increased perivascular spaces appear in presymptomatic (8 weeks) SOD1G93A mice spinal cords. Immunofluorescence for vascular (Col4a1) and astrocyte (Lama1) basement membranes. Scale bar, 10 μm. w, weeks. g, Transmission electron microscopy of increased perivascular spaces in 14-week SOD1G93A mice. Astrocyte (red) and vascular (blue) basement membranes are indicated with lines. Perivascular space is indicated with an asterisk. Scale bar, 5 μm. h, COL6A1 and SPP1 accumulate within increased perivascular spaces (outlined with COL4A1) in the spinal cords of patients with sALS. Two-color histochemistry. Scale bar, 10 μm. i, Quantifications of COL6A1 and SPP1 immunostainings from h. sALS and Ctrl n = 4 individuals (two-tailed Student’s t-test P value). j, Quantifications of perivascular space increase in SOD1G93A mice from g (SOD1G93A and BL/6 n = 4 mice) and in patients with sALS from h (sALS and Ctrl n = 4 individuals; two-tailed Student’s t-test P value). k, Schematic representation of perivascular fibroblast activity and enlarged perivascular spaces in ALS.

Article Snippet: The following antibodies were used for immunostaining: podocalyxin (AF1556 R&D; 1:250 dilution), collagen IV (2150-1470 Serotec; 1:250 dilution), SPP1 (HPA027541, Atlas Antibodies 1:250 dilution), COL6A1 (HPA019142, Atlas Antibodies, 1:250 dilution) and LAMA1 (L9393, Sigma, 1:500 dilution).

Techniques: Marker, Control, Two Tailed Test, Immunofluorescence, Staining, Transmission Assay, Electron Microscopy, Activity Assay

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Figure 6. Rarb relays the signal of glycemic variability to Runx3/Col6a1 via histone modification. (A) Sketch view of chromatin immunoprecipitation (ChIP) assay. (B) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H2AK119ub, H3K27me3, H3K4me3, and H3K27ac in hyperglycemic and hypoglycemic PDAC cells. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown; H, hyperglycemic cells; L, hypoglycemic cells. (C) Quantitative RT-PCR analysis shows expression of Rarb, Runx3, and Col6a1 after 10 mmol/L, 20 mmol/L SAHA treatment for 24 hours. Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, *P < .05. (D) ChIP analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and SAHA treated hypoglyemic (L) PDAC cells. Data are presented as mean ± standard deviation, and data of 2 independent experiments are shown.

Article Snippet: For Col6a1 overexpression transfection, hypoglycemic PDAC cells were transfected with 1 mg of either mouse cDNA ORF clone (MG223027; OriGene, Herford, Germany) or control ORF clone (PS100010; OriGene).

Techniques: Chromatin Immunoprecipitation, Standard Deviation, Quantitative RT-PCR, Expressing, Control

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Figure 7. Rarb relays the signal of glycemic variability to Runx3/Col6a1 via promoter methylation. (A) Methylation- specific PCR analysis shows the methylation status of Rarb gene in hypoglycemic PDAC cells, but not in hyperglycemic PDAC cells; M.SssI, CpG methyltransferase. (B) Quantitative RT-PCR analysis shows the expression of Rarb, Runx3, Col6a1, and Egr2 after decitabine treatment in hypoglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, *P < .05. (C) Western blot analysis shows expression of Runx3 and Col6a1 in hypoglycemic 399 (left panel) and 634 (right panel) cells after restoration of hyperglycemic culturing conditions.

Article Snippet: For Col6a1 overexpression transfection, hypoglycemic PDAC cells were transfected with 1 mg of either mouse cDNA ORF clone (MG223027; OriGene, Herford, Germany) or control ORF clone (PS100010; OriGene).

Techniques: Methylation, Quantitative RT-PCR, Expressing, Standard Deviation, Western Blot

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Figure 8. PDAC tissues of diabetic patients show increased expression of COL6A1. (A and B) Representative IHC pictures and statistical analysis show positive rate of RUNX3 and RARB in diabetic and non-diabetic PDAC slides; scale bar, 50 mm. (C) Representative COL6A1 IHC pictures and statistical analysis show positivity rate of COL6A1 in diabetic and non-diabetic PDAC slides, scale bar, 50 mm. (D) Representative H&E pictures (left panel) and metastatic area quantification (right panel) show metastatic colonization of PDAC cells in normal (Con) and diabetic samples (STZ). Data are presented as mean ± standard deviation, and data from 3 independent experiments are shown. Unpaired t test is applied, *P < .05.

Article Snippet: For Col6a1 overexpression transfection, hypoglycemic PDAC cells were transfected with 1 mg of either mouse cDNA ORF clone (MG223027; OriGene, Herford, Germany) or control ORF clone (PS100010; OriGene).

Techniques: Expressing, Standard Deviation

Journal: Cellular and molecular gastroenterology and hepatology

Article Title: Glycemic Variability Promotes Both Local Invasion and Metastatic Colonization by Pancreatic Ductal Adenocarcinoma.

doi: 10.1016/j.jcmgh.2018.07.003

Figure Lengend Snippet: Figure 9. Activity of the Rarb/Runx3/Col6a1 signal axis is inhibited by metformin. Quantitative RT-PCR results show expression of Rarb, Runx3, and Col6a1 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hypergly- cemic PDAC cells. (B) Western blot analysis shows expression of Rarb, Runx3, Col6a1, and phospho-AMPKaThr172 after met- formin and 2DG treatment for 48 hours in hyperglycemic PDAC cells. (C) Quantitative RT-PCR analysis shows expression of Runx1, Runx2, Rara, Rarg, and Egr2 after metformin (20 mmol/L) and 2DG (10 mmol/L) treatment for 48 hours in hyperglycemic PDAC cells. (D) Glucose uptake assay shows reduced cellular uptake after metformin treatment for 24 hours. (E) Representative H&E pictures (left panel) and metastatic area quantification (right panel) show metastatic colonization in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation; data of 3 independent experiments are shown. Unpaired t test is applied, *P < .05. (F) Chromatin immunoprecipitation (ChIP) analysis of Rarb gene shows percentage input of H3K9me2, H3K27me3, and H3K27ac in control and metformin treated hyperglycemic PDAC cells. All data are presented as mean ± standard deviation, and data from 2 independent experiments are shown. H, hyperglycemic cells; Met, metformin.

Article Snippet: For Col6a1 overexpression transfection, hypoglycemic PDAC cells were transfected with 1 mg of either mouse cDNA ORF clone (MG223027; OriGene, Herford, Germany) or control ORF clone (PS100010; OriGene).

Techniques: Activity Assay, Quantitative RT-PCR, Expressing, Western Blot, Control, Standard Deviation, Chromatin Immunoprecipitation