Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: Five NRDEGs were identified based on GEO and TCGA database. ( A ) Volcano plot of differentially expressed genes from GSE28735. ( B ) Volcano plot of differentially expressed genes from GSE15471. ( C ) Venn diagram of the intersection of DEGs and necroptosis-related genes. ( D ) Differential expression of 13 NRDEGs in the TCGA database. * means p < 0.05, *** means p < 0.001 and ns means no sense. ( E – I ) Kaplan–Meier curves of NRDEGs: ( E ) KRT7 ( p < 0.001), ( F ) IGF2BP3 ( p = 0.026), ( G ) PKM ( p = 0.007), ( H ) KRT19 ( p = 0.008), ( I ) CXCL5 ( p = 0.047).

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques: Quantitative Proteomics

Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: A necroptosis-related risk prognostic model was successfully constructed. ( A ) Ten-time cross-validation for tuning parameter selection in the LASSO model. ( B ) LASSO coefficient profiles. ( C ) The risk score, survival status, and heat map of model genes in patients with PC. ( D ) Kaplan–Meier curves showed that high-risk group had a worse prognosis than the low-risk group. ( E , F ) PCA and t-SNE visualization of low- and high-risk clusters. ( G ) ROC curves of KRT7, KRT19, CXCL5 and IGF2BP2. ( H ) ROC curves of prognostic model for 1, 3, 5 years, ( I ) ROC curves of risk score and clinicopathological characteristics. ( J , K ) Univariate and multivariate Cox regression analysis of clinicopathological features of PC associated with OS. ( L ) Clinical characteristics and prognostic model were used to establish a predictive nomogram. * means p < 0.05, ** means p < 0.01 ( M ) Heatmap of model genes and clinical parameters.

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques: Construct, Biomarker Discovery, Selection

Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: Model genes were involved in EMT of PC. ( A ) The correlation heat map between model genes and EMT-related genes. ( B ) KRT7 expression in primary as well as metastatic tumors in the EMTome database. ( C ) KRT19 expression in primary as well as metastatic tumors in EMTome database. ( D ) Gene mutations of KRT7 in various cancers in EMTome database. ( E ) Gene mutations of KRT19 in various cancers in EMTome database.

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques: Expressing

Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: Model genes regulated immunity of PC. ( A – D ) The correlation of model genes with various immune cells: ( A ) KRT7, ( B ) KRT19, ( C ) CXCL5, ( D ) IGF2BP3. ( E – H ) Histogram of correlation between model genes and immune cells (Th2, TFH cells). ( I – L ) Scatter plot of correlation between model genes and immune cells (Th2, TFH cells). ( M ) Expressions of immune checkpoints between the high- and low-risk groups. * means p < 0.05, ** means p < 0.01, *** means p < 0.001 and ns means no sense.

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques:

Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: Model genes were upregulated in clinical PC samples and PC cell lines. ( A ) HE, Masson and IHC staining of model genes including KRT7, KRT19, IGF2BP3 and CXCL5 in clinical PC tissues and corresponding adjacent tumor tissues. ( B ) Western blot and histograms of model genes in hTERT-HPNE, PANC-1 and PL45. ( C ) Immunofluorescence (×400) of model genes in hTERT-HPNE, PANC-1 and PL45. hTERT-HPNE is normal human pancreatic ductal cell line, and PANC-1 and PL45 are human pancreatic ductal carcinoma cell lines. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. hTERT-HPNE.

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques: Immunohistochemistry, Western Blot, Immunofluorescence

Journal: Biomedicines

Article Title: Necroptosis-Related Prognostic Model for Pancreatic Carcinoma Reveals Its Invasion and Metastasis Potential through Hybrid EMT and Immune Escape

doi: 10.3390/biomedicines11061738

Figure Lengend Snippet: Prognostic model genes for PC reveals its invasion and metastasis potential through hybrid EMT and immune escape. Hypoxic pancreatic cancer cells are more likely to suffer necroptosis to induce inflammation and immune responses in tumor tissues, which further enhance fibrosis and heterogeneity of tumor, thus the fibrotic and inflammatory circumstance together with immune evasion induced by Th2 assist PC cells to escape from immune elimination. Furthermore, our model genes promote tumor invasion and metastasis by promoting EMT especially hybrid EMT of PC cells that KRT7 and KRT19 are involved in EMT as epithelial components, while CXCL5 and IGF2BP3 act as regulatory factors to regulate EMT. Ultimately, the above effects together promote tumor invasion and metastasis.

Article Snippet: Primary antibodies against KRT7 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), KRT19 (1:50, Wuhan Boster Bio-Engineering Limited Company, Wuhan, China), IGF2BP3 (1:50, Signalway Antibody, MD, USA), and CXCL5 (1:50, Sangon Biotechnology, Shanghai, China) were added to these tissue sections, followed by incubation at 4 °C overnight.

Techniques:

Journal: Heliyon

Article Title: AHR regulates liver enlargement and regeneration through the YAP signaling pathway

doi: 10.1016/j.heliyon.2024.e37265

Figure Lengend Snippet: Activation of AHR promoted liver enlargement and regeneration in wild-type mice. (A) Wild-type mice were treated with vehicle or YH439 (100 mg/kg/d) for 10 days. (B) The relative liver/body weight ratios (n = 5). (C) Representative liver photos of vehicle or YH439-treated mice. (D) CTNNB1 staining, H&E staining were performed to measure the cell size around CV area. Quantification of cell size (n = 5). (E)CK19/Ki67 staining was performed to measure the cell proliferation around PV area. Quantification the number of Ki67 + cells (n = 5). Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test. Scale bar = 50 μm.

Article Snippet: For immunohistochemical staining, Anti-Ki67 (1:100 dilution, BOSTER, Cat# M00254-8, RRID: AB_3081764 ), Anti-CTNNB1(1:100 dilution BOSTER, Cat#PA1212, RRID: AB_3082601 ), Anti-CK19(1:100 dilution BOSTER, Cat#PB9715, RRID: AB_3082234 )/Anti- Ki67 (1:100 dilution), CTNNB1(1:100 dilution)/Ki67(1:100 dilution), Anti-Sox9(1:100 dilution BOSTER, Cat#BM4268, RRID: AB_2941988 )/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti-HNF4α (1:100 dilution, Santa Cat#sc-374229, RRID: AB_10989766 ) were used as primary antibody.

Techniques: Activation Assay, Staining

Journal: Heliyon

Article Title: AHR regulates liver enlargement and regeneration through the YAP signaling pathway

doi: 10.1016/j.heliyon.2024.e37265

Figure Lengend Snippet: AHR activation-induced liver enlargement and regeneration is abolished in Ahr ΔHep mice. (A) Ahr fl/fl.ALB-CreERT2 and Ahr ΔHep mice were treated with vehicle or YH439 (100 mg/kg/d) for 10 days. (B) The relative liver/body weight ratios (n = 5). (C) Representative liver photos of vehicle or YH439-treated mice. (D) CTNNB1 staining, H&E staining were performed to measure the cell size around CV area. Quantification of cell size (n = 5). (E)CK19/Ki67 staining was performed to measure the cell proliferation around PV area. Quantification the number of Ki67 + cells (n = 5). Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test. Scale bar = 50 μm.

Article Snippet: For immunohistochemical staining, Anti-Ki67 (1:100 dilution, BOSTER, Cat# M00254-8, RRID: AB_3081764 ), Anti-CTNNB1(1:100 dilution BOSTER, Cat#PA1212, RRID: AB_3082601 ), Anti-CK19(1:100 dilution BOSTER, Cat#PB9715, RRID: AB_3082234 )/Anti- Ki67 (1:100 dilution), CTNNB1(1:100 dilution)/Ki67(1:100 dilution), Anti-Sox9(1:100 dilution BOSTER, Cat#BM4268, RRID: AB_2941988 )/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti-HNF4α (1:100 dilution, Santa Cat#sc-374229, RRID: AB_10989766 ) were used as primary antibody.

Techniques: Activation Assay, Staining

Journal: Heliyon

Article Title: AHR regulates liver enlargement and regeneration through the YAP signaling pathway

doi: 10.1016/j.heliyon.2024.e37265

Figure Lengend Snippet: AHR activation promotes liver regeneration (A) Ahr fl/fl.ALB-CreERT2 and Ahr ΔHep mice were treated with vehicle or YH439 (100 mg/kg/d) for 2 days following PHx. (B) (B) CTNNB1 staining, H&E staining were performed to measure the cell size around CV area. Quantification of cell size (n = 5). Scale bar = 50 μm. (C)CK19/Ki67 staining was performed to measure the cell proliferation around PV area. Quantification the number of Ki67 + cells (n = 5). Scale bar = 20 μm. Data are expressed as means ± SD; *p < 0.05 and **p < 0.01; Student's t-test.

Article Snippet: For immunohistochemical staining, Anti-Ki67 (1:100 dilution, BOSTER, Cat# M00254-8, RRID: AB_3081764 ), Anti-CTNNB1(1:100 dilution BOSTER, Cat#PA1212, RRID: AB_3082601 ), Anti-CK19(1:100 dilution BOSTER, Cat#PB9715, RRID: AB_3082234 )/Anti- Ki67 (1:100 dilution), CTNNB1(1:100 dilution)/Ki67(1:100 dilution), Anti-Sox9(1:100 dilution BOSTER, Cat#BM4268, RRID: AB_2941988 )/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti-HNF4α (1:100 dilution, Santa Cat#sc-374229, RRID: AB_10989766 ) were used as primary antibody.

Techniques: Activation Assay, Staining

Journal: Heliyon

Article Title: AHR regulates liver enlargement and regeneration through the YAP signaling pathway

doi: 10.1016/j.heliyon.2024.e37265

Figure Lengend Snippet: YAP-TEAD interaction is involved in AHR-induced liver enlargement and regeneration. (A) Corn oil-treated mice and verteporfin-treated mice were administered vehicle or YH439 (100 mg/kg/d) for 5 days. (B) The relative liver/body weight ratios (n = 5). (C) Representative liver photos of vehicle or YH439-treated mice. (D) CTNNB1 staining, H&E staining were performed to measure the cell size around CV area. Quantification of cell size (n = 5). (E)CK19/Ki67 staining were performed to measure the cell proliferation around PV area. Quantification the number of Ki67 + cells (n = 5). Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test. Scale bar = 50 μm.

Article Snippet: For immunohistochemical staining, Anti-Ki67 (1:100 dilution, BOSTER, Cat# M00254-8, RRID: AB_3081764 ), Anti-CTNNB1(1:100 dilution BOSTER, Cat#PA1212, RRID: AB_3082601 ), Anti-CK19(1:100 dilution BOSTER, Cat#PB9715, RRID: AB_3082234 )/Anti- Ki67 (1:100 dilution), CTNNB1(1:100 dilution)/Ki67(1:100 dilution), Anti-Sox9(1:100 dilution BOSTER, Cat#BM4268, RRID: AB_2941988 )/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti-HNF4α (1:100 dilution, Santa Cat#sc-374229, RRID: AB_10989766 ) were used as primary antibody.

Techniques: Staining

Journal: Heliyon

Article Title: AHR regulates liver enlargement and regeneration through the YAP signaling pathway

doi: 10.1016/j.heliyon.2024.e37265

Figure Lengend Snippet: Yap is involved in AHR-induced liver enlargement and regeneration. (A) Yap fl/fl.ALB-CreERT2 and Yap ΔHep were treated with vehicle or YH439 (100 mg/kg/d) for 10 days. (B) The relative liver/body weight ratios (n = 5). (C) Representative liver photos of vehicle or YH439-treated mice. (D) CTNNB1 staining, H&E staining were performed to measure the cell size around CV area. Quantification of cell size (n = 5). (E)CK19/Ki67 staining was performed to measure the cell proliferation around PV area. Quantification the number of Ki67 + cells (n = 5). Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test. Scale bar = 50 μm.

Article Snippet: For immunohistochemical staining, Anti-Ki67 (1:100 dilution, BOSTER, Cat# M00254-8, RRID: AB_3081764 ), Anti-CTNNB1(1:100 dilution BOSTER, Cat#PA1212, RRID: AB_3082601 ), Anti-CK19(1:100 dilution BOSTER, Cat#PB9715, RRID: AB_3082234 )/Anti- Ki67 (1:100 dilution), CTNNB1(1:100 dilution)/Ki67(1:100 dilution), Anti-Sox9(1:100 dilution BOSTER, Cat#BM4268, RRID: AB_2941988 )/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti- Ki67 (1:100 dilution), Anti-Sox9(1:100 dilution)/Anti-HNF4α (1:100 dilution, Santa Cat#sc-374229, RRID: AB_10989766 ) were used as primary antibody.

Techniques: Staining

Journal: Stem Cell Research & Therapy

Article Title: Heme oxygenase-1-modified bone marrow mesenchymal stem cells combined with normothermic machine perfusion to protect donation after circulatory death liver grafts

doi: 10.1186/s13287-020-01736-1

Figure Lengend Snippet: Expression of CK19 in biliary epithelial cells of the transplanted liver. A Immunofluorescence staining for CK19 (× 200, red tag) showed that the percentage of CK19-positive cells among biliary epithelial cells in the HBP group was significantly higher than that in the other groups. B Expression of CK19 in the transplanted liver analyzed by western blotting. C Relative expression of the CK19 protein (CK19/β-actin) showed that the relative expression of CK19 in the HBP group was significantly higher than that in the other groups

Article Snippet: Samples were dehydrated, embedded in paraffin, sectioned, and subjected to HO-1 and cytokeratin 19 (CK19) staining (Proteintech, Wuhan, China).

Techniques: Expressing, Immunofluorescence, Staining, Western Blot

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: Keratin 19 knockout cells display an elongated phenotype . (a) Whole cell lysates of parental (p) control and two different clones (KO1 and KO2) of KRT19 KO cell lines were harvested, and immunoblotting was performed with antibodies against the indicated proteins. Molecular weights in kDa. (b) Immunostaining of K18 (green) in P and KRT19 KO cells. Nuclei are shown in blue. Bar, 20 µm. (c) Phase-contrast images of representative rounded and elongated shapes. Bar, 20 µm. (d) Percentages of P and KRT19 KO cells with rounded or elongated cell shape. Data from three experimental repeats are shown as mean ± SEM. Student’s t-test: *p < 0.05; **p < 0.001. Chi-square test: p < 0.001. (e) Minor/Major axis ratios of P and KRT19 KO cells. Data from four experimental replicates are shown as mean ± SEM. Student’s t-test: *p < 0.05; **p < 0.001. (f) Phase pseudoheight maps of P and KRT19 KO (KO2) cells collected by digital holographic microscopy (DHM). Sum of squared deviation (SSD) shows the distance of individual cells from the population mean. Representative P and KRT19 KO cells with the smallest SSD of 17 phase parameters collected shown. A color bar indicates cell phase height. Bar, 10 µm. Histograms of (g) circularity and (h) eccentricity of P and KRT19 KO cells from DHM analyses

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Knock-Out, Control, Clone Assay, Western Blot, Immunostaining, Digital Holographic Microscopy

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: Weakened cell-cell adhesion in KRT19 KO cells . (a) Phase-contrast images of representative cells engaged in high, medium and low adhesions. Arrows indicate high cell-cell adhesions and arrowheads indicate low cell-cell adhesions. Bar, 20 µm. (b) Percentage of parental (p) control and KRT19 KO cells in high, medium or low adhesions. Data from five experimental repeats are shown as mean ± SEM. Student’s t-test: *p < 0.05; **p < 1 × 0.001. Chi-square test: p < 0.0001. (c) Phase-contrast images of cell clusters formed by P and KRT19 KO cells. Arrows indicate high cell-cell adhesions and arrowheads indicate low cell-cell adhesions. Bar, 20 µm. (d) Number of cells per cluster. Clusters classified into three different ranges: 2–3 cells/cluster; 4–5 cells/cluster; and >6 cells/cluster. Data from three experimental repeats are shown as mean ± SEM. Any statistically significant difference between P and KRT19 KO cells is denoted by * (p < 0.05, Student’s t-test) above the cluster of KRT19 KO cells. Chi-square test: p < 0.0001. (e) Ratio of cell-cell contact length to cell perimeter per cell. Data from four experimental repeats normalized to that of the parental control are shown as mean ± SEM. Student’s t-test: *p < 0.05; **p < 1 × 0.001. (f) Monolayer fragment numbers of P and KRT19 KO cells from the dispase assay. Data from three experimental repeats, each with three replicates, are shown as mean ± SEM. Student’s t-test: *p < 0.05

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Control

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: Defective regulation of E-cadherin in KRT19 K O cells . (a) Whole cell lysates of parental (p) control and KRT19 KO cells were prepared and immunoblotting was performed with indicated antibodies. Molecular weights in kDa. (b) Signal intensities of plakoglobin, E-cadherin and β-catenin from ( A ) were quantified and normalized to those of the GAPDH loading control. Data from at least three experimental repeats normalized to that of the parental control are shown as mean ± SEM. Student’s t-test: ns: not significant; *p < 0.05. (c) Immunostaining of E-cadherin (green) in P and KRT19 KO (KO2) cells. Nuclei are shown in blue. Arrows indicate E-cadherin localization. Bar, 20 µm. (d) Streptavidin beads were used to pulldown P and KRT19 KO (KO2) cells either treated (+) or untreated (-) with biotin for cell surface labeling of proteins. Immunoprecipitates and inputs were subjected to SDS-PAGE and immunoblotting was performed with antibodies against the indicated proteins. Molecular weights in kDa. (e) Signal intensities of E-cadherin in immunoprecipitates of biotin-treated samples from ( D ) were quantified and normalized to those of E-cadherin in input. Data from at least three experimental repeats normalized to that of the parental control are shown as mean ± SEM. Student’s t-test: *p < 0.05. (f) Co-IP of β-catenin with E-cadherin. IP with anti-β-catenin antibody or IgG control was perform in P and KRT19 KO (KO2) cells. Immunoprecipitates and inputs were subjected to SDS-PAGE and immunoblotting was performed with antibodies against the indicated proteins. Molecular weights in kDa. (g) Signal intensities of E-cadherin in IP from ( F ) were quantified and normalized to those of β-catenin in IP. Data from at least three experimental repeats normalized to that of the parental control are shown as mean ± SEM. Student’s t-test: *p < 0.05. (h) Phase-contrast images of P and KRT19 KO (KO2) cells. Cells were either grown in normal growth condition (Normal media), placed in calcium-free media for 6 h, then left unstimulated (-Calcium) or stimulated (+Calcium) with CaCl 2 for 4 h. Arrows indicate high cell-cell adhesions and arrowheads indicate low cell-cell adhesions. Bar, 20 µm

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Control, Western Blot, Immunostaining, Labeling, SDS Page, Co-Immunoprecipitation Assay

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: Inhibiting internalization rescues cell-cell adhesion of KRT19 KO cells . Parental and KRT19 KO (KO2) cells were either (a) incubated with labeled transferrin (red) for 30 min, then immunostained for E-cadherin (green) or (b) co-immunostained for E-cadherin (green) and LAMP1 (red). Arrows indicate colocalization between E-cadherin and transferrin. Nuclei are shown in blue. Insets: areas in KRT19 KO cells rich in E-cadherin localization. Bar, 20 µm. (c) Parental and KRT19 KO (KO2) cells treated with dynamin inhibitor (Dynasore) or DMSO control for 2 h were subjected to dispase assay. Data from at least three experimental repeats, each with three replicates, are shown as mean ± SEM. Student’s t-test: ns: not significant; *p < 0.05. (d) Schematic of how K19 influences E-cadherin localization. While E-cadherin is localized to cell surface in the presence of K19 (left panel), it is internalized and accumulated in early/recycling endosomes in the absence of K19 (right panel)

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Incubation, Labeling, Control

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: K19 re-expression rescues defects associated with KRT19 KO cells . (a) KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 were co-immunostained for GFP (green) and F-actin (red). Nuclei are shown in blue. Bar, 20 µm. (b) Percentage of cells from ( A ) with rounded or elongated cell shape. Data from five experimental repeats are shown as mean ± SEM. N = 74 for GFP-expressing cells and N = 115 for GFP-K19-expressing cells. Student’s t-test: *p < 0.005. Chi-square test: p < 0.0001. (c) Number of monolayer fragments formed by KRT19 KO (KO2) cells stably expressing vector control (v) or K19 from the dispase assay. Data from at least three experimental repeats, each with three replicates, are shown as mean ± SEM. Student’s t-test: *p < 0.05. (d) Whole cell lysates of KRT19 KO (KO2) cells stably expressing vector control (V) or K19 were prepared and immunoblotting was performed with indicated antibodies. Molecular weights in kDa. (e) Signal intensities of plakoglobin and E-cadherin from ( D ) were quantified and normalized to those of the GAPDH loading control. Data from at least three experimental repeats normalized to that of the vector control are shown as mean ± SEM. Student’s t-test: *p < 0.05. (f) Co-IP of β-catenin with E-cadherin in KRT19 KO (KO2) cells stably expressing GFP or GFP-K19. IP with anti-β-catenin antibody or IgG control was perform. Immunoprecipitates and inputs were subjected to SDS-PAGE and immunoblotting was performed with antibodies against the indicated proteins. Molecular weights in kDa. Signal intensities of E-cadherin in IP from were quantified and normalized to those of β-catenin in IP and GFP control

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Expressing, Stable Transfection, Plasmid Preparation, Control, Western Blot, Co-Immunoprecipitation Assay, SDS Page

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: K19 inhibits cell migration and invasion . (a) Wound closure of parental (p) control and KRT19 KO cells. Phase-contrast images of wound area at 0 and 30 h after scratch. Bar, 0.5 mm. (b) Wound areas from ( A ) were quantitated using the ImageJ software and normalized to that at 0 h. (c) Wound closure of KRT19 KO (KO2) cells stably expressing vector control (v) or K19. Wound areas were quantitated using the ImageJ software and normalized to that at 0 h. Transwell migration of (d) P and KRT19 KO (KO2) cells or (e) KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 in the presence of either 0.1 or 10% serum as chemoattractant. Transwell invasion of (f) parental and KRT19 KO (KO2) cells or (g) KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 in the presence of either 0.1 or 10% serum as chemoattractant. Migrated/invaded cells were identified either by staining nuclei with propidium iodide or using GFP signals under a fluorescent microscope. For (d-g), Number of migrated or invaded cells per high-power field were quantified the ImageJ software. For (b-g), data from three experimental repeats, each with three replicates, are shown as mean ± SEM. Student’s t-test: ns: not significant; * p < 0.05; **p < 1 × 0.005

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Migration, Control, Software, Stable Transfection, Expressing, Plasmid Preparation, Staining, Microscopy

Journal: Cell Adhesion & Migration

Article Title: Keratin 19 maintains E-cadherin localization at the cell surface and stabilizes cell-cell adhesion of MCF7 cells

doi: 10.1080/19336918.2020.1868694

Figure Lengend Snippet: K19 is required for the anchorage-independent growth of MCF7 cells . (a) Mammosphere formation of parental (p) control and KRT19 KO cells. Phase-contrast images of mammospheres grown in ultra-low attachment plates for 7 days. Bar, 100 µm. (b) Mammosphere areas from ( A ) were quantitated using the ImageJ software. (c) Phase contrast and immunofluorescence images of mammospheres formed by KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 in ultra-low attachment plates. Bar, 100 µm. (d) Mammosphere areas from ( C ) were quantitated using the ImageJ software. (e) Anchorage-independent growth of P and KRT19 KO cells. Phase-contrast images of colonies grown in soft agar for 7 days. Bar, 200 µm. (f) Colony areas from using ( E ) were quantitated using the ImageJ software. (g) KRT19 KO (KO2) cells stably expressing GFP or GFP-K19 in soft agar for 7 days and colony areas were measured using the ImageJ software. For (B), (D), (F) and (G), data from three experimental repeats, each with three replicates, are shown as mean ± SEM. Student’s t-test: * p < 0.05; **p < 0.001

Article Snippet: For MCF7 KRT19 KO cells stably expressing GFP or GFP-K19, human KRT19 was cloned out of pMRB101 plasmid (courtesy of Dr. Bishr Omary, Rutgers University) and cloned into pLenti CMV/TO hygro (Addgene, Cambridge, MA) using an In-Fusion HD cloning system (Takara, Mountain View, CA) with oligonucleotides: 5ʹ-TCAGTCGACT GGATCC ATGGTGAGCAAGGGCGAGG-3ʹ (BamHI site underlined) and 5ʹ-GAAAGCTGGG TCTAG TCAGAGGACCTTGGAGGCAG-3ʹ (XbaI site underlined), following the manufacturer’s protocol.

Techniques: Control, Software, Immunofluorescence, Stable Transfection, Expressing