Journal: Aging (Albany NY)

Article Title: Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma

doi: 10.18632/aging.104091

Figure Lengend Snippet: KRT6A may participate in regulating TAMs in PDAC. ( A ) Venn diagram of common DEGs between PDAC and ANT and between the M0-high group and the M0-low group. ( B ) Correlation of gene expression between KRT6A and various TAM-related genes in PDAC. ( C ) Heatmap of TAM-related gene expression patterns of the KRT6A-high group and KRT6A-low group in PDAC. ( D ) KEGG (left) and GO (right) pathway enrichment analyses of DEGs between the KRT6A-high group and the KRT6A-low group. ( E , F ) Protein-protein interaction network related to KRT6A using STRING ( E ) and GeneMANIA ( F ). ( G ) Survival analysis of the KRT6A-high group and the KRT6A-low group in TCGA PAAD data (left) and GSE57495 (right).

Article Snippet: For immunofluorescence staining, frozen tissue section samples of PDAC and ANT were incubated with a mixture of rabbit anti-ITGAM (Proteintech; 1:50) and mouse anti-KRT6A (Proteintech; 1:50) antibodies overnight at 4°C after dewaxing, hydrating, antigen retrieval and blocking, followed by incubation with a mixture of Alexa Fluor 488 or Alexa Fluor 555 fluorescence-conjugated secondary antibodies (Abcam; 1: 200) for 1 hour and DAPI for 10 minutes.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma

doi: 10.18632/aging.104091

Figure Lengend Snippet: Immunofluorescence and immunohistochemistry (IHC) staining by integrating KRT6A and ITGAM expression. ( A ) Immunofluorescence staining of KRT6A (green) and ITGAM (red) in PDAC and ANT frozen tissue sections (100×). ( B ) Pearson's correlation of KRT6A and ITGAM expressing cells in PDAC and ANT. ( C – F ) IHC staining of KRT6A (c) and ITGAM (e) in PDAC and ANT (200×). Column charts were shown in ( D ) for KRT6A and ( F ) for ITGAM. *p<0.001.

Article Snippet: For immunofluorescence staining, frozen tissue section samples of PDAC and ANT were incubated with a mixture of rabbit anti-ITGAM (Proteintech; 1:50) and mouse anti-KRT6A (Proteintech; 1:50) antibodies overnight at 4°C after dewaxing, hydrating, antigen retrieval and blocking, followed by incubation with a mixture of Alexa Fluor 488 or Alexa Fluor 555 fluorescence-conjugated secondary antibodies (Abcam; 1: 200) for 1 hour and DAPI for 10 minutes.

Techniques: Immunofluorescence, Immunohistochemistry, Expressing, Staining

Journal: Aging (Albany NY)

Article Title: Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma

doi: 10.18632/aging.104091

Figure Lengend Snippet: The molecular mechanism by which KRT6A regulates TAMs. ( A ) CEMiTool was used to find gene modules affecting TAMs through KRT6A by dividing TCGA PAAD mRNA-seq data into five groups by the level of KRT6A expression: Grade 0 (G0), Grade 1 (G1), Grade 2 (G2), Grade 3 (G3), and Grade 4 (G4), 6A expression where G0 expressed the lowest amounts of KRT6A (average RPKM = 0) and G4 expressed the highest amounts of KRT6A (average RPKM = 193.24). ( B ) Expression of a gene module (Module 1) in each PDAC patient. ( C ) Major genes and the number of genes in each module. ( D ) Transcription factor PPI network and pathway enrichment of genes in Module 1. ( E ) Cell type enrichment of genes in Module 1.

Article Snippet: For immunofluorescence staining, frozen tissue section samples of PDAC and ANT were incubated with a mixture of rabbit anti-ITGAM (Proteintech; 1:50) and mouse anti-KRT6A (Proteintech; 1:50) antibodies overnight at 4°C after dewaxing, hydrating, antigen retrieval and blocking, followed by incubation with a mixture of Alexa Fluor 488 or Alexa Fluor 555 fluorescence-conjugated secondary antibodies (Abcam; 1: 200) for 1 hour and DAPI for 10 minutes.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Alteration of tumor-associated macrophage subtypes mediated by KRT6A in pancreatic ductal adenocarcinoma

doi: 10.18632/aging.104091

Figure Lengend Snippet: Genes that were expressed similarly to KRT6A in PDAC were identified using the STEM tool.

Article Snippet: For immunofluorescence staining, frozen tissue section samples of PDAC and ANT were incubated with a mixture of rabbit anti-ITGAM (Proteintech; 1:50) and mouse anti-KRT6A (Proteintech; 1:50) antibodies overnight at 4°C after dewaxing, hydrating, antigen retrieval and blocking, followed by incubation with a mixture of Alexa Fluor 488 or Alexa Fluor 555 fluorescence-conjugated secondary antibodies (Abcam; 1: 200) for 1 hour and DAPI for 10 minutes.

Techniques:

Journal: bioRxiv

Article Title: Excessive Polyamine Generation in Keratinocytes Promotes Self-RNA Endosomal Sensing by Dendritic Cells in Psoriasis

doi: 10.1101/2020.03.09.984658

Figure Lengend Snippet: ( A ) Schematic of Pp6 deletion in K5. Pp6 fl/fl mice. ( B ) Pp6 genotyping of the indicated tissue samples derived from WT, Pp6 fl/fl and K5. Pp6 fl/fl mice. The red arrow shows K5-Cre-mediated epidermal-specific deletion of Pp6 . ( C ) Immunoblot analysis of Pp6 expression in primary murine keratinocytes from Pp6 fl/fl and K5. Pp6 fl/fl mice. ( D ) Immunofluorescent staining of Krt5 and Pp6 in thymic sections from Pp6 fl/fl and K5. Pp6 fl/fl mice. The dotted lines indicate the border between the cortex and the medulla; the scale bars represent 50 μm. ( E ) Phenotypic representation of splenomegaly and lymphadenopathy observed from diseased K5. Pp6 fl/fl mice in contrast to Pp6 fl/fl controls. ( F ) Immunofluorescent labelling of Ki67 in mouse skin samples. ( G ) Flow cytometric analysis of immune cell-specific markers in mouse skin (n = 5∼6). ( H ) TEWL of mouse skin (n = 4). ( I ) Immunofluorescent labelling of Krt6 in mouse skin samples. ( J and K ) Immunofluorescent staining and quantitation of dermal CD80 + cells of mouse skin samples (n = 4). ( L ) Toluidine blue staining for mast cells in skin sections from Pp6 fl/fl and K5. Pp6 fl/fl mice. ( M ) Eosinophil peroxidase (EPX) staining for eosinophils in skin sections from Pp6 fl/fl and K5. Pp6 fl/fl mice. A skin section from a patient diagnosed with atopic dermatitis (AD) served as a positive control. ( N ) Serum IgE levels in Pp6 fl/fl and K5. Pp6 fl/fl mice, as measured by ELISA (n = 11∼12). Boxes show twenty-fifth to seventy-fifth percentiles; whiskers show minimum to maximum; and lines show medians; ns: not significant, two-tailed Student’s t -test. ( O ) GSEA of DEGs in K5. Pp6 fl/fl skin relative to Pp6 fl/fl skin with upregulated (AD UP) or downregulated (AD DOWN) DEGs in human AD (GSE12511) enriched. ( P ) H&E staining of skin sections from K5. Pp6 fl/fl mice treated with Halometasone or vehicle. Pp6 fl/fl , healthy skin sample from control mice; K5. Pp6 fl/fl -N, unaffected skin from K5. Pp6 fl/fl mice; K5. Pp6 fl/fl -L, affected skin from K5. Pp6 fl/fl mice. The data ( D, E, I, L and M ) are representative of > 5 mice in each group. The data ( P ) are representative of two independent experiments with two mice in each group. For ( F , I , J , L , M and P ), the dotted lines indicate the border between the epidermis and the dermis; e, epidermis; d, dermis; the scale bars represent 50 μm ( F , I , J , M and P ) or 100 μm ( L ). * p < 0.05, *** p < 0.001, **** p < 0.0001, ns: not significant, two-tailed Student’s t -test (means ± SEM).

Article Snippet: The slices were blocked with 1% BSA/PBS for 1 h and stained with anti-Krt5 (Invitrogen cat. MA5-17057, 1:100 dilution), anti-PPP6C (Abcam cat. 131335, 1:50 dilution), anti-mouse Ki67 (Servicebio cat. GB13030-2, 1:50 dilution), anti-Krt6 (Proteintech cat. 10590-1-AP, 1:2000 dilution), anti-mouse CD80 (BioLegend cat. 104702, 1:50 dilution), anti-eosinophil peroxidase (EPX, Santa Cruz cat. 19148, 1:50 dilution), anti-C/EBPβ (Santa Cruz cat. 7962, 1:100 dilution), anti-p-C/EBPβ (Thr235) (Cell Signaling Technology cat. 3084, 1:100 dilution), anti-ARG1 (Proteintech cat. 66129-1-Ig, 1:1000 dilution), anti-COX4I1 (Proteintech cat. 11242-1-AP, 1:100 dilution), anti-ASS1 (Proteintech cat. 16210-1-AP, 1:100 dilution), anti-human CD4 (BioLegend cat. 317402, 1:50 dilution) and anti-human Integrin α-X (Santa Cruz cat. 1185, 1:50 dilution) overnight at 4℃.

Techniques: Derivative Assay, Western Blot, Expressing, Staining, Quantitation Assay, Positive Control, Enzyme-linked Immunosorbent Assay, Two Tailed Test