Journal: JCI Insight

Article Title: Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

doi: 10.1172/jci.insight.87680

Figure Lengend Snippet: Immunofluorescence of (A) retinaldehyde dehydrogenase 2 (RALDH2), p75 neurotrophin receptor (p75NTR), and PDGFRβ; (B) β3-tubulin and RALDH2; (C and D) p75NTR; (E) p75NTR and CD21; (F) CD21 and CXCL13; and (G) CD3ε, CD21, and B220 in aged kidneys after ischemic reperfusion injury (IRI). Arrows indicate TLT localization. Arrowheads indicate the localization of the B cell area. Aged kidneys were analyzed 14 (B and C), 30 (A), and 45 (D–G) days after IRI. (H) Retinoic acid (RA) induces p75ntr mRNA expression in C3H10T1/2 mouse embryonic fibroblasts and FACS-sorted PDGFRβ+ cells (n = 3 per group). The data are presented as dot plots (mean ± SD). (I) Activation-induced cytidine deaminase (Aid) mRNA levels of kidneys 45 days after various ischemic time IRI in young and aged mice (n = 4 per group) and correlation with TLT size (n = 16, aged mice only). The expression levels were normalized to those of Gapdh and expressed relative to those of controls or young mouse kidney at day 0 (IRI). *P < 0.001 versus controls. A 2-tailed Student’s t test was used to analyze data from FACS-sorted PDGFRβ+ cells; 1-way ANOVA with Tukey’s post-hoc analysis was used for other experiments. Correlation was determined by Pearson’s correlation analysis. The box corresponds to the first quartile, median (horizontal bar in the box), and third quartile, and the whiskers extend from minimum to maximum values. Scale bars: (A, C, and D) 100 μm, (E and G) 50 μm, (B and F) 10 μm.

Article Snippet: The following primary antibodies were used in the mouse immunohistochemistry and immunofluorescence experiments: anti-αSMA (catalog C6198; Sigma-Aldrich), -AID (catalog 4959; Cell Signaling), -GFP (catalog ab13970; Abcam), -Ki67 (catalog 14-5698; eBioscience), -CD45 (catalog 14-0451; eBioscience), –CD11c-PE (catalog 12-0114; eBioscience), –PDGFR-β (catalog 14-1402; eBioscience), –β3tubulin-FITC (catalog A488-435L; Covance), -Cre (catalog 69050-3; Novagen), –LYVE-1 (catalog ab14917; Abcam), -CD21 (catalog ab75985; Abcam), –ER-TR7 (catalog T-2109; BMA Biomedicals), -CXCL13 (catalog AF470; R&D Systems), -CCL19 (catalog AF880; R&D Systems), -CCL21 (catalog AF457; R&D Systems), - p75NTR (catalog AF1157; R&D Systems), -CD3ε (catalog 550275; BD PharMingen), -B220 (catalog 557390; BD PharMingen), –PD-1 (catalog 114102; BioLegend), -CD4 (catalog 100726; BioLegend), -CD8 (catalog 100425; BioLegend), -PNAd (catalog 120801; BioLegend), and -ALDH1A2 (catalog HPA010022; Sigma-Aldrich) antibodies.

Techniques: Immunofluorescence, Expressing, Activation Assay

Journal: JCI Insight

Article Title: Heterogeneous fibroblasts underlie age-dependent tertiary lymphoid tissues in the kidney

doi: 10.1172/jci.insight.87680

Figure Lengend Snippet: (A–L) Histological analyses of aged human kidney samples. (A and B) Periodic acid-Schiff (PAS) staining and immunofluorescence analysis of (C) CD20 and CD3ε; (E) Ki67 and CD3ε/CD20 (arrowheads indicate double-positive cells); (G) CXCL13 and CD21; (J) CXCL13 and CD45; (K) CD21, α-smooth muscle actin (αSMA), and CD45; and (L) p75 neurotrophin receptor (p75NTR) and CD21, and immunohistochemical analysis of (D) peripheral lymph node addressin (PNAd); (F) CD21 (arrowheads indicate the localization of CD21+ follicular dendritic cell [FDC] networks); (H) CD20, activation-induced cytidine deaminase (AID), CD21, and retinaldehyde dehydrogenase 2 (RALDH2) (serial sections); and (I) RALDH2 (arrowheads indicate the localization of B cell follicles). The outlined region in (H) is magnified in (I). Arrows indicate TLT localization. Scale bars: (A, D, H, K, and L) 50 μm, (B, C, and F) 100 μm, (E, G, I, and J) 10 μm. (M) Quantitative analysis of TLT frequencies in human samples (n = 56; young = 13, aged = 43). #P < 0.05 (Pearson’s χ2 test).

Article Snippet: The following primary antibodies were used in the mouse immunohistochemistry and immunofluorescence experiments: anti-αSMA (catalog C6198; Sigma-Aldrich), -AID (catalog 4959; Cell Signaling), -GFP (catalog ab13970; Abcam), -Ki67 (catalog 14-5698; eBioscience), -CD45 (catalog 14-0451; eBioscience), –CD11c-PE (catalog 12-0114; eBioscience), –PDGFR-β (catalog 14-1402; eBioscience), –β3tubulin-FITC (catalog A488-435L; Covance), -Cre (catalog 69050-3; Novagen), –LYVE-1 (catalog ab14917; Abcam), -CD21 (catalog ab75985; Abcam), –ER-TR7 (catalog T-2109; BMA Biomedicals), -CXCL13 (catalog AF470; R&D Systems), -CCL19 (catalog AF880; R&D Systems), -CCL21 (catalog AF457; R&D Systems), - p75NTR (catalog AF1157; R&D Systems), -CD3ε (catalog 550275; BD PharMingen), -B220 (catalog 557390; BD PharMingen), –PD-1 (catalog 114102; BioLegend), -CD4 (catalog 100726; BioLegend), -CD8 (catalog 100425; BioLegend), -PNAd (catalog 120801; BioLegend), and -ALDH1A2 (catalog HPA010022; Sigma-Aldrich) antibodies.

Techniques: Staining, Immunofluorescence, Immunohistochemical staining, Activation Assay

Journal: PloS one

Article Title: Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

doi: 10.1371/journal.pone.0093362

Figure Lengend Snippet: Figure 1. PU.1 binds specifically to KLF4 promoter during differentiation of myeloid progenitors to macrophages. A) PU/ER(T) cells were grown in presence of 95% Ethanol or 100 nM Tamoxifen for time periods as indicated. Cytosolic and nuclear protein fractions were prepared and translocation of PU.1 from cytosol to nucleus was detected by immunoblotting with anti PU.1 rabbit polyclonal antibodies. Purity of cytoplasmic and nuclear fractions was analyzed by immunoblotting with a-Tubulin and HDAC2 proteins. B) Nuclear extracts were prepared from differentiating bone marrow derived macrophages on day 1 and 7 and in vitro binding to biotin labeled 76 bp oligonucleotide probe representing 2118/2113 PU.1 binding element in the KLF4 promoter was determined by EMSA as described in methods. C) PU/ER(T) cells were treated with Ethanol or 100 nM Tamoxifen for 1, 4 or 24 h and nuclear extracts were prepared. An equal amount of nuclear protein extract from Ethanol and Tamoxifen treatment was analyzed for DNA binding activity using biotin labeled 76 bp oligonucleotide probe representing 2118/2113 PU.1 binding element in the KLF4 promoter. Specificity of PU.1-KLF4 DNA complex in experiment B & C was determined by the ability of the PU.1-KLF4 DNA complex to form supershift with anti PU.1 antibody. D) Specificity of PU.1-KLF4 DNA complex was determined by including 5, 15, 25 and 100 picomole of excess unlabelled KLF4 promoter oligo in the DNA binding reaction. E) In vivo binding of PU.1 to KLF4 promoter was determined by Chromatin-immuno precipitation in differentiating BMDM on day 1, 2 and 7 and in F) PU/ER(T) cells treated with Tamoxifen for 1 and 24 h. Error bars represent standard deviation and * is used where ever p value is #0.05 and ** is used when p#0.005. doi:10.1371/journal.pone.0093362.g001

Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM603), respectively.

Techniques: Translocation Assay, Western Blot, Derivative Assay, In Vitro, Binding Assay, Labeling, Activity Assay, In Vivo, Chromatin Immunoprecipitation, Standard Deviation

Journal: PloS one

Article Title: Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

doi: 10.1371/journal.pone.0093362

Figure Lengend Snippet: Figure 4. PU.1 transcriptionally regulates KLF4 expression that is sensitive to promoter methylation. A) PU/ER(T) cells were electroporated with 1.6 kb pGL3-KLF4 vector, after 16 h of electroporation treated with ethanol or 100 nM Tamoxifen for 24 h and analyzed for Luciferase reporter activity B) HEK293 cells were transfected with empty pGL3, 1.6 kb KLF4-pGL3, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. C) HEK293 cells were transfected with empty pGL3, 1.6 kb mutant pGL3-KLF4, pCMV-PU.1 or pCMV control vectors separately or in combination as indicated in figure, and after 36 h cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. D) PU/ER(T) cells were treated with Tamoxifen or ethanol for indicated time periods and expression of KLF4 was determined by immunoblotting. E) PU/ER(T) cells were electroporated with either control pCMV or KLF4 over expression vector and grown in IMDM for 72 h and analyzed for surface expression of F4/80. F) The biotin labeled KLF4 promoter oligo was methylated using M.SssI in presence of S-Adenosyl Methionine and used as a probe to determine the DNA binding activity in the Ethanol and Tamoxifen treated nuclear extracts as described in figure 1. G) Empty pGL3 vector, pGL3-KLF4 Luciferase vector were in vitro methylated using M.SssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector. After 36 h, cell extracts were prepared in Promega cell lysis buffer and analyzed for luciferase activity. H) Empty pcpgf-basic, pcpgf- KLF4 were in vitro methylated using M.sssI in presence of S-Adenosyl Methionine and electroporated along with control pCMV or pCMV-PU.1 expression vector in to HEK293 cells and grown in HEK293Blue detection medium for 36 h. Relative promoter activity was compared by mSEAP reporter activity as measured by the absorbance of the HEK293 Blue growth medium at 630 nm. For figures 4 B, C, G, H reporter gene activities of pGL3-KLF4/pGL3-mutant KLF4/pcpg-KLF4 were not significantly different when co-transfected with the pCMV control plasmid. Figures 4 A to 4 C and 4 G, H means were compared and * is used where ever p value is #0.05 and ** is used when p#0.005. Error bars represent standard deviation. doi:10.1371/journal.pone.0093362.g004

Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM603), respectively.

Techniques: Expressing, Methylation, Plasmid Preparation, Electroporation, Luciferase, Activity Assay, Transfection, Control, Lysis, Mutagenesis, Western Blot, Over Expression, Labeling, Binding Assay, In Vitro, Standard Deviation

Journal: PloS one

Article Title: Krüppel like factor 4 promoter undergoes active demethylation during monocyte/macrophage differentiation.

doi: 10.1371/journal.pone.0093362

Figure Lengend Snippet: Figure 6. AICDA is essential for KLF4 promoter demethylation. A) The relative methylation index of the proximal M1 CpG region in KLF4 promoter was compared on 1st and 7th day of differentiating BMDM from wild type and GADD45a knock out macrophages as described in Figure 3 C&D. B) Knockdown of AICDA protein in AICDA-shRNA or control-shRNA electroporated PU/ER(T) cells. C) Methylation index of the proximal KLF4 promoter M1 CpG region was compared in AICDA shRNA or control shRNA electroporated PU/ER(T) cells as described in Figure 3C&D. D) Expression of F4/80 was analyzed in control shRNA or AICDA-shRNA electroporated PU/ER(T) cells treated with or without tamoxifen for 72 h. E) Morphological changes in ethanol/tamoxifen treated PU/ER(T) cells were analyzed in AICDA depleted cells in comparison with control cells. Representative histograms of flow cytometry, western blots and cell staining were presented. Figure 6A and C, * is used where ever p value is #0.05 and ** is used when p#0.005. Error bars represent standard deviation. doi:10.1371/journal.pone.0093362.g006

Article Snippet: Myc tagged AICDA and FLAG tagged human KLF4 expression vectors were purchased from Origene (RC202949) and SABiosciences (DAM603), respectively.

Techniques: Methylation, Knock-Out, Knockdown, shRNA, Control, Expressing, Comparison, Flow Cytometry, Western Blot, Staining, Standard Deviation