Journal: Journal of cell science

Article Title: FAK, PIP5KIgamma and gelsolin cooperatively mediate force-induced expression of alpha-smooth muscle actin.

doi: 10.1242/jcs.044008

Figure Lengend Snippet: Fig. 2. FAK activation after mechanical force is independent of RhoA. (A) Top panels: immunoblot analysis of Tyr397FAK-P (pY397FAK) in collagen-coated- bead-associated proteins after force application for the indicated time intervals. β1-integrin immunoblots show loading for each bead preparation. Bottom left panels: cells were incubated with collagen beads and force was applied for 60 minutes, the optimal time for phosphorylation of Tyr397FAK. Lia1/2 β1-integrin- blocking antibody was co-incubated with collagen beads prior to force application and was used here as a β1-integrin control to test for specificity. Fibronectin and BSA-coated beads were used as alternative controls in cells treated with force for 60 minutes. F, force. Bottom center panels: immunoblots show force-induced collagen-coated-bead association of talin and Arp2 after 60 minutes of force. NF, no force. Bottom right panels: activation of Src by force was determined by immunoblots of Tyr527Src-P (pY527-Src) and Tyr416Src (pY416-Src). Note that pre-incubation of cells with the Src inhibitor SU6656 at 1 μM does not inhibit force-induced phosphorylation of Tyr397FAK. (B) Confocal microscopy after force application shows Tyr397FAK-P around collagen beads. DIC images of the same cells show the location of beads. BSA-coated beads with force application were used as non-specific controls. Arrows show the area shown at higher magnification in the insets. Line graphs show fluorescence intensity across bound beads. (C) Membrane-anchored, dominant-negative Tyr397Phe FAK (DN FAK) was transfected into cells and protein recruitment to collagen-coated beads was analyzed in bead-associated proteins. After force application, immunoblots show increased levels of Tyr397FAK-P and β-actin in bead-associated proteins in vehicle (Veh)-treated cells but not in DN-FAK-treated cells. Ratios of β-actin to integrin were computed from densitometry measurements of immunoblots (mean ± s.e.m.). FAK knockdown by siRNA showed decreased Tyr397FAK-P in relation to the amount of FAK present. (D) Cells treated with Rho inhibitor, Y27632, were assessed for phosphorylation of Tyr397FAK. (E) Rho activation was measured in cells with or without siRNA knockdown using rhotekin-binding assay and immunoblotting for RhoA. Ratios of active Rho A to total Rho A were computed from densitometry measurements of immunoblots (mean ± s.e.m.).

Article Snippet: Antibodies to Arp2, Tyr527Src-P and Tyr416Src-P were purchased from Cell Signaling Technology (Danvers, MA).

Techniques: Activation Assay, Western Blot, Incubation, Phospho-proteomics, Blocking Assay, Control, Confocal Microscopy, Fluorescence, Membrane, Dominant Negative Mutation, Transfection, Knockdown, Binding Assay

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 3. mRNA and protein expression of novel cytoskeletal molecules in PAN rats. In relation to GAPDH, the mRNA and protein expression of Tagln (transgelin) (A, A′), Birc5 (survivin) (B, B′), Actr2 (arp2) (C, C′), Krt2–7 (cytokeratin7) (D, D′) and Vcl (vinculin) (E, E′) increased significantly at Day 10, then decreased at Day 15 in PAN rats compared to controls (Con). Data are presented as mean ± SD. N = 6. ∗P < 0.05 versus Con; ∗∗P < 0.01 versus Con.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Expressing

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 4. Immunofluorescence staining of novel cytoskeletal molecules in PAN rats. Transgelin (A), survivin (D), arp2 (G) and cytokeratin7 (J) were labelled green and showed a weak staining in normal rat glomeruli (Control). Double-labelling assays showed that transgelin (C), survivin (F), arp2 (I) and cytokeratin7 (L) colocalized with podocyte marker synaptopodin, labelled red (Synap: B, H, K), displaying a linear pattern along the glomerular capillary loops. Vinculin (M), labelled red, showed colocalization with the podocyte molecule α-actinin-4, labelled green (N), displaying a dotted, linear pattern in glomeruli (O). Bar = 20 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Immunofluorescence, Staining, Control, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 5. Immunofluorescence staining of novel cytoskeletal molecules in human kidneys. Transgelin (A1, a1, a4, a7), arp2 (C1, c1, c4, c7), and cytokeratin7 (D1, d1, d4, d7) are green. Vinculin is red (E1, e1, e4, e7). Their staining was intense in the glomeruli of patients with MCNS (a1, c1, d1, e1), FSGS (a4, d4, e4) and MN (a7, c7, d7, e7) as compared to normal glomeruli (Control: A1, C1, D1, E1), except for arp2 in FSGS glomeruli (c4). Transgelin, arp2, and cytokeratin7 colocalized with the podocyte marker synaptopodin, labelled red, in glomeruli of MCNS (a3, c3, d3), FSGS (a6, d6) and MN (a9, c9, d9). Vinculin showed complete colocalization with the podocyte molecule podocin, labelled green in control (E3), MCNS (e3), FSGS (e6) and MN (e9). The staining of survivin was negative in glomeruli of normal controls (B1), but intensive staining was revealed in MCNS (b1), FSGS (b4) and MN (b7), and mainly distributed to the perinuclear area of glomeruli and tubules of MCNS (b3), FSGS (b6) and MN (b9). Bar = 20 µm.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Immunofluorescence, Staining, Control, Marker

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: Newly identified cytoskeletal components are associated with dynamic changes of podocyte foot processes.

doi: 10.1093/ndt/gfp338

Figure Lengend Snippet: Fig. 6. (A) Distribution of novel cytoskeletal molecules in cultured podocytes. Cell nuclei were stained blue with Hoechst. In normal podocytes (Control: a–e), the staining of transgelin, survivin, arp2 and cytokeratin7 was weak and mainly distributed in cytoplasm, whereas their fluorescence intensity increased, especially along the cell membrane, in puromycin aminonucleoside-treated podocytes (PAN: f–j). The distribution of vinculin was obviously altered, displaying a dotted- and a plaque-like pattern along cell membranes. Bar = 30 µm. (B) Protein expression of novel cytoskeletal molecules in cultured podocytes. Transgelin (k), survivin (l), arp2 (m), cytokeratin7 (n) and vinculin (o) increased in PAN-treated cells compared to controls. Data are presented as mean ± SD. N = 3. Values were normalized to GAPDH. ∗P < 0.05 versus Con.

Article Snippet: The following primary antibodies were used: rabbit anti-podocin (1:800, a gift from Professor Corinne Antignac), rabbit anti-α-actinin, transgelin, arp2, cytokeratin7 (1:100, Santa Cruz, USA), survivin (1:800, Cell Signaling, USA), mouse anti-vinculin (1:500, Abcam, USA) and mouse antisynaptopodin (ready to use, Progen, Germany).

Techniques: Cell Culture, Staining, Control, Fluorescence, Membrane, Expressing

Journal: Open Biology

Article Title: Acquisition of the dorsal structures in chordate amphioxus

doi: 10.1098/rsob.160062

Figure Lengend Snippet: Distribution pattern of active Arp2/3 complex in unfertilized and fertilized egg and its co-localization with microtubules and actin filaments. Partial rendering lateral images near maximum diameter in all but ( d (iv­–vi)), which are viewed vegetally near vegetal pole. ( a ) Eggs are oriented by the aid of polar body (pb) and/or vegetal tuft-like structure (tu). ( b (i–vi)) Change in location of cortical pArp2 immunopositive signals after sperm fusion from ubiquitous in cortical region in unfertilized egg ( b (i)) to on the one side of egg ( b (vi)) passing through animal pole ( b (iii,iv)) (arrowheads). ( c (i–iii)) Co-localization of F-actin and pArp2 immunopositive signals in late fertilized egg. ( d (i­–vi)) Co-localization of microtubules and pArp2 immunopositive signals in late fertilized egg. Note a tuft-like structure at the vegetal pole in immunostain for pArp2 and for tubulin ( d (ii)). Scale bar, 100 µm.

Article Snippet: The antibodies were anti-Arp2 (Thr237/Thr238) phospho-specific antibody (AP3871, ECM Biosciences, KY) for detecting active Arp 2/3 complex and anti-phospho-Smad1 (Ser463/465) antibody (06-702, Merck Millipore, Germany) for Bmp signalling.

Techniques:

Journal: Open Biology

Article Title: Acquisition of the dorsal structures in chordate amphioxus

doi: 10.1098/rsob.160062

Figure Lengend Snippet: Distribution patterns of maternal nodal mRNA and Arp2/3 complex. ( a (i) –h (i)) Maternal nodal mRNA (red) and pArp2 immunopositive signals (green) are co-localized from one-cell to blastula stage shown as partial rendering images near maximum diameter. Animal pole to the top in ( a (i)), ( d (i)) and ( h (i)). ( a (ii) –h (ii)) Relative fluorescent intensity curve at section denoted by white arrow that indicates direction of x -axis. Red for nodal and green for pArp2. Note a tuft-like immunopositive for anti-pArp2 antibody in a cell at 16- and 64-cell stage (arrowheads) ( e (i), g (i)). ( i (i–iii)) Expression pattern of nodal in CK666-treated blastula to late gastrula stage. ( j (i–iii)) Expression pattern of lefty in CK666-treated blastula to late gastrula stage. Note expression at median furrow (arrowheads) and a half of embryo in both genes. ( k (i–iii)) Co-localization of pArp2 immunopositive signals and nodal mRNA in CK666-treated blastula. Bl, blastula; bp, blastopore; iG, initial gastrula; lG, late gastrula. Scale bar, 100 µm.

Article Snippet: The antibodies were anti-Arp2 (Thr237/Thr238) phospho-specific antibody (AP3871, ECM Biosciences, KY) for detecting active Arp 2/3 complex and anti-phospho-Smad1 (Ser463/465) antibody (06-702, Merck Millipore, Germany) for Bmp signalling.

Techniques: Expressing

Journal: Frontiers in Cell and Developmental Biology

Article Title: Feedback-Driven Mechanisms Between Phosphorylated Caveolin-1 and Contractile Actin Assemblies Instruct Persistent Cell Migration

doi: 10.3389/fcell.2021.665919

Figure Lengend Snippet: Actin protrusive network is upregulated by AMPK-Rac1-PAK1-Cofilin signaling cascade in cells lacking CAV-1. (A) Immunofluorescence microscopy analysis demonstrating that more pronounced endogenous ARPC2 colocalizes with F-actin (visualized by Alexa 568 phalloidin) on the lamellipodia protrusions in CAV-1 deficient cells. Magnified regions of cell edges on the right show the distribution of ARPC2 in WT and CAV-1 KO cells. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). Quantification of width of lamellipodial protrusions are shown on the right. n = 16 regions from 16 cells for each group. (B) Representative images of membrane ruffling by time-lapse microscope. 1-pixel-wide areas were cut out to generate a 300 frame 2 s interval kymograph. Yellow dashed lines indicate the track of cell movement. An enlarged region is displayed on the right, vertical dashed lines show the membrane protrusion distance, while horizontal dashed lines mark the duration of protrusion. Bars, 10 μm (in cell images) and 2 μm (in the magnified box). Quantification of protrusion rate are shown on the right. n = 16 regions from 16 cells for each group. (C) P-AMPK (Thr172) and total AMPK were detected from the lysates of each group by western blotting. Please note that CAV-1(Y14F)-mEGFP can’t be detected by using phospho-CAV-1(Tyr14) antibody. Asterisk denotes the non-specific band. Quantification of P-AMPK (Thr172) levels (compared to total AMPK) from each group was shown on the right panel. n = 3. (D) Pull-down assays were performed for WT, CAV-1 KO, and CAV-1 KO; CAV-1-mEGFP re-expressed cells. Proteins bound to GST-PAK binding domain were analyzed by western blots and further quantified (compared to total Rac1) based on the band’s intensity. n = 3. (E) Western blot analysis and quantification (compared to total PAK1 and Cofilin) of the levels of phosphorylated PAK1 (Thr423) and Cofilin (Ser3) in WT and CAV-1 KO cell lysates. n = 3. (F) Immunostaining and quantification of endogenous P-Cofilin (Ser3) and F-actin distribution in WT and CAV-1 KO cells. A 16 μm length line was used to generate a line profile to illustrate the co-localization of P-Cofilin (Ser3) and F-actin. The lamellipodia region was enlarged on the right, and 2 μm width region was chosen to analyze the mean intensity of P-Cofilin (Ser3) on the leading edge. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (G) Immunostaining and quantification of endogenous P-cofilin (Ser3) distribution upon compound C treatment in WT and CAV-1 KO cells. Magnified regions represent the lamellipodia region. Bars, 10 μm (in cell images) and 5 μm (in the magnified box). n = 18 regions from 18 cells for each group. (H) Western blot analysis and quantification of the phosphorylated levels of AMPK (Thr172), PAK1 (Thr423), Cofilin (Ser3) and activity of Rac1 and RhoA upon compound C treatment. n = 3. In panel (C–E,H) , the obtained intensity value from wild-type cells was set to 1. All the data are presented as mean ± SD. In (C,G,H) , *** P < 0.001; * P < 0.05; N.A., not significant (one-way ANOVA). In (D–F) , *** P < 0.001; ** P < 0.01 (unpaired t -test). All the data are from three independent experiments.

Article Snippet: The following antibodies were used in this study: CAV-1 (D46G3) rabbit antibody (1:1,000 dilution for WB, 1:200 for IF; #3267, Cell Signaling, Beverly, MO, United States); Phospho-CAV-1 (Tyr14) rabbit antibody (dilution 1:1,000 for WB; #3251, Cell signaling); AMPK rabbit antibody (dilution 1:500 for WB; #SAB4502329, Sigma, St. Louis, MO, United States); P-AMPK (Thr172) rabbit antibody (dilution 1:500 for WB, 1:100 for IF; #2531S, Cell Signaling); Cofilin (E-8) mouse antibody (dilution 1:1,000 for WB; #sc-376476, Santa Cruz, Dallas, TX, United States); Phospho-Cofilin (Ser3) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #3313, Cell signaling); p190RhoGAP rabbit antibody (dilution 1:2,000 for WB; #26789, Proteintech, Rosemont, IL, United States); FAK rabbit antibody (dilution 1:1,000 for WB; #3285, Cell Signaling); Phospho-FAK (Tyr397) rabbit antibody (dilution 1:1,000 for WB; #3283, Cell Signaling); PAK1 rabbit antibody (dilution 1:1,000; #2602, Cell Signaling); Phospho-PAK1 (Thr423)/PAK2 (Thr402) rabbit antibody (dilution 1:1,000 for WB, 1:200 for IF; #2601, Cell Signaling); Tpm4.2 (LC24) mouse antibody (dilution 1:500 for WB and IF; a kind gift from Peter W. Gunning, UNSW Australia); Phospho-myosin light chain 2 (Thr18/Ser19) rabbit antibody (dilution 1:500 for WB, 1:200 for IF; #3674, Cell Signaling); Myosin light chain mouse antibody (dilution 1:1,000 for WB; #M4401, Sigma); Vinculin mouse antibody (dilution 1:100 for IF; #V9131, Sigma); ARPC2 rabbit antibody (dilution 1:1,000 for WB and IF; #15058, Proteintech, Rosemont, IL, United States); Myosin-18B rabbit antibody (dilution 1:500 for WB; #HPA000953, Sigma); Rab8 rabbit antibody (dilution 1:100 for IF; #R5530, Sigma); and GAPDH mouse polyclonal antibody (dilution 1:1,000 for WB; #G8795, Sigma).

Techniques: Immunofluorescence, Microscopy, Membrane, Western Blot, Binding Assay, Immunostaining, Activity Assay

Journal: Biomolecules

Article Title: Identification of a Fibroblast-Related Prognostic Model in Glioma Based on Bioinformatics Methods

doi: 10.3390/biom12111598

Figure Lengend Snippet: Correlation between prognostic gene expression and CNV: ( A ) correlation scatter plot of ANGPTL2 expression and CNV; ( B ) correlation scatter plot of CDK4 expression and CNV; ( C ) correlation scatter plot of MEX3A expression and CNV; ( D ) correlation scatter plot of NEGR1 expression and CNV; ( E ) correlation scatter plot of PBK expression and CNV; ( F ) correlation scatter plot of SLC2A3 expression and CNV; and ( G ) correlation scatter plot of TMEM100 expression and CNV.

Article Snippet: ANGPTL2 (#12316-1-AP, rabbit pAb), PBK (#16110-1-AP, rabbit pAb), and TMEM100 (#25581-1-AP, rabbit pAb) were obtained from Proteintech (Rosemont, IL, USA).

Techniques: Gene Expression, Expressing

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