Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Rpkm values are plotted for FOSL1 [left] and FOSL2 [right] RNA at different time points of activation (Th0) or Th17-polarization, using published RNA-seq data ( Oncotarget ). ( B ) Immunoblot images (lower panel) show FOSL1 [left] and FOSL2 [right] protein levels in Th0 and Th17-polarizing cells, over a time-course. Actin was used as loading control. Blots from three biological replicates were quantified using ImageJ and the corresponding FOSL intensity values (normalized to actin) are plotted as a line graph in the above panel. ( C ) Flow cytometry analysis of FOSL1 [left] and FOSL2 [right] expression in naive CD4+ T cells cultured for 24h, under conditions of activation (Th0), Th17-polarization, or activation in presence of the Th17-cytokines (used either alone or in combination). Bar plot shows median fluorescence intensity (MFI) values normalized to Th0, for three biological replicates. Statistical significance was calculated by comparing each condition to Th0. ( D ) Flow cytometry analysis of FOSL1 [left] and FOSL2 [right] protein levels in non-targeting (SCR) versus STAT3 KD Th17 cells, at 72h of polarization. Graph shows MFI values normalized to SCR for four biological replicates. ( E ) Naive CD4 + T cells were silenced for FOSL1 [left] or FOSL2 [right] using two different siRNAs each, and further polarized to Th17-fate for 24h. Knockdown was analyzed using immunoblotting. Representative blots for three biological replicates are shown. ( F ) ELISA was used to estimate IL-17A secretion in supernatants of FOSL1 [left] and FOSL2-silenced [right] Th17 cells, at 72h of polarization. Values were first normalized to live cell count, followed by normalization with SCR. Data represents four or five biological replicates, as indicated. Graphs in the above panels show mean ± standard error of the mean (SEM). Statistical significance is calculated using two-tailed Student’s t test (*p < 0.05; **p < 0.01, ***p < 0.001).

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Activation Assay, RNA Sequencing Assay, Western Blot, Flow Cytometry, Expressing, Cell Culture, Fluorescence, Enzyme-linked Immunosorbent Assay, Cell Counting, Two Tailed Test

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Immunoblot images show FOSL1 (above) and FOSL2 (below) protein levels in naive CD4 + T cells cultured under activation (Th0) or Th17 differentiation conditions, for the indicated time points. Actin has been used as loading control. Data represents biological replicates for . ( B ) UCSC genome browser snapshots indicate the binding of STAT3 over the promoter of FOSL2 (above panel) and not FOSL1 (below panel), in Th17 cells cultured for 0.5 h and 72h. Figures were derived using bed files of STAT3 ChIP-seq data from Tripathi et al., 2017 Cell Reports. ( C ) Nucleofection workflow for FOSL1/FOSL2 knockdown (KD). Naive CD4 + T cells were treated with FOSL1- or FOSL2-targeting siRNAs, rested for 36-40 h, and further cultured under Th17-polarizing conditions (IL-6, IL-1β and TGF-β) for 72h. ( D ) Immunoblots depict FOSL1 and FOSL2 protein levels in naive CD4 + T cells that were silenced for the respective factors and cultured under Th17-polarizing conditions for 24h. Non-targeting siRNA (Scramble or SCR) was used as nucleofection control and actin was used as loading control. Blots shown are biological replicates for .

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Western Blot, Cell Culture, Activation Assay, Binding Assay, Derivative Assay, ChIP-sequencing

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Naive CD4 + T cells were silenced for FOSL1, FOSL2 or both factors in parallel (double KD; DKD), and cultured under Th17-polarizing conditions for 24h. Total FOSL1 [left] or FOSL2 [right] protein was stained (Alexa-647) and analysed by flow cytometry. Non-targeting siRNA (SCR) was used as nucleofection control. Representative histograms for four biological replicates are shown. ( B ) Naive CD4 + T cells were treated with in-vitro transcribed GFP-FOSL1 RNA, GFP-FOSL2 RNA or both (double OE; DOE). After resting the cells for 18–20h, total FOSL1 [left] or FOSL2 [right] protein was stained (Alexa-647) and analysed by flow cytometry. GFP RNA were used as nucleofection control. Representative histograms for four biological replicates are shown. ( C and D ) Bar plot shows ELISA results for secreted IL-17A levels in supernatants of FOSL KD/DKD (panel C) or FOSL OE/DOE Th17 cells (panel D), at 72h of polarization. Values were first normalized to live cell count, and then to the respective control condition (SCR or GFP). Data represent four biological replicates. ( E and F ) qRT-PCR analysis for measurement of IL-17A [left] and IL-17F [right] RNA levels in FOSL KD/DKD (panel E) or FOSL OE/DOE Th17 cells (panel F), at 72h of polarization. Fold-change normalized to the respective controls (SCR or empty GFP) was plotted for four biological replicates. For panels C-F, plots show mean ± SEM. Statistical significance is calculated using two-tailed Student’s t test (*p < 0.05, **p < 0.01, ***p < 0.001, ****p<0.0001).

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Cell Culture, Staining, Flow Cytometry, In Vitro, Enzyme-linked Immunosorbent Assay, Cell Counting, Quantitative RT-PCR, Two Tailed Test

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A and ) FOSL KD/DKD (panel A) and FOSL OE/DOE (panel B) Th17 cells were labelled (Alexa-647) for total FOSL1 and FOSL2 protein at 24h of polarization. Expression of the corresponding factors was analyzed using flow cytometry and overlay histograms were plotted [FOSL1, left; FOSL2, right]. Figure shows biological replicates for and .

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Expressing, Flow Cytometry

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A and B ) Heatmap in panel A shows the DE genes that are more profoundly altered in FOSL DKD Th17 cells relative to the single KD controls, at 24h (above) and 72h (below) of polarization. Panel B includes the DE genes that show enhanced changes in FOSL DOE Th17 cells as compared to the single OE controls at 72h of polarization. Genes with Th17-relevance are highlighted; upregulated genes are in red, and downregulated ones are in blue. Log2fold-change was calculated relative to the respective control conditions (i.e., SCR or GFP). ( C and D ) Ingenuity pathway analysis was used to identify signaling pathways that are altered upon FOSL DKD (panel C) or DOE (panel D). ( E and F) Genome-wide expression analysis of FOSL DKD and FOSL DOE Th17 cells. Volcano plots in Panel E highlight the Th17-associated transcripts that are differentially expressed upon co-depletion of FOSL1 and FOSL2, at 24h [left] and 72h [right] of Th17 polarization. Panel F highlights the Th17-associated genes that are differentially expressed upon parallel over-expression of FOSL1 and FOSL2, at 72h of Th17 polarization. Targets with FDR ≤ 0.1 and |fold-change| ≥ 1.8 have been plotted. Upregulated genes are in red, and the downregulated ones are in blue. ( G ) Heatmap depicts the DE genes that show opposite expression changes in FOSL DKD versus DOE conditions, at the indicated time points of Th17 polarization. Th17-relevant genes have been highlighted.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Genome Wide, Expressing, Over Expression

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Immunofluorescence images showing nuclear localization of FOSL1 (red, above panel) and FOSL2 (red, below panel) in Th17 cells polarized for 72h. Lamin A/C (in green) marks the nuclear periphery, whereas phalloidin (in blue) stains the cytoplasmic actin. ( B ) ChIP-seq analysis was performed for FOSL1 and FOSL2 using Th17 cells cultured for 72h. Figures on the left show distribution of FOSL1 and FOSL2 binding sites relative to the position of the closest transcription start site (TSS). TSS is defined to be at position zero. The adjoining figure on the right is an overlay plot that compares the binding profiles of the two factors. ( C ) The topmost consensus sequences for FOSL1 and FOSL2 genomic-binding were identified using de-novo motif enrichment analysis by Homer. FOSL1 [left] and FOSL2 [right] peaks were further enriched for known TF motifs and the top six motifs identified by Homer are shown. Peaks with IDR p < 0.01 were used for motif discovery. ( D ) ChIPpeakAnno was used to determine the overlap in the genomic binding sites of FOSL1 and FOSL2 (overlap represents peaks sharing 200 bp or more). Genes neighboring to these overlying sites and differentially expressed under DKD or DOE conditions (FDR ≤ 0.1, |fold-change| ≥ 1.5) were assigned as the shared-direct targets of FOSL1 and FOSL2. Adjoining volcano plots show the logarithmic fold changes for selected shared targets (DKD [left] and DOE [right]). Downregulated genes are in blue, and upregulated ones are in red. Targets with FOSL occupancy over promoter regions (5-kb window around TSS) are highlighted in yellow. ( E ) Integrative Genomics Viewer (IGV) track snapshots show the binding overlap of FOSL1 and FOSL2 over selected Th17 genes.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Immunofluorescence, ChIP-sequencing, Cell Culture, Binding Assay

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Bar plot depicts peak-annotation results for binding sites of FOSL1 and FOSL2 in 72h Th17-polarized cells. ( B ) Genes that were co-regulated (i.e., DE under DKD or DOE conditions) and showed co-localized genomic-binding of FOSL1 and FOSL2, were annotated as their shared direct targets. Venn diagram in the figure highlights (in bold) the shared targets [DKD, left; DOE, right] that are bound by FOSL factors over putative-promoter regions (5-kb around TSS). Out of these, the Th17-relevant targets have been marked in the volcano plots of .

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Binding Assay

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Heatmap on the top shows logarithmic fold-change values for the DE genes that are oppositely regulated in FOSL DKD and BATF KD Th17 cells, at the indicated time points of polarization. Heatmap in the bottom panel depicts the DE genes that are similarly altered in FOSL DOE and BATF KD Th17 cells. Th17-relevant genes are highlighted in red. ( B ) Comparing the ChIP-seq profiles of FOSL1, FOSL2 and BATF in Th17 cells. Graph (above) shows the overlay between the peak distribution profiles of the three TFs. Bar plot (below) depicts peak-annotation results for their identified binding sites. ( C ) Heatmap with k-means clustering shows the ChIP-seq signal intensities ± 2-kb around the centers of the genomic-binding regions of FOSL1, FOSL2 and BATF. Th17-associated genes in the vicinity of the binding sites are highlighted within the respective clusters. ( D ) Venn diagram shows an overlap between the genomic binding sites of FOSL1, FOSL2 and BATF (overlap represents peaks sharing 200 bp or more). Adjoining heatmap depicts fold-change values for the gene targets that are co-bound and oppositely regulated by FOSL proteins and BATF. Genes showing shared occupancy of the three factors over putative-promoters have been marked (*asterisk). Th17-relevant targets are highlighted. ( E ) IGV track snapshots illustrate the co-localization of FOSL1, FOSL2 and BATF over selected Th17 genes. Profile of H3K27ac marks around the shared sites is shown. ( F ) Bar plot depicts immunoblot-based expression analysis of STAT4 in FOSL DKD [left] and BATF KD [right] Th17 cells, cultured for 72h. Data shows mean ± SEM for three or four biological replicates, as indicated. Statistical significance is calculated using two-tailed Student’s t test (*p < 0.05). Adjoining IGV track shows the binding overlap of FOSL1, FOSL2 and BATF, flanked by H3K27ac marks near the STAT4 locus.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: ChIP-sequencing, Binding Assay, Western Blot, Expressing, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Figure illustrates the common binding partners of FOSL1 and FOSL2 in Th17 cells (72h), based on data acquired from a parallel study of our lab ( bioRxiv ). Interactors having reported roles in T-cell function are shown. ( B ) STRING network analysis of human BATF. Width of lines between the nodes indicate confidence values for each protein-protein association. Only interactions with a minimum score of 0.7 are shown (high confidence). ( C and D ) Immunoprecipitated BATF was analyzed for its interaction with selected common binding partners of FOSL1 and FOSL2 (JUNB, SIRT-1, JUN and RUNX1), using western blotting (panel C). Additionally, BATF-interaction with STAT3 and IRF4 was analyzed to validate their previously-known association in mouse (panel D). Data is shown for three biological replicates.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Binding Assay, Cell Function Assay, Immunoprecipitation, Western Blot

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Enrichment of disease-associated SNPs (or their proxies in Caucasian populations) within FOSL1, FOSL2 and BATF genomic-binding sites, relative to random sets of background SNPs. ( B ) SNPs relevant to the study were shortlisted. Out of these, the SNPs that were functionally validated in DNA-affinity precipitation assays (DAPA) have been shown. ( C and D ) DAPA reveals the SNPs that alter the binding of FOSL1, FOSL2 or BATF to their genomic sites that were identified by ChIP-seq analysis. Wildtype (WT) oligonucleotides containing the binding motifs of these TFs (at different genomic loci), and mutant oligonucleotides harboring a SNP within the corresponding motif, were used as baits. For experimental controls, an oligonucleotide with a conserved binding sequence for BATF (BATF WT), and the corresponding mutated sequence which is known to disrupt BATF occupancy (BATF MUT) were used. Immunoblot results for the SNPs unique to FOSL1, FOSL2 and BATF (panel C), and the ones common across the three factors (panel D) are shown. Data is representative of three biological replicates.

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Binding Assay, Affinity Precipitation, ChIP-sequencing, Mutagenesis, Sequencing, Western Blot

Journal: bioRxiv

Article Title: The AP-1 factors FOSL1 and FOSL2 co-regulate human Th17 responses

doi: 10.1101/2021.04.26.441472

Figure Lengend Snippet: ( A ) Table illustrates information on the autoimmune-linked SNPs that are harbored within consensus AP-1 motifs at the shared genomic-binding sites of FOSL1, FOSL2 and BATF. The sequence logos shown have been derived from the respective TF ChIP-seq peaks using Homer. ( B and C ) DAPA analysis was performed to test the effect of selected SNPs on the DNA-binding abilities of FOSL1, FOSL2 and BATF. The immunoblot images in panels B & C show biological replicates (R1, R2, R3) for and respectively

Article Snippet: To generate linearized vectors for the IVT reaction, the T7 promoter containing plasmids: empty pGEM-GFP64A, pCMV6-AC-GFP-FOSL1 (Origene, Cat no. RG202104) and pCMV6-AC-GFP-FOSL2 (Origene, Cat no. RG204146), were in vitro digested using the restriction enzymes Spe1 (NEB, Cat no. R0133), Xma1 (NEB, Cat no. R0180) and Ssp1 (NEB, Cat no. R3132), respectively.

Techniques: Binding Assay, Sequencing, Derivative Assay, ChIP-sequencing, Western Blot

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Roles of different Ras downstream effectors in the induction of Fra-1. (A) Scheme of the activity of the Ras (V12) effector mutants. The C40, G37, and S35 effector loop mutations enable the Ras (V12) oncoprotein to activate selectively the PI3-kinase (C40)-, the RalGDS (G37)-, or the Raf (S35)-dependent cascade. (B) Immunoblotting and RT-PCR analysis of the activity of Ras (V12) effector mutants on the expression of fra-1 in thyroid cells. The fra-1/β-globin reporter construct (10 μg) was cotransfected with the empty vector (pCDNA3) or the indicated Ras expression vector (5 μg) in FRTL-5 cells. As a control of activity in the transformed cell line, the reporter construct was transfected in FRTL-5Kras cells. The total DNA was kept to 20 μg, and 3 μg of the pCMV-CAT reporter was cotransfected as an internal control for transfection efficiency. After 36 h cell extracts or total RNA was prepared. For immunoblotting (upper panel), 50 μg of cell extracts was processed as described in Materials and Methods and was probed with anti-Fra-1 antibody (Santa Cruz Biotechnology, Inc.). As a control for equal loading, the blotted proteins were stained with Red-Ponceau (not shown). For the RT-PCR (lower panel), total RNA was reverse transcribed and the 76-bp β-globin transcript was coamplified with the 346-bp CAT mRNA in the presence of [α32-P]dCTP and was analyzed by 5% PAGE. (C) Diagram of PhosphorImager quantitation (ImageQuant software) of the RT-PCR data. The relative activity of the Ras effector double mutants is expressed as a percentage of the activity of the Ras (V12) construct which resulted in the maximal stimulation of the fra-1/β-globin reporter. In the right-hand diagram the results are shown as fold induction of the reporter gene relative to its activity in FRTL-5 cells. These experiments were repeated three times with similar results. (D) In vitro MBP phosphorylation assay of ERK activation by Ras (V12) effector mutants. The Ras expression constructs or the empty vector were cotransfected in FRTL-5 cells along with the vector encoding the epitope-tagged ERK2 (pcDNA3-ERK2-HA). After 24 h cells were collected and equal amounts of cell lysates were immunoprecipitated (IP) with α-HA antibody or nonimmune serum and were subjected to in vitro phosphorylation reaction as described in Materials and Methods. Reaction products were analyzed by SDS-10% PAGE.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Activity Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Expressing, Construct, Plasmid Preparation, Transformation Assay, Transfection, Staining, Quantitation Assay, Software, In Vitro, Phosphorylation Assay, Activation Assay, Immunoprecipitation

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Effect of chemical inhibition of the MEK/ERK pathway on Fra-1 expression. FRTL-5KRas cells were treated with 10 μM U0126 (Promega) for 4, 8, or 12 h. Total RNA or whole-cell extracts were prepared from untreated FRTL-5 (lane 1) and FRTL-5KRas (lane 2) cells or from FRTL-5KRas cells treated with U0126 (lanes 3 to 5). (A) For Northern blotting, 30 μg of total RNA was analyzed by hybridization to the radiolabeled rat fra-1 cDNA probe as indicated in Materials and Methods. The ethidium bromide staining of rRNAs (28S and 18S) was utilized as a control for RNA loading. (B) For immunoblotting analysis, 50-μg samples of cell extracts were processed as described in Materials and Methods. The same membrane was first incubated with the α-Fra-1 antibody and subsequently was stripped and reprobed with α-P-ERK1/2 (New England Biolabs) as a control for the inhibitory activity of the drug. Finally, the blot was incubated with α-ERK1 antibody (New England Biolabs) as a control for equal protein loading. For the in vitro dephosphorylation reaction (B, right-hand panel) the cell extracts from untreated FRTL-5KRas cells were incubated for 2 h at 37°C with or without 20 U of CIP prior to SDS-10% PAGE and immunoblotting. Northern blotting and immunoblotting data were confirmed by multiple experiments, and similar results were obtained by using the PD 98059 MEK inhibitor.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Inhibition, Expressing, Northern Blot, Hybridization, Staining, Western Blot, Incubation, Activity Assay, In Vitro, De-Phosphorylation Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Effect of chemical inhibition of the MEK/ERK pathway on Fra-1 stability and DNA binding activity. (A) Pulse-chase analysis of the Fra-1 half-life in FRTL-5KRas cells treated with the MEK inhibitor. Cells pretreated for 30 min with 10 μM U0126 were subjected to pulse-chase labeling, as described in Materials and Methods, alone or in the presence of U0126. Control cells were treated with the vehicle (dimethyl sulfoxide). Cells were collected at the indicated time points. Whole-cell extracts were immunoprecipitated with the α-Fra-1 antibody and were analyzed by SDS-PAGE. −ab, without antibody. (B) Diagram of densitometric quantitation of the results (QuantityOne software) showing the kinetics of decay of distinct Fra-1 electrophoretic isoforms in untreated cells (upper, middle, and lower bands) compared to that of U0126-treated cells (middle and lower bands) expressed as the relative optical density of the autoradiographic image. Similar results were obtained in four different pulse-chase experiments. (C) Immunoblotting analysis of Fra-1 following DNA affinity chromatography of nuclear extracts subjected to in vitro dephosphorylation. The upper left-most panel shows 25 μg of nuclear extract from FRTL-5KRas cells incubated with 5 μg of fra-1 TRE DNA-agarose beads in the absence of competitor oligonucleotide (no competitor) or after preincubation with a 25-fold molar excess of fra-1 TRE or mut fra-1 TRE competitor oligonucleotides. The eluted TRE-bound material was subjected to SDS-10% PAGE along with the same amount of untreated proteins (input). The two upper right panels show 25 μg of nuclear extract subjected to in vitro dephosphorylation (+CIP) and compared to the untreated control (−CIP). The lower panel is on off-rate analysis of an equivalent untreated sample (25 μg) subjected to DNA affinity chromatography, as described in Materials and Methods. A 25-fold molar excess of competitor fra-1 TRE oligonucleotide was added, and the DNA-agarose-bound complex was allowed to dissociate at room temperature during the indicated time course prior to elution and SDS-PAGE. The diagram was obtained by quantitation of the chemiluminescence signal by use of the Gel-Doc image acquisition apparatus and QuantityOne software (Bio-Rad). The data, representing the average of two independent experiments, were normalized by attributing the 100% value to the first time point for each of the two electrophoretic isoforms (upper and lower). IP, immunoprecipitation.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Inhibition, Binding Assay, Activity Assay, Pulse Chase, Labeling, Immunoprecipitation, SDS Page, Quantitation Assay, Software, Western Blot, Affinity Chromatography, In Vitro, De-Phosphorylation Assay, Incubation

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Characterization of the Ras-responsive element of the fra-1 gene. (A) Schematic representation of the fra-1/β-globin reporter constructs. The −710 to +2741 region of the rat fra-1 gene was fused to a portion of the rabbit β-globin gene (exons 2 and 3, black arrow) to generate a stable chimeric transcript. The fra-1-derived sequence includes the (−710) promoter region, the first exon, the first intron, and part of the second exon (white boxes). The first intron contains two AP-1-like sites (empty circles) and an AP-1 consensus element (fra-1 TRE, black circle) deleted in the mutated version (fra-1/β-globin-Δ). For stable clones, FRTL-5 and FRTL-5KRas cell lines were transfected with the DNA vector encoding the selectable marker (pCMV-Neo), alone (MOCK clones) or in combination with the linearized fra-1/β-globin wild type (wt) or the fra-1/β-globin-Δ construct. After selection in G418 (800 μg/ml; Calbiochem), pools of stably transfected cell clones were derived from a similar number (∼80 to 100) of G418-resistant colonies. The comparable copy number of stably integrated constructs in each pool of G418-resistant transfectants was verified by Southern blot hybridization with a rabbit β-globin probe detecting an internal 3.8-kb EcoRI fragment (data not shown). (B) RT-PCR analysis of the basal and serum-inducible expression of the chimeric transcript in the FRTL-5- and FRTL-5KRas-derived pools of transfected cell clones. Cells were maintained in normal growth conditions before stimulation with 20% fetal calf serum for 3 h. Total RNA was prepared from MOCK cell clones (lanes 1 and 6) and from cell clones expressing the wild type (lanes 2 to 3 and 7 to 8) or the deletion-containing (lanes 4 to 5 and 9 to 10) chimeric minigene. For RT-PCR, 2 μg of DNase-treated RNA was reverse transcribed as described in Materials and Methods and the 76-bp β-globin cDNA fragment was amplified together with the 370-bp HPRT cDNA as an internal control. PCR products were resolved by 4% agarose gel electrophoresis. (C and D) Serum induction of fra-1 and mRNA and protein binding to fra-1 TRE. FRTL-5 and FRTL-5KRas cells were maintained in complete medium or were switched into a medium containing 0.5% FBS for 48 h, and then 20% FBS was added 3 h prior to harvest. Total RNA or nuclear proteins were extracted from cycling (lanes 1 and 4), serum-arrested (lanes 2 and 5), and serum-stimulated (lanes 3 and 6) cells. (C) For Northern blot analysis, 30 μg of total RNA/sample was hybridized to the radiolabeled rat fra-1 cDNA probe, as indicated in Materials and Methods. Equal loading was verified by ethidium bromide staining of rRNAs (bottom panel). (D) EMSA of the serum-induced binding to the fra-1 TRE. Nuclear proteins were incubated with the 5′-end-labeled fra-1 TRE oligonucleotide before 5% polyacrylamide gel retardation. The arrows indicate the oligoncleotide/AP-1 complex. All results were confirmed in at least three independent experiments.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Construct, Derivative Assay, Sequencing, Clone Assay, Transfection, Plasmid Preparation, Marker, Selection, Stable Transfection, Southern Blot, Hybridization, Reverse Transcription Polymerase Chain Reaction, Expressing, Amplification, Agarose Gel Electrophoresis, Protein Binding, Northern Blot, Staining, Binding Assay, Incubation, Labeling, Electrophoretic Mobility Shift Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: In vitro analysis of the fra-1 TRE binding complex in the cell clones expressing the MEK and Rac constitutive derivatives. (A) EMSA of AP-1 binding to the fra-1 TRE oligonucleotide in the FRTL-5-derived cell clones expressing the constitutively active form of MEK and/or Rac (FRTL-5MEK, FRTL-5Rac, and FRTL-5MEK/Rac). Nuclear proteins (3 μg) were incubated with the labeled fra-1 TRE oligonucleotide before PAGE. (B) Immunoblotting analysis of Fra-1 and Jun proteins in the normal (FRTL-5), Ha-ras- or Ki-ras-transformed (FRTL-5Hras and FRTL-5KRas), and stably transfected cell lines (FRTL-5MEK, FRTL-5Rac, and FRTL-5MEK/Rac). Nuclear proteins were separated by SDS-PAGE (20 μg/lane) and were transferred to a polyvinylidene difluoride membrane. Western blots were sequentially incubated with anti-Fra-1, anti-c-Jun, anti-JunB, and anti-JunD followed by anti-α-tubulin antibodies as a control for equal loading. The arrows indicate the major isoforms of Fra-1 (38 kDa), c-Jun (39 kDa), and JunB (38 kDa) and the two isoforms of JunD (41 and 37 kDa). (C) Antibody supershift analysis of the complex bound to the fra-1 TRE oligonucleotide. After protein binding to the labeled oligonucleotide, nuclear extracts were incubated for 3 h with the indicated antibodies before gel retardation. Different autoradiographic exposures were chosen for different panels (FRTL-5, 24 h; FRTL-5MEK and FRTL-5 MEK/Rac, 16 h; FRTL-5Kras, 8 h) to allow the optimal visualization of supershifted complexes. The arrows indicate the AP-1/oligonucleotide complex, while the asterisks refer to the supershifted ternary complexes. In vitro binding and supershift assays were repeated at least twice with comparable results.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: In Vitro, Binding Assay, Clone Assay, Expressing, Derivative Assay, Incubation, Labeling, Western Blot, Transformation Assay, Stable Transfection, Transfection, SDS Page, Protein Binding, Electrophoretic Mobility Shift Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: In vivo occupancy of the fra-1 TRE in normal and ras-transformed cells. (A) Radioactive PCR on naked DNA from normal and transformed cells as a control for the amplification products. Chromosomal DNA extracted from FRTL-5 and FRTL-5KRas was analyzed by radioactive PCR. The fra-1-Int primers amplified a 594-bp DNA region containing the intronic fra-1 TRE, while the primers for the HPRT gene gave rise to a 558-bp PCR product. (B) ChIp of the +178- to +772-nucleotide region encompassing the fra-1 TRE. Following in vivo formaldehyde cross-linking, chromatin extracted from FRTL-5 and FRTL-5KRas cells was immunoprecipitated with anti-Fra-1 or anti-acetylated histone H3 antibodies (α-AcH3). Following reversal of cross-linking, the purified DNA fragments were amplified by radioactive PCR with the fra-1-Int and HPRT primers as a control for equal input. The products were resolved by native gel electrophoresis and were detected by autoradiography. (C) The radioactive signal of the amplification products was quantified by PhosphorImager (with ImageQuant software), normalized for the HPRT internal control and expressed as relative to the no-antibody (−Ab)/FRTL-5 control sample. The reproducibility of these results was confirmed by repeating the ChIp experiments two times independently.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: In Vivo, Transformation Assay, Amplification, Immunoprecipitation, Purification, Nucleic Acid Electrophoresis, Autoradiography, Software

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Analysis of the Fra-1-dependent transactivation in response to the ras oncogene and transactivation activity of Gal4/Fra-1 fusion proteins. The reporter plasmid FrLuc (5 μg) was coexpressed with the vectors (10 μg) expressing the Gal4/Fra-1, GAL4/Fra-1-ΔZip, GAL4/c-Fos, or GAL4/c-Fos-insZip chimeric protein, along with the pCDNA3-Ras (V12) expression vector (5 μg) or the pCDNA3 empty vector. As an internal control, a vector encoding Renilla Luciferase (0.5 μg) was cotransfected. Thirty-six hours after transfection cells were harvested and assayed for both firefly and Renilla luciferase activities (Dual-Luciferase Reporter Assay system) to allow for normalizing of the FrLuc reporter activity for variations of transfection efficiency. The diagram shows the relative luciferase activity in the absence (hatched boxes) or presence (black boxes) of pCDNA3-Ras (V12). The results represent the average of three independent experiments, with the error bars indicating the standard errors.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Activity Assay, Plasmid Preparation, Expressing, Luciferase, Transfection, Reporter Assay

Journal:

Article Title: Accumulation of Fra-1 in ras -Transformed Cells Depends on Both Transcriptional Autoregulation and MEK-Dependent Posttranslational Stabilization

doi: 10.1128/MCB.23.12.4401-4415.2003

Figure Lengend Snippet: Model of multistep positive autoregulation of Fra-1 in ras-transformed cells. The thickness of the arrows originating from the constitutively GTP-bound oncogene (Ras*) indicates the relative contribution of different Ras-dependent pathways in the activation of the fra-1 promoter. Following ERK-dependent phosphorylation, Fra-1 is stabilized in transformed cells and interacts with Jun family partners. The Jun/Fra-1 heterodimer can activate fra-1 gene transcription by binding the autoregulatory site in its first intron (fra-1 TRE) and recruiting a transcriptional coactivator (CBP/p300). White boxes, fra-1 exons; thick black line, fra-1 5′-flanking and first intron; grey line, fra-1 TRE; trash bin, degradation apparatus.

Article Snippet: Binding of annealed, double-stranded biotinylated fra-1 TRE oligonucleotide to streptavidin beads (Pierce) was performed according to the manufacturer's instructions. (ii) fra-1 TRE DNA affinity chromatography. fra-1 TRE DNA affinity chromatography was performed as follows.

Techniques: Transformation Assay, Activation Assay, Binding Assay

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: (A) MCF10A, MDA-MB-468, and MDA-MB-231cells were subjected to serum starvation, then stimulated with serum for the times indicated in the figure (h). Cells were harvested and protein levels were detected using western blot. X = exponentially growing cells. The figure is representative of more than three independent experiments. (B) Fra-1 protein levels were analyzed in a panel of TNBC cell lines. X = exponential growth 0 = serum starvation for 48 hours. 8 = 8 hours of serum stimulation.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Western Blot

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: Colon, lung, prostate, and melanoma cancer cell lines were analyzed for Fra-1 protein by western blotting. X = exponential growth in complete medium. 0 = serum starvation for 48 hours. 8 = 8 hours of serum stimulation.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Western Blot

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: MDA-MB-231 cells were infected with a retroviral vector encoding a doxycycline inducible Flag-AFos gene as described in material and methods. Transfected cells were divided into two groups; non-induced Dox (−) or induced Dox (+). (A) A-Fos and Fra-1 protein levels were examined by western blotting. The figure is representative of 3 independent experiments (B) Cells were treated +/− Dox then subjected to serum starvation and nocodazole treatment as described in . The data shown is representative of three independent experiments.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Infection, Plasmid Preparation, Transfection, Western Blot

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: (A) The effects of A-Fos expression on the ability of the MDA-MB-231 cells to grow in the presence and absence of serum were analyzed for several passages. Cells were plated at density of 3×10 5 with and without Dox and/or serum. After 72 hours cells were counted and re-plated at the same density, and this process was repeated for 3 passages. (B) The effect of A-Fos on the migration of MDA-MB-231 cells in the presence and absence of serum using the wound healing assay *= significant (p<0.05), the supplementary image represents migration in presence of serum. (C) Cells were infected with a retroviral vector encoding a scrambled sequence (Scramble shRNA) or Fra-1 shRNA (Fra-1 shRNA-8). Protein lysates were analyzed by western blot to detect Fra-1 expression (left). Cell proliferation was compared in absence (96 hour SF) and presence (96 hours of serum) of serum using CCK8 cell counting kit. The (0 hour) represents the absorbance at time of plating (Right).

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Expressing, Migration, Wound Healing Assay, Infection, Plasmid Preparation, Sequencing, shRNA, Western Blot, Cell Counting

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: (A) Using a transwell system MCF10A cells were cultured in the upper chamber with MDA-MB-231 cells or serum free medium in the lower chamber, then the levels of Fra-1 protein in MCF10A cells were examined. (B) Conditioned medium from serum starved MDA-MB-231 cells was added to serum starved MCF10A cells for 6 hours then Fra-1 protein levels were examined. Blots were quantified using Image J software. (C) MDA-MB-468 cells were incubated with CM or cocultured with MDA-MB-231 cells in a transwell chamber as described in materials and methods, then Fra-1 expression was examined. (D) CM from MDA-MB-231/Flag-AFos cells incubated in the presence (Dox(+)) and absence (Dox(−)) of doxycycline was added to MCF10A cells, and Fra-1 protein levels were examined. The results shown are representative of 3 experiments. (E) MCF10A cells were incubated with MDA-MB-231 CM with and without PD98059 and U0126 (MEK inhibitors). The cells were harvested after 6 hours and the level of Fra-1 was detected by western blotting.

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Cell Culture, Software, Incubation, Expressing, Western Blot

Journal: bioRxiv

Article Title: The role of AP-1 in self-sufficient proliferation and migration of cancer cells and its potential impact on an autocrine/paracrine loop

doi: 10.1101/271536

Figure Lengend Snippet: (A) MCF10A cells were cultured on an 8μm pore transwell insert with or without MDA-MB-231 cells in the lower chamber and migrated MCF10A cells on the lower side of the membrane were fixed and stained as described in materials and methods. (B) MCF10A cells were cultured in the upper chamber of transwell inserts in MDA-MB-231 CM versus serum free medium (left graph), or MCF10A cells were cultured in the upper chamber in serum free medium with the lower chamber filled with either CM or serum free medium (right graph). Cells were counted per HPF. from 3 independent experiments and a representative picture of the migrated cells is shown. (C) MDA-MB-468 cells were co-cultured with MDA-MB-231/Flag-AFos cells in a transwell system in the presence (Dox(+)) and absence (Dox(−)) of doxycycline. (D) A wound healing migration assay was used to measure the effect of CM on MCF10A cell migration. The graph shows percent closure after 24 hours. (E) MCF10A cells were infected with scrambled shRNA virus (vector) or two different Fra-1 shRNA viruses and Fra-1 levels were compared (Left). A wound healing assay was carried out to measure the effect of conditioned medium on MCF10A migration (Right).

Article Snippet: The following antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA): Anti-Fra-1 (sc-183) (sc-605x) (sc-28310), anti-c-Fos (sc-52), anti-c-Jun (sc-44) (sc-74543), anti-Jun D (sc-74) (sc-271938), anti-α-Tubulin (sc-8035), normal rabbit IgG (sc-2027), and mouse-IgG k BP-HRP (sc-516102).

Techniques: Cell Culture, Staining, Migration, Infection, shRNA, Plasmid Preparation, Wound Healing Assay

Journal: International Journal of Molecular Sciences

Article Title: Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression

doi: 10.3390/ijms23041955

Figure Lengend Snippet: FOSL1 mediates A1AT knockdown-induced syncytialization of trophoblasts. ( a ) Venn diagram showing the numbers of upregulated DEGs and of genes encoding proteins that may be capable of binding to the ERVFDR-1 or CGB promoter. ( b ) Immunoblotting showing the protein levels of FOSL1 in lysates from A1AT-KD and A1AT-OE BeWo cells. GAPDH served as the loading control. ( c ) Expression of FOSL1 in FOSL1-OE BeWo cells by Immunoblotting (left) and qPCR (right). Results shown are the means ± SEMs of three independent experiments. ** p < 0.01. ( d ) Expression of syncytialization markers in FOSL1-OE BeWo cells treated with Db (0.5 μM) by qPCR. Values are means ± SEMs of three independent experiments. ** p < 0.01. ( e ) Visualization of syncytialization by immunostaining cells with anti-E-cadherin antibody (red) and DAPI (blue). Representative pictures are shown, with syncytialized cells marked with a stippled line. Scale bar = 50 μm. ( f ) Expression of mRNAs encoding inflammatory cytokines in FOSL1-OE BeWo cells. GAPDH was used as the loading control. Results are reported as the means ± SEMs of three independent experiments. * p < 0.05, ** p < 0.01. ( g ) Expression of mRNAs encoding inflammatory cytokines in A1AT-KD BeWo cells treated with SP600125 (SP, 20 μM) or SB203580 (SB, 20 μM) for 24 h. GAPDH was used as the loading control. Results are reported as the means ± SEMs of three independent experiments. ** p < 0.01.

Article Snippet: The FOSL1 expression vector pRP (pDNA VB900007-4466mvj) was purchased from VectorBuilder (Chicago, IL, USA).

Techniques: Binding Assay, Western Blot, Expressing, Immunostaining

Journal: International Journal of Molecular Sciences

Article Title: Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression

doi: 10.3390/ijms23041955

Figure Lengend Snippet: Schematic diagram illustrating the proposed A1AT/FOSL1 signaling in trophoblasts. A1AT controls the phosphorylation of p38MAPK and JNK, which may result in upregulation of several inflammatory cytokines, whereas p38MAPK regulates syncytialization by inducing FOSL1. P: phosphorylation. Red arrow: expression levels.

Article Snippet: The FOSL1 expression vector pRP (pDNA VB900007-4466mvj) was purchased from VectorBuilder (Chicago, IL, USA).

Techniques: Expressing

Journal: International Journal of Molecular Sciences

Article Title: Alpha 1 Antitrypsin Regulates Trophoblast Syncytialization and Inflammatory Factor Expression

doi: 10.3390/ijms23041955

Figure Lengend Snippet: Primers for real-time qPCR analyses.

Article Snippet: The FOSL1 expression vector pRP (pDNA VB900007-4466mvj) was purchased from VectorBuilder (Chicago, IL, USA).

Techniques: Sequencing

Journal: Gastroenterology

Article Title: Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

doi: 10.1053/j.gastro.2018.05.041

Figure Lengend Snippet: (A) Small interfering RNA (siRNA) knockdown of FOSL1. Western blot analysis showing FOSL1 knockdown and its effect on PAF1 in CSE-treated and untreated HPNE and Capan1 cells. GAPDH was used as loading control. (B) Chromatin immunoprecipitation (ChIP) assays were performed using chromatin from CSE exposed cells and the control IgG or phospho-FOSL1 antibodies. Phospho-FOSL1 enriched DNA was used in PCR assay using primers specific to FOSL1 or AP1 binding sites (see Supplementary Figure 8) on the promoter region of PAF1 gene. Chip DNA PCR product was resolved on 2% agarose gel, and the DNA bands for BS1-9 were shown. (C) Left: Representative images of immunohistochemistry for FOSL1 in pancreatic tissues obtained from cigarette smoke-exposed control and KrasG12D Pdx-Cre mice. Scale bar, 100 μm. Middle: Immunofluorescence staining for PAF1 (stained in red) and p-FOSL1 (stained in green) in cigarette smoke exposed control and KrasG12D Pdx-Cre tissues (Nuclei were stained with DAPI). Scale bar, 50 μm. Right: Bar chart represents the H score of FOSL1 staining. Data represent mean ± SD (n=6). (p values were calculated by Student’s t test). *p < 0.05, **p < 0.01. (D) Left: Immunohistochemical staining for FOSL1 and PAF1 in human PDAC tissues (with and without smoking history) and in normal pancreas. Scale bar, 100 μm. Right: Confocal images showing the co-expression of FOSL1 (stained in red) and PAF1 (stained in green) in these tissues. Scale bar, 100 μm. Nuclei were stained in blue using DAPI. Bar charts below show quantification of FOSL1 and PAF1 staining in normal pancreas (n=15), PDAC without (n=15) and with (n=15) smoking history. Data represent mean ± SD. (p values were calculated by Student’s t test). ***p < 0.001. (E) Immunoblotting assays for CHRNA7, p-ERK1/2, ERK1/2, p-FOSL1, FOSL1, p-cJun and cJun signaling molecules in CSE-treated cells as compared to untreated controls. (F) Immunoblotting assays for p-ERK1/2, ERK1/2, p-FOSL1, FOSL1, PAF1 in CSE exposed HPNE and Capan1 cells with or without ERK1/2 inhibition using PD98059. (G) Small interfering RNA (siRNA) knock down of nACHRα7 in CSE treated cells. Western blot analysis showing the effect of nACHRα7 knockdown on p-FOSL1 and PAF1. (E–G) β-actin was used as loading control. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

Article Snippet: FOSL1 (ID 8061) Trilencer-27 Human siRNA , Origene , SR322327.

Techniques: Small Interfering RNA, Knockdown, Western Blot, Control, Chromatin Immunoprecipitation, Binding Assay, Agarose Gel Electrophoresis, Immunohistochemistry, Immunofluorescence, Staining, Immunohistochemical staining, Expressing, Inhibition

Journal: Gastroenterology

Article Title: Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

doi: 10.1053/j.gastro.2018.05.041

Figure Lengend Snippet: HPNE and Capan1 cells were left untreated or treated with Nicotine (2μM), NNK (2μM) and NNN (2μM) for 80 days. (A) Left: Flow cytometry analysis of AF content. Right: Percentage of AF+ population in HPNE and Capan1 cells exposed to cigarette smoke components as compared to respective controls. Data represent mean ± SD (n=3) (p values were calculated by Student’s t test). *p < 0.05, **p < 0.01. (B) Sphere formation assay was performed on HPNE and Capan1 cells exposed to cigarette smoke components. 2000 cells/well were seeded in 96 well ultra-low attachment plates in stem cell medium. Left: Morphology of 10-day old spheres. Scale bar: 200μm. Right: Number of spheres per 2000 cells in HPNE and Capan1 cells exposed to cigarette smoke components as compared to untreated controls. Data represent mean ± SD (n=6). (p values were calculated by Student’s t test). ***p < 0.001. (C–D) Immunoblotting assay for CHRNA7, p-FOSL1, PAF1, KLF4 and SOX9. β-actin was used as loading control.

Article Snippet: FOSL1 (ID 8061) Trilencer-27 Human siRNA , Origene , SR322327.

Techniques: Flow Cytometry, Tube Formation Assay, Western Blot, Control

Journal: Gastroenterology

Article Title: Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

doi: 10.1053/j.gastro.2018.05.041

Figure Lengend Snippet: (A) HPNE and Capan1 cells were left untreated or treated with CSE (1%) for 80 days. Immunoblot analysis showing the protein expression levels of PAF1 complex (PAF1C) molecules along with serine 2 phosphorylated RNA polymerase II (S2-Phos RNA Pol II) and PHF5A. β-actin was used as loading control. (B) Left: Representative images of IHC for PHF5A in pancreatic tissues obtained from 20 weeks cigarette smoke-exposed control and KrasG12D Pdx-Cre mice. Scale bar, 100 μm. Right: Immunofluorescence staining for PAF1 (stained in red) and PHF5A (stained in green) in pancreas of cigarette smoke-exposed control and KrasG12D Pdx-Cre mice models (Nuclei were stained with DAPI). Scale bar, 50 μm. Bar chart below (C) represents the H score of PHF5A staining in the pancreas of cigarette smoke exposed mice. Data represent mean ± SD (n=6). (p values were calculated by Student’s t test). *p < 0.05, **p < 0.01. (D) Immunofluorescence staining for PAF1 and PHF5A on HPNE and Capan1 cells exposed to CSE. Co-expression of PAF1 with PHF5A was shown. Nuclei were stained with DAPI. (E) Immunoblots showing that PAF1 interacts with PHF5A in untreated and CSE treated HPNE and Capan1 cells. Pull down was performed using PAF1 antibody, and immunoprecipitates were probed with PHF5A antibody. IgG control and input, 10% of total lysate, were used as negative and positive controls, respectively. (F) Schematic showing overall mechanism involved in the cigarette smoke mediated induction of pancreatic stemness. Exposure of human pancreatic ductal cells and cancer cells to cigarette smoke and its components increases induce stemness by increasing PAF1 through CHRNA7-ERK1/2-AP1 (FOSL1-cJUN) signaling pathway. Cigarette smoke induced PAF1 and PHF5A interacts and form PAF1-PHF5A complex, required for the activation of stemness or cancer stemness genes.

Article Snippet: FOSL1 (ID 8061) Trilencer-27 Human siRNA , Origene , SR322327.

Techniques: Western Blot, Expressing, Control, Immunofluorescence, Staining, Activation Assay

Journal: Gastroenterology

Article Title: Cigarette Smoke Induces Stem Cell Features of Pancreatic Cancer Cells via PAF1

doi: 10.1053/j.gastro.2018.05.041

Figure Lengend Snippet:

Article Snippet: FOSL1 (ID 8061) Trilencer-27 Human siRNA , Origene , SR322327.

Techniques: DC Protein Assay, Sample Prep, Staining, Blocking Assay, Plasmid Preparation, Software, Imaging

Journal: Cellular and molecular life sciences : CMLS

Article Title: TRPM7 transactivates the FOSL1 gene through STAT3 and enhances glioma stemness.

doi: 10.1007/s00018-023-04921-6

Figure Lengend Snippet: Fig. 6 ChIP-qPCR analysis of STAT3 direct binding to the FOSL1 promoter. ChIP-qPCR results were analyzed by evaluating signal of enrichment over noise normalized to input. A172 cells (A) and PDX-L14 cells (B) were transduced with STAT3-CA and vector. DNA levels were normalized to the relative inputs (n = 3 independent experiments; **p < 0.001 among groups by one-way ANOVA). The representative nuclear staining of FOSL1 in A172 cells was shown in C, magnification ×40

Article Snippet: FOSL1 human shRNA lentivirus particles (sh FOSL1 lentiviral particles) were purchased from Origene (TL312944V, Rockville, MD).

Techniques: ChIP-qPCR, Binding Assay, Transduction, Plasmid Preparation, Staining