Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of TNFSF13B mRNA and TNFSF13B pre-mRNA in THP1 cells that were either untreated (Control) or treated with IR (5 Gy) and analyzed 6 days later. The levels of ACTB mRNA, encoding the housekeeping protein ACTB (β-Actin), were measured and used for data normalization. ( B ) Gene Ontology terms enriched after proteomic analysis of THP-1 cells that were rendered senescent by IR relative to untreated control cells. Proteomic analysis is available as . GO terms were sorted by p-value ranking (EnrichR analysis). ( C ) Schematic of conserved TF-binding sites on the TNFSF13B promoter, as analyzed using ECR browser and rVista 2.0. ( D ) Schematic of the interferon response triggered by DNA damage, highlighting the targets affected by the different interferon inhibitors used in our study ( ; ; ), created using BioRender. ( E ) RT-qPCR analysis of the levels of TNFSF13B mRNA, TNFSF13B pre-mRNA, and IRF1 mRNA in THP-1 cells after treatment with the inhibitors of the interferon pathway indicated in ( D ) [Ruxolitinib (Ruxo, 1 μM), MRT67307 (MRT67, 5 μM), and BX-795 (5 μM)] or with control vehicle DMSO for 5 days after treatment with IR. IRF1 mRNA was included as a positive control of interferon-stimulated mRNAs. ACTB mRNA levels were measured and used for data normalization. ( F ) RT-qPCR analysis of the expression levels of TNFSF13B and p21 mRNAs and other interferon-regulated transcripts ( IRF1 , IRF2 , IRF8 , and BLIMP1 mRNAs) in THP-1 cells that were left untreated or were treated with IR and cultured for the indicated times. ACTB mRNA levels were used for data normalization. ( G ) Western blot analysis of the levels of IRF1 and IRF2 in the cytoplasmic and nuclear fractions of THP-1 cells that were left untreated or were irradiated and assayed 6 days later. The cytoplasmic protein tubulin, the nuclear protein PARP1, and the senescence-associated protein p21 were included in the analysis. Ponceau staining of the transfer membrane was included to monitor differences in loading and transfer among samples. ( H ) ChIP-qPCR analysis of endogenous IRF1 in control (proliferating) or IR-treated THP-1 cells (5 Gy IR, assayed 72 hr later). The purified DNA was analyzed by qPCR using primers binding to TNFSF13B promoter, GAPDH promoter (negative control) and MX1 promoter (positive control). Data are presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative 2 –∆∆Ct method normalized to the percentage of the input. ( I ) RT-qPCR analysis of the levels of IRF1 mRNA and TNFSF13B pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 hr after either no treatment (-) or treatment with IR. ACTB mRNA levels were used for data normalization. ( J ) RT-qPCR analysis of the levels of TNFSF13B pre-mRNA in WI-38 cells transfected with CTRLsi or IRF1si and either left untreated (-) or treated with IR (10 Gy) and assayed 5 days later. ACTB mRNA levels were measured and used for data normalization. Significance (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001) was assessed by Student’s t -test. Figure 2—source data 1. Proteomic analysis performed in control THP-1 cells treated with IR. Figure 2—source data 2. Uncropped western blot images for .

Article Snippet: Pools #1 ( ) were from Santa Cruz Biotechnology (IRF1 siRNA, cat. sc-35706, and control siRNA, cat. sc-37007) and pools #2 ( ) were from Horizon Discovery (IRF1 siRNA, cat. M-011704-01-0005, and control siRNA, cat. D-001206-14-20).

Techniques: Quantitative RT-PCR, Control, Binding Assay, Positive Control, Expressing, Cell Culture, Western Blot, Irradiation, Staining, Membrane, ChIP-qPCR, Purification, Negative Control, Transfection

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) Measurement of mRNA stability in THP-1 cells. The levels of TNFSF13B mRNA ( top ) and labile control MYC mRNA ( bottom ) remaining (% of untreated) after treating cells with the inhibitor of RNA polymerase II actinomycin D (ActD) (2 μg/ml); THP-1 cells were either untreated or had been treated with 5 Gy IR 6 days earlier, and mRNA levels were measured by RT-qPCR analysis of total RNA collected at the times shown after addition of ActD. RNA levels in each sample were normalized to 18 S rRNA levels. Half-lives are estimated as the time needed for each mRNA to reach one-half (50%, discontinuous line) of their abundance at time 0 hr. ( B ) RT-qPCR analysis of the levels of TNFSF13B mRNA and pre-mRNA in WI-38 cells that were either untreated or treated with a single dose of IR (10 Gy) and cultured for 10 days ( top ) or in WI-38 cells that were proliferating (PDL 24) or replicatively senescent (PDL50) ( bottom ). ACTB mRNA levels were calculated and used for sample normalization. ( C,D ) Stability of TNFSF13B mRNA ( top ) and MYC mRNA ( bottom ) as determined following treatment with ActD as explained in ( A ), in WI-38 cells rendered senescent by exposure to IR (10 Gy) and culture for 10 days ( C ) or by replicative exhaustion (PDL50) ( D ). 18 S rRNA levels were calculated and used for sample normalization. ( E ) RT-qPCR analysis of the levels of IRF1 mRNA and TNFSF13B pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 hr after either no treatment (-) or treatment with IR. ACTB mRNA levels were used for sample normalization. ( F ) RT-qPCR analysis showing the levels of TNFSF13B and IRF1 mRNAs in WI-38 cells treated with IR (10 Gy) and collected at the indicated times. ( G ) Western blot analysis of the levels of IRF1 protein in WI-38 fibroblasts that were proliferating (PDL24), rendered senescent following exposure to IR (10 Gy) and cultured for 10 days, or rendered senescent through replicative exhaustion (PDL50); p21 was used as positive marker for senescence and GAPDH as loading control. Significance (*, p<0.05; **, p<0.01; ***, p<0.001) was assessed with Student’s t test. Figure 2—figure supplement 1—source data 1. Uncropped western blots.

Article Snippet: Pools #1 ( ) were from Santa Cruz Biotechnology (IRF1 siRNA, cat. sc-35706, and control siRNA, cat. sc-37007) and pools #2 ( ) were from Horizon Discovery (IRF1 siRNA, cat. M-011704-01-0005, and control siRNA, cat. D-001206-14-20).

Techniques: Control, Quantitative RT-PCR, Cell Culture, Transfection, Western Blot, Marker

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: Proposed model for the regulation and role of BAFF in senescence (created using BioRender). Following DNA damage, the TF interferon-regulated factor IRF1 induces the transcription of TNFSF13B mRNA. The protein BAFF is translated and inserted into the plasma membrane, where it can be further processed into a secreted form. Both forms of BAFF are increased in senescence, and both have been previously reported to be functional and capable of activating BAFF receptors (BAFFR, TACI, BCMA), which in turn stimulate stress-related pathways in a cell type-dependent manner, with a predominant activation of the NF-κB pathway in monocytic-like cells, and the p53 pathway in primary fibroblasts. Therefore, BAFF may have pleiotropic actions on senescence-associated phenotypes in different cell types. We propose that BAFF is a novel biomarker of senescence and a regulator of different senescence traits.

Article Snippet: Pools #1 ( ) were from Santa Cruz Biotechnology (IRF1 siRNA, cat. sc-35706, and control siRNA, cat. sc-37007) and pools #2 ( ) were from Horizon Discovery (IRF1 siRNA, cat. M-011704-01-0005, and control siRNA, cat. D-001206-14-20).

Techniques: Clinical Proteomics, Membrane, Functional Assay, Activation Assay, Biomarker Discovery

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet:

Article Snippet: Pools #1 ( ) were from Santa Cruz Biotechnology (IRF1 siRNA, cat. sc-35706, and control siRNA, cat. sc-37007) and pools #2 ( ) were from Horizon Discovery (IRF1 siRNA, cat. M-011704-01-0005, and control siRNA, cat. D-001206-14-20).

Techniques: Recombinant, Over Expression, Plasmid Preparation, Sequencing, Control, Staining, Luminex, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Electroporation, Software

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of TNFSF13B mRNA and TNFSF13B pre-mRNA in THP1 cells that were either untreated (Control) or treated with IR (5 Gy) and analyzed 6 days later. The levels of ACTB mRNA, encoding the housekeeping protein ACTB (β-Actin), were measured and used for data normalization. ( B ) Gene Ontology terms enriched after proteomic analysis of THP-1 cells that were rendered senescent by IR relative to untreated control cells. Proteomic analysis is available as . GO terms were sorted by p-value ranking (EnrichR analysis). ( C ) Schematic of conserved TF-binding sites on the TNFSF13B promoter, as analyzed using ECR browser and rVista 2.0. ( D ) Schematic of the interferon response triggered by DNA damage, highlighting the targets affected by the different interferon inhibitors used in our study ( ; ; ), created using BioRender. ( E ) RT-qPCR analysis of the levels of TNFSF13B mRNA, TNFSF13B pre-mRNA, and IRF1 mRNA in THP-1 cells after treatment with the inhibitors of the interferon pathway indicated in ( D ) [Ruxolitinib (Ruxo, 1 μM), MRT67307 (MRT67, 5 μM), and BX-795 (5 μM)] or with control vehicle DMSO for 5 days after treatment with IR. IRF1 mRNA was included as a positive control of interferon-stimulated mRNAs. ACTB mRNA levels were measured and used for data normalization. ( F ) RT-qPCR analysis of the expression levels of TNFSF13B and p21 mRNAs and other interferon-regulated transcripts ( IRF1 , IRF2 , IRF8 , and BLIMP1 mRNAs) in THP-1 cells that were left untreated or were treated with IR and cultured for the indicated times. ACTB mRNA levels were used for data normalization. ( G ) Western blot analysis of the levels of IRF1 and IRF2 in the cytoplasmic and nuclear fractions of THP-1 cells that were left untreated or were irradiated and assayed 6 days later. The cytoplasmic protein tubulin, the nuclear protein PARP1, and the senescence-associated protein p21 were included in the analysis. Ponceau staining of the transfer membrane was included to monitor differences in loading and transfer among samples. ( H ) ChIP-qPCR analysis of endogenous IRF1 in control (proliferating) or IR-treated THP-1 cells (5 Gy IR, assayed 72 hr later). The purified DNA was analyzed by qPCR using primers binding to TNFSF13B promoter, GAPDH promoter (negative control) and MX1 promoter (positive control). Data are presented as fold enrichment of the antibody signal versus the negative control IgG using the comparative 2 –∆∆Ct method normalized to the percentage of the input. ( I ) RT-qPCR analysis of the levels of IRF1 mRNA and TNFSF13B pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 hr after either no treatment (-) or treatment with IR. ACTB mRNA levels were used for data normalization. ( J ) RT-qPCR analysis of the levels of TNFSF13B pre-mRNA in WI-38 cells transfected with CTRLsi or IRF1si and either left untreated (-) or treated with IR (10 Gy) and assayed 5 days later. ACTB mRNA levels were measured and used for data normalization. Significance (ns, not significant; *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001) was assessed by Student’s t -test. Figure 2—source data 1. Proteomic analysis performed in control THP-1 cells treated with IR. Figure 2—source data 2. Uncropped western blot images for .

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Quantitative RT-PCR, Control, Binding Assay, Positive Control, Expressing, Cell Culture, Western Blot, Irradiation, Staining, Membrane, ChIP-qPCR, Purification, Negative Control, Transfection

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) Measurement of mRNA stability in THP-1 cells. The levels of TNFSF13B mRNA ( top ) and labile control MYC mRNA ( bottom ) remaining (% of untreated) after treating cells with the inhibitor of RNA polymerase II actinomycin D (ActD) (2 μg/ml); THP-1 cells were either untreated or had been treated with 5 Gy IR 6 days earlier, and mRNA levels were measured by RT-qPCR analysis of total RNA collected at the times shown after addition of ActD. RNA levels in each sample were normalized to 18 S rRNA levels. Half-lives are estimated as the time needed for each mRNA to reach one-half (50%, discontinuous line) of their abundance at time 0 hr. ( B ) RT-qPCR analysis of the levels of TNFSF13B mRNA and pre-mRNA in WI-38 cells that were either untreated or treated with a single dose of IR (10 Gy) and cultured for 10 days ( top ) or in WI-38 cells that were proliferating (PDL 24) or replicatively senescent (PDL50) ( bottom ). ACTB mRNA levels were calculated and used for sample normalization. ( C,D ) Stability of TNFSF13B mRNA ( top ) and MYC mRNA ( bottom ) as determined following treatment with ActD as explained in ( A ), in WI-38 cells rendered senescent by exposure to IR (10 Gy) and culture for 10 days ( C ) or by replicative exhaustion (PDL50) ( D ). 18 S rRNA levels were calculated and used for sample normalization. ( E ) RT-qPCR analysis of the levels of IRF1 mRNA and TNFSF13B pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 hr after either no treatment (-) or treatment with IR. ACTB mRNA levels were used for sample normalization. ( F ) RT-qPCR analysis showing the levels of TNFSF13B and IRF1 mRNAs in WI-38 cells treated with IR (10 Gy) and collected at the indicated times. ( G ) Western blot analysis of the levels of IRF1 protein in WI-38 fibroblasts that were proliferating (PDL24), rendered senescent following exposure to IR (10 Gy) and cultured for 10 days, or rendered senescent through replicative exhaustion (PDL50); p21 was used as positive marker for senescence and GAPDH as loading control. Significance (*, p<0.05; **, p<0.01; ***, p<0.001) was assessed with Student’s t test. Figure 2—figure supplement 1—source data 1. Uncropped western blots.

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Control, Quantitative RT-PCR, Cell Culture, Transfection, Western Blot, Marker

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of TNFSF13B mRNA in THP-1 cells transfected with CTRLsi or BAFFsi, irradiated 18 hr later (5 Gy), and assessed 72 hr after that. ACTB mRNA levels were measured and used for data normalization. Bottom , schematic of the timeline of BAFF silencing and exposure to IR in THP-1 cells. ( B ) Venn diagram of mRNAs identified by RNA-seq analysis as being differentially upregulated in THP-1 cells transfected with a control siRNA (CTRLsi) or BAFF siRNA (BAFFsi) following exposure to IR (5 Gy, collected 72 hr later). Red circle: mRNAs upregulated in CTRLsi exposed to IR (CTRLsi IR) relative to non-irradiated cells (CTRLsi). Gray circle: mRNAs less induced in BAFFsi cells exposed to IR (BAFFsi IR) relative to CTRLsi exposed to IR (CTRLsi IR). A complete list of genes from the RNA-seq analysis is available (GSE213993 and ); padj <0.05. ( C ) Molecular Signatures Database (MSigDB) hallmark gene set summarizing the differentially expressed genes obtained from ( B ); diagram was created with EnrichR and gene sets were ordered by p value. ( D ) Gene ontology (Biological Processes, Molecular Function and Cellular Component) of the differentially upregulated mRNAs identified in ( B ). ( E ) Heatmap of the differentially upregulated genes identified in ( B ) and included in the GO terms ‘Leukocyte activation’, and ‘Immune effector process’, as well as those present in the MSigDB Hallmark term ‘Inflammatory Response’. Values are averages of duplicates. Top genes upregulated in IR were sorted according to their greater reduction after BAFF silencing. Data are shown as Log2FC in gene expression relative to untreated cells (CTRLsi: log2FC = 0). ( F ) Reactome network showing the most highly enriched categories of proteins differentially upregulated in THP-1 cells transfected with control (CTRLsi) or BAFF-directed (BAFFsi) siRNAs and the next day exposed to 5 Gy IR or left untreated and collected 72 hr later (complete proteomic datasets are in ; Cutoff fold change was 1.3). Two pathways (nodes) are connected if they share 10% or more proteins. Darker nodes are more significantly enriched protein sets; larger nodes represent larger protein sets. Thicker edges represent more overlapped proteins. ( G ) Heatmap of the top differentially upregulated proteins between CTRLsi and BAFFsi. Top proteins increased after IR were sorted according to their greater reduction after BAFF silencing. Data are shown as fold change between PSM (peptide spectrum matches) relative to the untreated (CTRLsi: FC = 1). Cutoff: ([FC]>1.3, protein with PSM above 15). A complete list of differentially upregulated proteins is available in . ( H ) Western blot analysis of representative proteins identified from the proteomic analysis in ( G ). p21 was included as a control for senescence and ACTB as a loading control. Diagrams in ( D,F ) were created with ShinyGO. Figure 3—source data 1. RNA-seq analysis performed in THP-1 cells transfected with CTRLsi or BAFFsi and treated with IR. Figure 3—source data 2. Proteomic analysis performed in THP-1 cells transfected with CTRLsi or BAFFsi and treated with IR. Figure 3—source data 3. Uncropped immunoblots for .

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Quantitative RT-PCR, Transfection, Irradiation, RNA Sequencing, Control, Activation Assay, Gene Expression, Western Blot

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) Venn diagram showing the mRNAs differentially downregulated in THP-1 cells transfected with control siRNA (CTRLsi) or BAFF siRNA (BAFFsi) upon IR (5 Gy IR, collected 72 hr later). Green circle: THP-1 cells exposed to IR (CTRLsi IR) relative to untreated cells (CTRLsi). Gray circle: BAFFsi THP-1 cells exposed to IR relative to CTRLsi THP-1 cells exposed to IR. A complete list of genes from the RNA-seq analysis is available (GSE213993 and ); cutoffs: padj <0.05. ( B ) Gene ontology analysis performed on the differentially downregulated mRNAs identified in ( A ). ( C ) Top left , Venn diagram showing the proteins increased after IR (red) and those less upregulated in IR after silencing BAFF (gray) in THP-1 cells. Cells were collected and processed for proteomics as described in Materials and methods. Cutoff for proteomic data: [fold change]>1.3. Right and bottom , GO analysis (Biological Processes, Molecular Function and Cellular Component) was performed on the differentially upregulated proteins. ( D ) Venn diagram showing the proteins reduced after IR (green) and those less downregulated in IR after BAFF silencing in THP-1 cells (gray). ( E ) Reactome network analysis highlighting the most highly enriched categories of proteins differentially reduced in CTRLsi or BAFFsi THP-1 cells 72 hr after treatment with IR (5 Gy). The plot shows the relationship between the enriched pathways. Two pathways (nodes) are connected if they share 10% or more proteins. Darker nodes are more significantly enriched protein sets; bigger nodes represent larger protein sets; thicker edges represent protein sets with greater overlap. ( F ) Heatmap of the top proteins differentially reduced in BAFFsi IR and CTRLsi IR. Data are shown as fold change between PSM relative to the control (CTRLsi: FC = 1). Cutoff: [FC]>1.3, proteins with detected PSM score above 5. Diagrams in ( C, E ) were created with ShinyGO.

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Transfection, Control, RNA Sequencing

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) SA-β-Gal activity assay in THP-1 cells transfected with a control siRNA (CTRLsi) or BAFF siRNA (BAFFsi), following exposure to IR (5 Gy, collected 72 hr later); SA-β-Gal-positive cells (% of total cells) were quantified by percentage (%) of positively stained cells as described (Materials and methods). Scale bars, 100 μm. ( B ) RT-qPCR analysis of a subset of differentially expressed mRNAs identified through RNA-seq analysis and encoding SASP factors. ACTB mRNA levels were measured and used for data normalization. ( C ) Relative levels (fold change) of secreted cytokines and chemokines in THP-1 cells processed as in ( A ), as measured by multiplex ELISA 72 hr after IR (5 Gy). ( D,E ) THP-1 cells were transfected for 16 hr with a control plasmid (pCTRL) or a plasmid to express BAFF (pBAFF) and then were either left untreated or treated with IR (5 Gy); 72 hr later, whole-cell lysates were studied by western blot analysis ( D ), and multiplex ELISA assay to detect BAFF and additional SASP factors ( E ). Secretion levels are shown as relative fold change compared to pCTRL group. ( F ) Cytokine array analysis performed on the media of THP-1 cells that were transfected with Control (pCTRL) or BAFF (pBAFF) plasmids, treated with IR (5 Gy) 18 hr later, and assayed 72 hr after that. Reference control spots are present on each individual array. Media were collected from 2×10 6 cells per sample. Significance (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001) was assessed by Student’s t- test. Figure 4—source data 1. Uncropped western blots for .

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Activity Assay, Transfection, Control, Staining, Quantitative RT-PCR, RNA Sequencing, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Western Blot

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: ( A ) Western blot analysis of BAFF receptors in THP-1 cells untreated or irradiated with 5 Gy and cultured for 6 days. Due to the low levels of TACI, contrast was increased on the acquired image. ( B ) Flow cytometry analysis of THP-1 cells expressing surface BAFF receptors 6 days after either no treatment or treatment with IR (5 Gy). ( C ) RT-qPCR analysis of the levels of representative SASP mRNAs in THP-1 cells transfected with CTRLsi or siRNA targeting BAFF receptors, irradiated 18 hr later (5 Gy), and assessed 72 hr after that. ACTB mRNA levels were measured and used for data normalization. ( D ) EnrichR analysis of the differentially abundant mRNAs obtained from . The TRRUST v2 analysis predicts the transcription factors potentially affected by BAFF silencing in THP-1 cells. ( E ) TransAM NF-κB Activation Assay (Materials and methods) performed using THP-1 nuclear extracts to evaluate the binding of different NF-κB subunits to a DNA consensus sequence ( top ), as measured at 450 nm. Each antibody-specific signal was normalized to the respective blank control. Finally, the basal activity of p65 in the untreated control sample (CTRLsi) was set at 100% and all other values were normalized to it. ( F ) Western blot analysis of p65 levels in nuclear and cytoplasmic fractions of THP-1 cells that were transfected overnight with CTRLsi or BAFFsi and the next day were either left untreated or irradiated and collected 72 hr later. Cytoplasmic and nuclear markers (Tubulin and PARP1, respectively) were included to monitor the fractionation procedure; Ponceau S staining served to assess equal loading and transfer. ( G ) Western blot analysis of the proteins in whole-cell extracts prepared from THP-1 cells that were transfected with either CTRLsi or BAFFsi, then exposed to IR (5 Gy) and assessed at the indicated times. Arrowheads point to signals showing differences in NF-κB kinetics after BAFF silencing. Significance (*, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001) was assessed by Student’s t- test. Figure 5—source data 1. Uncropped western blots for .

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Western Blot, Irradiation, Cell Culture, Flow Cytometry, Expressing, Quantitative RT-PCR, Transfection, Activation Assay, Binding Assay, Sequencing, Control, Activity Assay, Fractionation, Staining

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet: Proposed model for the regulation and role of BAFF in senescence (created using BioRender). Following DNA damage, the TF interferon-regulated factor IRF1 induces the transcription of TNFSF13B mRNA. The protein BAFF is translated and inserted into the plasma membrane, where it can be further processed into a secreted form. Both forms of BAFF are increased in senescence, and both have been previously reported to be functional and capable of activating BAFF receptors (BAFFR, TACI, BCMA), which in turn stimulate stress-related pathways in a cell type-dependent manner, with a predominant activation of the NF-κB pathway in monocytic-like cells, and the p53 pathway in primary fibroblasts. Therefore, BAFF may have pleiotropic actions on senescence-associated phenotypes in different cell types. We propose that BAFF is a novel biomarker of senescence and a regulator of different senescence traits.

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Clinical Proteomics, Membrane, Functional Assay, Activation Assay, Biomarker Discovery

Journal: eLife

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.7554/eLife.84238

Figure Lengend Snippet:

Article Snippet: Sequence-based reagent , IRF1 siRNA , SCBT , sc-35706 , .

Techniques: Recombinant, Over Expression, Plasmid Preparation, Sequencing, Control, Staining, Luminex, Multiplex Assay, Enzyme-linked Immunosorbent Assay, Electroporation, Software

Journal: bioRxiv

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.1101/2022.10.25.513730

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of BAFF mRNA and BAFF pre-mRNA in THP1 cells that were either untreated (Control) or treated with IR (5 Gy) and analyzed 6 days later. ( B ) Terms enriched by Gene Ontology analysis after proteomic analysis of THP-1 cells that were rendered senescent by IR relative to untreated control cells. Proteomic analysis is available as Figure 2-Source Data 1 . GO terms were sorted by p-value ranking (EnrichR analysis). ( C ) Schematic of conserved TF binding sites on the BAFF promoter, as analyzed using ECR browser and rVista 2.0. ( D ) Schematic of the interferon response triggered by DNA damage, highlighting the targets affected by the different interferon inhibitors used in our study ( ; ; ); created using BioRender. ( E ) RT-qPCR analysis of the levels of BAFF mRNA, BAFF pre-mRNA, and IRF1 mRNA in THP-1 cells after treatment with the inhibitors of the interferon pathway indicated in (D) [Ruxolitinib (Ruxo, 1 μM), MRT67307 (MRT67, 5 μM), and BX-795 (5 μM)] or with control vehicle DMSO for 5 days after treatment with IR. IRF1 mRNA was included as a positive control of interferon-stimulated mRNAs. ( F ) RT-qPCR analysis of the levels of expression of BAFF and p21 mRNAs and other interferon-regulated transcripts ( IRF1, IRF2, IRF8 , and BLIMP1 mRNAs) in THP-1 cells that were left untreated or were treated with IR and cultured for the indicated times. ( G ) Western blot analysis of the levels of IRF1 and IRF2 in the cytoplasmic and nuclear fractions of THP-1 cells that were left untreated or were irradiated and assayed 6 days later. The cytoplasmic protein tubulin, the nuclear protein PARP1, and the senescence-associated protein p21 were included in the analysis. Ponceau staining of the transfer membrane was included to monitor differences in loading and transfer among samples. ( H ) RT-qPCR analysis of the levels of IRF1 mRNA and BAFF pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 h after either no treatment (-) or treatment with IR. (I) RT-qPCR analysis showing the levels of BAFF and IRF1 mRNAs in WI-38 cells treated with IR (10 Gy) and collected at the indicated times. ( J ) Western blot analysis of the levels of BAFF protein in WI-38 fibroblasts that were proliferating (PDL24), senescent following exposure to IR (10 Gy) and cultured for 10 days, and senescent through replicative exhaustion (PDL50); p21 was used as positive marker for senescence and GAPDH as loading control. ( K ) RT-qPCR analysis of the levels of BAFF pre-mRNA in WI-38 cells transfected with CTRLsi or IRF1si and either left untreated (-) or treated with IR (10 Gy) and assayed 5 days later. Significance (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001) was assessed with Student’s t -test. Source Data Files for : Figure 2—Source Data 1. Proteomic analysis performed in control THP-1 cells treated with IR. Figure 2—Source Data 2. Uncropped western blot images for .

Article Snippet: IRF1 pool siRNAs and control siRNAs were from Santa Cruz Biotechnology (sc-35706, sc-37007).

Techniques: Quantitative RT-PCR, Control, Binding Assay, Positive Control, Expressing, Cell Culture, Western Blot, Irradiation, Staining, Membrane, Transfection, Marker

Journal: bioRxiv

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.1101/2022.10.25.513730

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of BAFF mRNA and BAFF pre-mRNA in THP1 cells that were either untreated (Control) or treated with IR (5 Gy) and analyzed 6 days later. ( B ) Terms enriched by Gene Ontology analysis after proteomic analysis of THP-1 cells that were rendered senescent by IR relative to untreated control cells. Proteomic analysis is available as Figure 2-Source Data 1 . GO terms were sorted by p-value ranking (EnrichR analysis). ( C ) Schematic of conserved TF binding sites on the BAFF promoter, as analyzed using ECR browser and rVista 2.0. ( D ) Schematic of the interferon response triggered by DNA damage, highlighting the targets affected by the different interferon inhibitors used in our study ( ; ; ); created using BioRender. ( E ) RT-qPCR analysis of the levels of BAFF mRNA, BAFF pre-mRNA, and IRF1 mRNA in THP-1 cells after treatment with the inhibitors of the interferon pathway indicated in (D) [Ruxolitinib (Ruxo, 1 μM), MRT67307 (MRT67, 5 μM), and BX-795 (5 μM)] or with control vehicle DMSO for 5 days after treatment with IR. IRF1 mRNA was included as a positive control of interferon-stimulated mRNAs. ( F ) RT-qPCR analysis of the levels of expression of BAFF and p21 mRNAs and other interferon-regulated transcripts ( IRF1, IRF2, IRF8 , and BLIMP1 mRNAs) in THP-1 cells that were left untreated or were treated with IR and cultured for the indicated times. ( G ) Western blot analysis of the levels of IRF1 and IRF2 in the cytoplasmic and nuclear fractions of THP-1 cells that were left untreated or were irradiated and assayed 6 days later. The cytoplasmic protein tubulin, the nuclear protein PARP1, and the senescence-associated protein p21 were included in the analysis. Ponceau staining of the transfer membrane was included to monitor differences in loading and transfer among samples. ( H ) RT-qPCR analysis of the levels of IRF1 mRNA and BAFF pre-mRNA in THP-1 cells transfected with control (CTRLsi) or IRF1-directed (IRF1si) siRNAs 72 h after either no treatment (-) or treatment with IR. (I) RT-qPCR analysis showing the levels of BAFF and IRF1 mRNAs in WI-38 cells treated with IR (10 Gy) and collected at the indicated times. ( J ) Western blot analysis of the levels of BAFF protein in WI-38 fibroblasts that were proliferating (PDL24), senescent following exposure to IR (10 Gy) and cultured for 10 days, and senescent through replicative exhaustion (PDL50); p21 was used as positive marker for senescence and GAPDH as loading control. ( K ) RT-qPCR analysis of the levels of BAFF pre-mRNA in WI-38 cells transfected with CTRLsi or IRF1si and either left untreated (-) or treated with IR (10 Gy) and assayed 5 days later. Significance (ns, not significant; *p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001) was assessed with Student’s t -test. Source Data Files for : Figure 2—Source Data 1. Proteomic analysis performed in control THP-1 cells treated with IR. Figure 2—Source Data 2. Uncropped western blot images for .

Article Snippet: IRF1 pool siRNAs and control siRNAs were from Santa Cruz Biotechnology (sc-35706, sc-37007).

Techniques: Quantitative RT-PCR, Control, Binding Assay, Positive Control, Expressing, Cell Culture, Western Blot, Irradiation, Staining, Membrane, Transfection, Marker

Journal: bioRxiv

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.1101/2022.10.25.513730

Figure Lengend Snippet: ( A ) RT-qPCR analysis of the levels of BAFF mRNA in THP-1 cells transfected with CTRLsi or BAFFsi, irradiated 18 hr later (5 Gy), and assessed 72 h after that. Bottom , schematic of the timeline of BAFF silencing and exposure to IR in THP-1 cells. ( B ) Venn diagram of mRNAs identified by RNA-seq analysis as being differentially upregulated in THP-1 cells transfected with a control siRNA (CTRLsi) or BAFF siRNA (BAFFsi) following exposure to IR (5 Gy, collected 72 h later). Red circle: mRNAs upregulated in CTRLsi exposed to IR (CTRLsi IR) relative to non-irradiated cells (CTRLsi). Gray circle: mRNAs less induced in BAFFsi cells exposed to IR (BAFFsi IR) relative to CTRLsi exposed to IR (CTRLsi IR). A complete list of genes from the RNA-seq analysis is available (GSE213993, reviewer token mbubswukttkhtsf, and Figure 3-Source Data 1 ); cutoffs in Source Data: padj<0.05, [fold change] >1.3. ( C ) Molecular Signatures Database (MSigDB) hallmark analysis performed on the differentially expressed genes obtained from (B); diagram was created by EnrichR analysis and gene sets ordered by p value. ( D ) Gene ontology analysis (Biological Processes, Molecular Function and Cellular Component) performed on the differentially upregulated mRNAs identified in (B). ( E ) Heatmap of the differentially upregulated genes identified in (B) and included in the GO terms ‘Inflammatory Response’, ‘Leukocyte activation’, and ‘Immune effector process’, as well as those present in the MSigDB Hallmark term ‘Inflammatory Response’. Values are averages of duplicates. Top genes upregulated in IR were sorted according to their greater reduction after BAFF silencing. Data are shown as Log2FC in gene expression relative to untreated cells (CTRLsi: log2FC=0). ( F ) RT-qPCR analysis of a subset of differentially expressed mRNAs identified in (B) and (E). Significance (*p < 0.05, **p < 0.01, ***p < 0.001, ****p <0.0001) was assessed with Student’s t -test. (G) Reactome network analysis showing the most highly enriched categories of proteins differentially upregulated in THP-1 cells transfected with control (CTRLsi) or BAFF-directed (BAFFsi) siRNAs and the next day exposed to 5 Gy IR or left untreated and collected 72 h later (complete proteomic datasets are in Figure 3-Source Data 2; Cutoff fold change was 1.3). Two pathways (nodes) are connected if they share 10% or more proteins. Darker nodes are more significantly enriched protein sets; larger nodes represent larger protein sets. Thicker edges represent more overlapped proteins. (H) Heatmap of the top differentially upregulated proteins between CTRLsi and BAFFsi. Top proteins increased after IR were sorted according to their greater reduction after BAFF silencing. Data are shown as fold change between PSM (peptide spectrum matches) relative to the untreated (CTRLsi: FC=1). Cutoff: [FC] >1.3, protein with PSM above 15). A complete list of differentially upregulated proteins is available in Figure 3-Source Data 2 . ( I ) Western blot analysis of representative proteins identified from the proteomic analysis in (G). p21 was included as a control for senescence and ACTB as a loading control. Diagrams in (D,G) were created with ShinyGO. Source Data Files for : Figure 3—Source Data 1. RNA-seq analysis performed in THP-1 cells transfected with CTRLsi or BAFFsi and treated with IR. Figure 3—Source Data 2. Proteomic analysis performed in THP-1 cells transfected with CTRLsi or BAFFsi and treated with IR. Figure 3—Source Data 3. Uncropped immunoblots for .

Article Snippet: IRF1 pool siRNAs and control siRNAs were from Santa Cruz Biotechnology (sc-35706, sc-37007).

Techniques: Quantitative RT-PCR, Transfection, Irradiation, RNA Sequencing, Control, Activation Assay, Gene Expression, Western Blot

Journal: bioRxiv

Article Title: Pleiotropic effects of BAFF on the senescence-associated secretome and growth arrest

doi: 10.1101/2022.10.25.513730

Figure Lengend Snippet: Model proposed for the regulation and role of BAFF in senescence (created using BioRender). Following DNA damage, the TF interferon-regulated factor IRF1 induces the transcription of BAFF mRNA. The protein BAFF is translated and inserted into the plasma membrane, where it can be further processed into a secreted form. Both forms of BAFF are increased in senescence, and both have been previously reported to be functional and capable of activating BAFF receptors (BAFFR, TACI, BCMA), which in turn stimulate stress-related pathways in a cell type-dependent manner, with a predominant activation of the NF-κB pathway in monocytic-like cells, and the p53 pathway in primary fibroblasts. Therefore, BAFF may have pleiotropic actions on senescence-associated phenotypes in different cell types. We propose that BAFF is a novel biomarker of senescence and a regulator of different senescence traits.

Article Snippet: IRF1 pool siRNAs and control siRNAs were from Santa Cruz Biotechnology (sc-35706, sc-37007).

Techniques: Clinical Proteomics, Membrane, Functional Assay, Activation Assay, Biomarker Discovery

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 1. CD47 levels in human lung adenocarcinoma cells were increased following tumor metastasis (A and B) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the lymph nodes. (C and D) Comparison of CD47 expression between primary lung adenocarcinoma and lung adenocarcinoma metastases in the liver. (A) and (C) showed representative IHC images for CD47 staining from 14 to 5 lung adenocarcinoma tissue samples, respectively. Data were presented as means ± SDs. **p < 0.01. Scale bar, 50 mm.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Comparison, Expressing, Staining

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 2. Induction of CD47 by IFN-g in human lung cancer cells (A) Flow cytometry analysis of CD47 surface expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Right: Representative image; left: quantitative analysis. (B) Immunofluorescence analysis of CD47 expression in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Scale bar, 20 mm. (C) Western blot analysis of CD47 expression in human lung cancer cell lines after IFN-g treatment. (D) qRT-PCR analysis of CD47 mRNA in human lung cancer cell lines upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Left: Representative image; right: quantitative analysis. Data from more than 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Flow Cytometry, Expressing, Incubation, Recombinant, Western Blot, Quantitative RT-PCR

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 3. Identification of genes involved in the IFN signaling pathway that upregulates CD47 expression (A) qRT-PCR analysis of various genes involved in the IFN signaling pathway in A549 cells upon incubation with re- combinant IFN-g (100 ng/mL, 24 h). (B) Normalized luciferase reporter expression of A549 cells transduced with different sets of lentiviral shRNA constructs. Upper: Schematic representation of the CD47-Prom-Firefly Lucif- erase-EF1AProm-Renilla luciferase constructs used to generate the reporter cell lines. Lower: Quantitative anal- ysis. (C) IRF1 protein levels in human lung cancer cells upon incubation with recombinant IFN-g (100 ng/mL, 24 h). Upper: Representative image; lower: quantitative anal- ysis. Data from more than 3 independent experiments were presented as means ± SDs. ***p < 0.001.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Expressing, Quantitative RT-PCR, Incubation, Luciferase, Transduction, shRNA, Construct, Recombinant

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 4. IFN-g induces CD47 expression in lung cancer cells through IRF-1 (A) Sequence of the CD47 promoter showing the position of the putative IRF-1-binding site. (B) Reporter assay for putative IRF-1 binding. The results were normalized to relative luciferase units (RLUs). (C) ChIP assay analyzing the CD47 promoter in A549 cells. The results were normalized to the input. (D and E) IRF-1 mRNA (D) and protein (E) levels in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment. (F and G) CD47 level in A549 cells transfected with IRF-1-specific shRNA or scramble shRNA after IFN-g treatment detected by flow cytometry (F) and immunofluorescence (G). Scale bar, 10 mm. (H) Western blot analysis of CD47 levels in A549 cells transfected with IRF-1-specific or scramble shRNA. Left: Representative image; right: quantitative analysis. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Expressing, Sequencing, Binding Assay, Reporter Assay, Luciferase, Transfection, shRNA, Cytometry, Western Blot

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 5. IFN-g promotes lung cancer cell metastasis by upregulating CD47 expression in vitro (A) Scratch-wound healing assay in A549 cells (A549-WT) and CD47-knockout A549 cells (A549-CD47-KO) were treated with/without IFN-g. (B) Transwell assay in A549 cells (A549-WT) and A549-CD47-KO cells were treated with/without IFN-g. Data from 3 independent experiments were presented as means ± SDs. **p < 0.01, ***p < 0.001, ****p < 0.0001. Scale bar, 200 mm.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Expressing, In Vitro, Wound Healing Assay, Knock-Out, Transwell Assay

Journal: Molecular therapy oncolytics

Article Title: Human lung adenocarcinoma CD47 is upregulated by interferon-γ and promotes tumor metastasis.

doi: 10.1016/j.omto.2022.04.011

Figure Lengend Snippet: Figure 6. IFN-g promotes human lung cancer cell metastasis in immunodeficient mice by upregulating CD47 (A) A549 cells (A549-WT) and A549-CD47-KO were engrafted in the lungs of the BALB/c-nude mice. Three weeks post-engraftment, the mice were randomly divided into 2 groups. One group was administered with IFN-g (10 ng/mouse, injected once every 2 days) (A549-WT + IFN-g; A549-CD47-KO + IFN-g), and the other group without IFN-g injection was served as control (A549-WT; A549-CD47-KO). After 5 weeks, the mice were sacrificed to analyze tumor growth and metastasis. (B and C) H&E staining and human CD47 immune staining in mouse lungs (B) and livers (C). In both (B) and (C), left: representative images; right: quantification of tumor nodules. Data were presented as means ± SDs. *p < 0.05, ***p < 0.001. NS, no significance.

Article Snippet: For the reporter analyses, CD47 promoter WT plasmid containing the IRF-1 binding site was generated by inserting the fragment (600 bp, obtained from the University of California, Santa Cruz [UCSC] database, http://genome-asia.ucsc.edu), from A549 genomic DNA into the multicloning site (MCS) of the PGL3 Basic Vector (Promega, Madison, WI, USA).

Techniques: Injection, Control, Staining