Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Exploration of PCIF1 target genes by genome-wide gene expression analysis. ( A ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) or two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2). ( B ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #2) with anti-PCIF1 and anti-b-actin antibodies. ( C , D ) Venn diagrams showing the overlap between the two indicated siRNA-mediated downregulated ( C ) and upregulated ( D ) genes identified by the gene expression profile analyzed by DNA microarray using a Human Genome U133 Plus 2.0 Array (Affymetrix). The condition for selecting differentially expressed genes are as follows: (1) cut-off condition: expression > 100, (2) fold change: >2, (3) p -value < 0.05.

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Genome Wide, Gene Expression, Quantitative RT-PCR, Isolation, Control, Western Blot, Microarray, Expressing

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Expression of RAB23 and CNOT6 is regulated by PCIF1 at both the mRNA and protein levels. ( A – C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3), using the specific primer set detecting PCIF1 ( A ), RAB23 ( B ), and CNOT6 ( C ) expression. ( D , E ) Immunoblotting analysis of total protein extracts from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) with the indicated antibodies. Signal intensities obtained from immunoblotting were quantified using ImageJ software version 1.52. The y-axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Control, Western Blot, Software, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant PCIF1 restores the normal levels of target mRNA expression. ( A , B ) RT-qPCR analysis of total RNAs isolated from HeLa cells transfected with a control empty vector (Vec) or a vector expressing siRNA-resistant PCIF1 (PCIF1siR) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1 #3), using the specific primer set detecting RAB23 ( A ) and CNOT6 ( B ) expression. The y -axis represents the fold change relative to the levels in HeLa cells treated with control siRNA. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between indicated pairs (Student’s t -test, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Isolation, Transfection, Control, Plasmid Preparation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 does not regulate expression of its target genes at the transcriptional level. ( A , E ) Schematic illustrations of the RAB23 ( A ) and CNOT6 ( E ) genes. The transcription start sites are indicated by arrows, the exons are shown as black boxes, and the polyadenylation signals are indicated by arrowheads. The positions of the PCR primer set for RT-qPCR amplification of the precursor and mature mRNAs are indicated by arrows. ( B – D , F – H ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated with control siRNA (siNC) and two distinct PCIF1-targeted siRNAs (siPCIF1 #1 and #3) using the indicated primer sets. ( I , J ) ChIP analyses of the RAB23 ( I ) and CNOT6 ( J ) gene promoters (position 2 in ) using antibodies against Pol II in HeLa cells treated with control siRNA (siNC) and two distinct PCIF1 targeted siRNAs (siPCIF1 #1 and #3). Normal rabbit IgG was used as the negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1 targeted siRNA (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Quantitative RT-PCR, Amplification, Isolation, Control, Negative Control, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 regulates the stability of target gene mRNAs in opposite ways. After HeLa cells were treated with a negative control siRNA (siNC) and two PCIF1-targeted siRNAs (siPCIF1 #1 and #3) for 72 h, actinomycin D was added to inhibit transcription. Cells were harvested at 0, 2, 4, 8, and 12 h after treatment, and total RNA was isolated. The amount of residual mRNA was analyzed by RT-qPCR at each time point, using the specific primer set detecting PCIF1 ( A ), CRAB23 ( B ), and CNOT6 ( C ) mRNAs. The relative value was calculated using the expression level of β-actin mRNA (ACTB) as a normalizer. The relative values at each time point were calculated relative to time zero. Asterisks represent statistically significant differences between the control siRNA treatment and the indicated PCIF1-targeted siRNA (Student’s t -test, * p < 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Negative Control, Isolation, Quantitative RT-PCR, Expressing, Control

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: Ectopic expression of siRNA-resistant wild-type but not methyltransferase-deficient mutant PCIF1 restored normal levels of target mRNA expression. ( A ) Immunoblotting analysis of total protein extracts from HeLa cells transfected with a control empty vector (Vec), a vector expressing siRNA-resistant wild-type PCIF1 (siR_wt), or methyltransferase-deficient mutant PCIF1 (siR_mut) under treatment with control siRNA (siNC) or PCIF1-targeted siRNAs (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression) with the indicated antibodies. ( B , C ) RT-qPCR analysis of total RNAs isolated from HeLa cells treated as in ( A ), using the specific primer set detecting RAB23 ( B ) and CNOT6 ( C ) expression. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, * p < 0.05, ** p < 0.01, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Expressing, Mutagenesis, Western Blot, Transfection, Control, Plasmid Preparation, Quantitative RT-PCR, Isolation, Standard Deviation

Journal: Cells

Article Title: Cap-Specific m 6 Am Methyltransferase PCIF1/CAPAM Regulates mRNA Stability of RAB23 and CNOT6 through the m 6 A Methyltransferase Activity

doi: 10.3390/cells13201689

Figure Lengend Snippet: PCIF1 suppression resulted in a significant decrease in m 6 A levels of both RAB23 and CNOT6 mRNAs. MeRIP-qPCR analysis was performed using HeLa cells treated with control siRNA (siNC) or PCIF1-targeted siRNA (siPCIF1: siPCIF1 #3 was used for PCIF1 suppression). RT-qPCR analysis of RNAs purified from the immunoprecipitates of HeLa cell extracts using the anti-m 6 A antibody, using the specific primer set detecting RAB23 ( A ), CNOT6 ( B ), and ACTB ( C ) mRNAs. The y -axis represents the fold change relative to the RNA levels in the immunoprecipitate by anti-m 6 A antibody from HeLa cells treated with control siRNA (siNC). Normal rabbit IgG (IgG) was used as a negative control. Data are expressed as the mean ± standard deviation of three independent experiments. Asterisks represent statistically significant differences between the indicated pairs (Student’s t -test, n.s. p > 0.05, *** p < 0.001).

Article Snippet: An amount of 1.5 μg of anti-m 6 A antibody (Abcam Limited, Cambridge, UK, ab151230) or normal rabbit IgG (Medical & Biological Laboratories Co.) was incubated with 30 μL of Dynabeads Protein G (Thermo Fisher Scientific) at room temperature for 1 h. Total RNA (50 μg) was added to the antibody-Dynabead complexes and incubated at 4 °C for 4 h. After washing three times, the bound RNA was purified from the beads using SepasolTM-RNA I Super G. The first strand of cDNA was synthesized from m 6 A-RNA using ReverTra AceTM qPCR RT Master Mix with gDNA Remover (Toyobo Co., Osaka, Japan) with random hexamer primers according to the manufacturer’s instructions. cDNA was quantified using GeneAce SYBRTM qPCR Mix II (Nippon Gene Co., Tokyo, Japan) and a Mx3000P real-time PCR system (Agilent).

Techniques: Control, Quantitative RT-PCR, Purification, Negative Control, Standard Deviation

Journal: Cell Host & Microbe

Article Title: SARS-CoV-2 spike L452R variant evades cellular immunity and increases infectivity

doi: 10.1016/j.chom.2021.06.006

Figure Lengend Snippet:

Article Snippet: Normal goat IgG (R&D Systems, cat# AB-108-C) was used as the negative control for this assay.

Techniques: Virus, Recombinant, Modification, Expressing, Reverse Transcription, Transfection, Luciferase, Mutagenesis, Gel Extraction, Cloning, Plasmid Preparation, Quantitative RT-PCR, Competitive Binding Assay, Sequencing, Random Hexamer, Software, Pore Size, Membrane

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) The left graph shows the X-ray crystal structure of a p50 / p65 heterodimer bound to DNA as published in (PDB 1kvx), while the right graph shows the entire p65 protein structure including the disordered C-terminal half as calculated by alphafold ( https://alphafold.ebi.ac.uk/entry/Q04206 ). Residues required for dimerization (Phe (F) 213, Leu (L) 215) or DNA binding (Glu (E) 39) are indicated in both structures. (B) Scheme of the HA-tagged p65-miniTurbo fusion proteins that were used to reconstitute p65-deficient HeLa cells under the control of a tetracycline-sensitive promoter. F213 and L215 in p65 wildtype (wt) were mutated to Asp (FL / DD) for dimerization-deficient p65 or E39 to Ile (E / I) for DNA-binding-deficient p65. (C) Principle of proximity-based biotin tagging. (D) Pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected (using branched Polyethyleneimine, PEI)) with the constructs shown in (B) and their expression was induced with doxycycline (1 µg / ml) for 17 h. At the end of this incubation, intracellular biotinylation was induced by adding 50 µM biotin for 70 minutes as indicated. Additionally, half of the samples were treated with IL-1α (10 ng / ml) for the last 60 minutes. Cell cultures expressing HA-miniTurbo only (empty vector, EV) or receiving only doxycycline or biotin served as negative controls (indicated by gray font). Parental HeLa cells (p) were included as further controls. Left panel: Cells were lysed and proteins were analyzed by Western blotting for the expression of p65-HA-miniTurbo and HA-miniTurbo using anti p65 and anti HA antibodies. Equal loading was confirmed by probing the blots with anti β-actin antibodies. Right panel: Biotinylated proteins from the same samples were purified on streptavidin agarose beads and biotinylation patterns were visualized by Western blotting using streptavidin-horseradish peroxidase (HRP) conjugates (representative images from two independent experiments). (E) Biotinylated proteins from the experiment shown in (C) and from a second biological replicate were identified by mass spectrometry. Volcano plots show the ratio distributions of Log 2 -transformed mean protein intensity values on the X-axes obtained with wild type p65 or the p65 mutants compared to the empty vector controls in the presence or absence of IL-1α treatment. Y axes show corresponding p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (F) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only (see ). This set of proteins was intersected with proteins enriched in cells expressing p65 mutant proteins (LFC ≥ 2, -log10 p ≥ 1.3). Venn diagrams show the numbers of p65 / RELA interactors and their overlaps before and after IL-1α-treatment, with values in the lower left corners indicating total numbers of interactors. (G) The six protein sets shown in (E) were subjected to parallel overrepresentation pathway analysis using Metascape software . The Venn diagrams show the overlap of the top 100 enriched pathway terms. For IL-1α samples, only 92 terms were enriched. Values in the lower left corners indicate total numbers of unique pathways. (H) The table shows the most strongly enriched pathway categories associated with the p65 / RELA wild type or mutant interactomes. Numbers in brackets indicate the total numbers of p65 / RELA interactors per condition that were subjected to overrepresentation analysis according to (E, F). The mass spectrometry data and bioinformatics analysis results are provided in Supplementary Table 1. See also and . rtTA, reverse tetracycline-controlled transactivator.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, Control, CRISPR, Transfection, Construct, Expressing, Incubation, Plasmid Preparation, Western Blot, Purification, Mass Spectrometry, Transformation Assay, Mutagenesis, Software

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Parental HeLa cells or pools of HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with empty vector (EV) encoding HA-miniTurbo (HA-mTb) or with p65 / RELA wild type (wt) fused C-terminally to HA-mTb (p65(wt)-HA-mTb) as described in the legend of . The expression of the constructs was induced with increasing concentrations of doxycycline for 17 h as indicated. At the end of the incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cell extracts were analyzed by Western blotting for the expression of the p65-HA-mTb fusion protein or HA-mTb using polyclonal antibodies raised against the C-terminus of p65 / RELA (sc-372) or a monoclonal antibody raised against N-terminal amino acids 1-286 of p65 / RELA (sc-8008), or an anti HA antibody, respectively. Note that the fusion protein is better recognized with the N-terminal antibody preparations. (B) HeLa cells with CRISPR / Cas9-based suppression of endogenous p65 / RELA (Δp65) were transiently transfected with the indicated constructs and their expression was induced with doxycycline at 1 µg / ml for 17 h. On the next day, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Total RNA was isolated and analyzed by RT-qPCR for expression of the indicated genes. Bar graphs show means ± s.d. from two biologically independent experiments. (C) Cells were transfected as in (A) and expression of the p65 / RELA fusion protein was induced 20 h later with doxycycline (10 ng / ml) for 4 h. In last period of this incubation, half of the cell cultures were treated with IL-1α (10 ng / ml) for 1 h. Cells were lysed and cytosolic (C), soluble nuclear (N1) and insoluble, chromatin nuclear fractions (N2) were analyzed by Western blotting for the expression and distribution of p65(wt)-HA-mTb. Antibodies against RNA polymerase II, tubulin and β-actin were used to control purity of fractions and equal loading.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: CRISPR, Transfection, Plasmid Preparation, Expressing, Construct, Incubation, Western Blot, Isolation, Quantitative RT-PCR, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Biotinylated proteins from the experiments shown in and from a second biological replicate were identified by mass spectrometry in the presence or absence of IL-1α treatment of cells. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 in the presence of doxycycline and biotin (wt) compared to the empty vector control (EV) or compared with conditions in which only biotin (wt(bio)) or doxycycline (wt(dox)) were added to the cell cultures, to determine false positive values in the absence of expression of fusion protein but facilitated biotinylation, or in the absence of biotinylation but induced expression of the fusion protein, respectively. X-axes show mean ratio value and Y-axes show p values from t-test results. Strong enrichment of the bait p65 / RELA proteins together with the core canonical NF-kB components is shown in red and blue colors, respectively (two biologically independent experiments and three technical replicates per sample). (B) Specific proteins binding to p65 / RELA wild type were defined by significant enrichment (LFC ≥ 2, -log 10 p ≥ 1.3) compared to HA-miniTurbo only and to cells exposed to doxycycline or biotin only as shown in (A). Venn diagrams show the total numbers of specific p65 / RELA interactors and their overlaps before and after IL-1α-treatment. The intersecting 279 (without IL-1α) and 310 (with IL-1α) interactors were pooled, resulting in the set of 366 specific p65 / RELA interactors that was used for further downstream analyses. Numbers in the left lower corner of the boxes indicate the total number of detected interactors.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Mass Spectrometry, Transformation Assay, Plasmid Preparation, Control, Expressing, Binding Assay

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Protein interaction network of the 46 known p65 / RELA interactors found by miniTurboID. Edge widths visualize the evidence for experimental interactions deposited in the STRING database . Nodes are colored in red and are arranged according to the enrichment found by proximity labeling in our study. (B) Venn diagram of p65 / RELA interactors in IL-1α or untreated cells revealing a total of 366 unique p65 / RELA interactors, of which 320 (87.4 %) have no documented protein interaction entries in STRING. (C) Overlap of the RELA interactome with 1639 human TFs and 801 epigenetic regulators . (D) Graphs visualizing the top 10 enriched epigenetic regulators. Volcano plots show the ratio distributions of Log 2 transformed mean protein intensity values obtained with wild type p65 / RELA (wt) or with p65 / RELA mutants (FL/DD, E/I) compared to empty vector controls (EV). Only 9 reader proteins were found. (E) Association of enriched epigenetic regulators with known epigenetic complexes according to the annotation provided by . Numbers in brackets show identified components per complex. (F) Venn diagram showing the overlap of enriched TFs in basal or IL-1α-stimulated conditions. (G) Volcano plots visualizing all TFs significantly enriched with wt p65 / RELA (LFC ≥ 2, -log 10 p ≥ 1.3) compared with empty vector control (EV) and the changes obtained with p65 mutants in basal conditions. (H) Distribution of TF families found to be associated with p65 / RELA in basal and IL-1α-stimulated conditions according to the annotation provided by (I) IL-1α-dependent enrichment of all TF belonging to ZBTB and ZNF families as identified by miniTurboID. (J) The top 10 pathway terms according to GO (BP, CC, MF), KEGG, Reactome, STRING clusters and WikiPathways data base entries and the top 10 subcellular localizations associated with the 366 p65 / RELA interactors. Annotations, number of components and false discovery rates (FDR) were retrieved using the STRING plugin of Cytoscape . The mass spectrometry data sets and bioinformatics analysis results are provided in Supplementary Table 1.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Labeling, Transformation Assay, Plasmid Preparation, Control, Mass Spectrometry

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Final list of top ranking high confidence interactors p65 / RELA selected for further studies. The heatmap shows the Log 2 transformed mean protein intensity values from technical triplicates of the two biological independent miniTurboID experiments, the enrichment ratio values compared to the empty vector (HA-miniTurbo) control (EV) and the regulation by IL-1α. With the exception of N4BP3, all proteins were identified by at least two peptides. (B) Graph showing that the top 38 p65 / RELA interactors are largely devoid of known protein interactions based on STRING entries. According to STRING, only two factors (CEBPD and FOSL1) interact with p65 /RELA. Node borders visualize the main functional annotations. (C) HeLa cells were transiently transfected for 48 h with 20 nM of siRNAs mixtures for 38 HCI and p65 / RELA, a siRNA targeting luciferase, transfection reagent alone or were left untreated (untr.). Half of the cells per plate were treated for 1 h with IL-1α (10 ng / ml) at the end of the incubation. cDNAs were transcribed in lysates and amplicons for three NF-kB target genes, two housekeeping genes and all 38 HCI p65 / RELA interactors were pre-amplified by linear PCR and then quantified by qPCR. Based on Ct values, mRNA levels were quantified and normalized against GUSB . The effects of knockdowns were calculated separately for basal and IL-1α-inducible conditions against the luciferase siRNA. The heatmap shows hierarchically Kmeans clustered mean ratio values derived from three biologically independent siRNA screens. As a positive control, RELA knockdowns were performed in parallel. Green colors highlight p65 / RELA interactors selected for further analysis. (D) The miniTurboID enrichment of six p65 / RELA interactors (green colors) chosen from (C) is shown. The complete set of data of the screen is provided in Supplementary Table 2. See also .

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Transformation Assay, Plasmid Preparation, Control, Functional Assay, Transfection, Luciferase, Incubation, Amplification, Derivative Assay, Positive Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Scheme illustrating the arrangement of siRNAs and controls on individual cell culture plates and the performance of RT-qPCR measurements in cell extracts without prior RNA purification. A linear PCR amplification step was included to pre-amplify specific transcripts. (B) Confirmation of knockdown of 38 HCI and of RELA mRNAs by RT-qPCR as shown in (A). Bar graphs show mean changes ± s.d. relative to the luciferase siRNA controls (siLuci) from three biologically independent experiments.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Cell Culture, Quantitative RT-PCR, Purification, Amplification, Knockdown, Luciferase

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Proximity-ligation assays coupled to immunofluorescence (IF) were performed with HeLa cells or Δp65 HeLa cells lacking endogenous p65 / RELA to demonstrate interactions of p65 / RELA with TFE3 (A), TFEB (B), GLIS2 (C) and ZBTB5 (D) using pairs of antibodies as indicated. PLA-spots are colored in red, while p65 IF is colored in green. Nuclear DNA is counterstained with Hoechst (blue signals). The images show representative fluorescence raw data and the violin plots on the right show quantification from the numbers of cells indicated in brackets. Samples omitting one of the two antibodies or both primary antibodies (ctr) served as negative controls. Solid lines indicate medians and dashed lines indicate 1 st and 3 rd quartiles. Asterisks indicate results from Kruskal-Wallis tests compared to the parental control (****p ≤ 0.0001). obtained by one-way ANOVA.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Ligation, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on basal p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 hours with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in untreated, basal conditions, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all constitutively expressed genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to analyze the influences of novel p65 / RELA interactors on IL-1α-regulated p65 / RELA target genes by combining siRNA-mediated knockdown with transcriptome analysis. (B) HeLa cells were transiently transfected for 48 h with 20 nM siRNA mixtures against RELA, ZBTB5, S100A8, S100A9 (series 1) or RELA, GLIS2, TFE3, TFEB (series 2) and an siRNA against luciferase (siLuc) as control. Half of the cells were treated with IL-1α (10 ng/ml) for 1 hour at the end of incubation, and Agilent microarray analyses were performed from total RNA. Normalized data were used to identify DEGs based on an LFC ≥ 1 with a -log 10 p value ≥ 1.3. Venn diagrams show the overlap of all DEGs that were affected at least twofold by siRNA knockdown in IL-1α-treated samples, with the ratio of siLuc to individual knockdown determined in each case. Red colors mark genes jointly regulated by knockdown of RELA and one of its interactors (two biologically independent experiments). (C) Violin plots show the distribution, medians, and interquartile ranges of normalized expression levels for all IL-1α-regulated genes and the corresponding changes in the gene subsets defined in that were affected by siRNA knockdown. The number of these genes is indicated in parentheses. Asterisks indicate significant changes as determined by a two-tailed Mann-Whitney test (*p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001). (D) Superimposed pairwise correlation analyses of the mean ratio changes of all genes (gray), IL-1α-regulated genes (blue), and gene sets significantly up- or down-regulated by siRNA knockdown (red). Ratio values from RELA knockdown conditions were compared with the knockdown of a RELA interactor in each case. Genes that are jointly regulated by knockdown of RELA and one of its interactors correspond to the Venn diagrams of (B) and are marked in red. Coefficients of correlation (Pearson’s r), corresponding p values and coefficients of determination (r 2 ) rare indicated for all comparisons. The complete set of data is provided in Supplementary Table 3.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Knockdown, Transfection, Luciferase, Control, Incubation, Microarray, Expressing, Two Tailed Test, MANN-WHITNEY

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to project the protein interactions of all target genes defined by knockdowns of p65 / RELA or its interactors in IL-1α-stimulated cells into combined functional networks. (B) Table summarizing the numbers of mapped IDs (= nodes) corresponding to the gene groups shown in , their protein interactions (= edges) and the protein interaction network enrichment p values as derived from STRING. (C) Cytoscape-derived PPI networks. Nodes are colored and arranged according to the deregulation of the corresponding genes by knockdown of p65 / RELA or its interactors. Edges visualize known protein interactions, including the small number of interactions reported for p65 / RELA, S100A8 / 9, and TFE3 / TFEB. No interactions were found for ZBTB5 and GLIS2.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Functional Assay, Derivative Assay, Knockdown

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: (A) Schematic illustrating the strategy to use p65 / RELA ChIPseq data for delineating chromatin recruitment of RELA together with its interactors on the basis of DNA motifs and three possible scenarios of interactions. (B) Windows of 1000 base pairs surrounding experimentally determined p65 / RELA ChIPseq peaks were searched for motifs of RELA and REL using matrices from the JASPAR data base. P values indicated significant enrichment compared to the whole genome. The Venn diagram shows the overlap and inserts show motif compositions. (C) Venn diagrams indicating the overlap of motifs found for RELA or the RELA interactors TFE3, TFEB or GLIS2 in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions. (D) All target genes that were significantly up- or downregulated under basal or IL-1α-stimulated conditions as shown in or were collected and were examined for their association with a p65 / RELA ChIPseq peak. The pie charts show the numbers of RELA, TFE3, TFEB and GLIS2 motifs detected in siRNA RELA target genes with an annotated p65 / RELA peak in their promoters or enhancers. (E) Overlap of all genes with a p65 / RELA peak in promoters or enhancers and at least one motif for the indicated transcription factors in IL_1a-stimulated conditions. (F) Genome browser view of the TNFAIP3 locus with p65 / RELA ChIPseq peaks, activated enhancers and promoters (H3K27ac), accessible chromatin (ATACseq) and mRNA production (RNAseq) before and after 1 h of IL-1α stimulation. Data sets were from GSE64224, GSE52470 and GSE134436 and are aligned to HG19 ( ; ). p65 / RELA binding regions of 1000 bp under p65 / RELA peaks and identified TF motifs are indicated by horizontal lines. (G) HeLa cells were left untreated or were starved for 24 h in HBSS. Half of the cells was treated with IL-1α (10 ng / ml) for 1 h before the end of the experiment. ChIP-qPCR was performed with the indicated antibodies or IgG controls and a primer pair covering the TNFAIP3 promoter region (marked with an arrow in ). Floating bar plots show percent input plus the mean of all values from three independent biological replicates performed with two technical replicates. The complete set of data is provided in Supplementary Table 4.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques: Binding Assay, ChIP-qPCR

Journal: bioRxiv

Article Title: The proximity-based protein interaction landscape of the transcription factor p65 NF-κB / RELA and its gene-regulatory logics

doi: 10.1101/2024.01.03.574021

Figure Lengend Snippet: Venn diagrams indicating the overlap of RELA motifs with motifs of ZBTB factors that were found by miniTurboID to interact with RELA, in chromosomal regions assigned to p65 / RELA ChIPseq peaks. P values indicated significant enrichment compared to the whole genome. Inserts show motif compositions.

Article Snippet: 1 μg of total RNA was prepared by column purification using the NucleoSpin® RNA Kit (Macherey-Nagel; #740955.250) and transcribed into cDNA using 0.5 μl RevertAid Reverse Transcriptase (Fisher Scientific #EP0441), 4 μl 5x reaction buffer, 0.5 μl Random Hexamer Primer, 0.5 mM dNTP mix (10 mM) in a total volume of 20 μl at 25°C for 10 min, 42°C for 1 h and 70°C for 10 min. 1 μl of the reaction mixture was used to amplify cDNA using Taqman® Gene Expression Assays (0.25 μl) (Applied Biosystems) primarily for ACTB (#Hs99999903_m1), GUSB (#Hs99999908_m1), GAPDH (#Hs02758991_g1), IL8 (#Hs00174103_m1), NFKBIA (#Hs00153283_m1), CXCL2 (# Hs00236966_m1), RELA (#Hs01042019_g1) and TaqMan® Fast Universal PCR Master Mix (Applied Biosystems; #4352042).

Techniques:

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: A) Size exclusion chromatography (SEC) of 100,000 x g supernatants of MCF-7 CCM following addition of RNase A (middle) or Ribonuclease Inhibitor (RI, bottom). Red, Blue: absorbance at 260 nm and 280 nm, respectively. B) The earlier RI was added to the CCM during sample preparation, the higher the P0 peak and the lower the P2 peak (left) and therefore the higher the P1 / P2 ratio (right). C) The P0 peak was purified after SEC and treated with RNase A, which partially reconstituted the P2 peak. D) Comparison of extracellular stabilities of the P0, P1and P2 peaks. E) Size distribution of reads mapping to the 5’ half of glycine tRNA (left) or to all tRNAs (right) in the P1 peak from MCF-7 cells either with (black) or without (red) addition of RI. F) Relative representation of reads mapping to different tRNA isoacceptors in the P1 peak of MCF-7 cells obtained after treatment (top) or without treatment (bottom) of RI. G) Analysis of the P1 peak either with (+) or without (-) RI treatment (left gel) or the P0 peak (right gel) in a denaturing (7M urea) 8% polyacrylamide gel. Sizes were estimated based on a MCF-7 RNA lysate (“cells”) and a RiboRuler Low Range small RNA ladder (left marks; the 33 nt mark was calculated based on Rf). H) Size distribution of small RNA sequencing reads mapping to rRNAs (red), tRNAs (violet) or other ncRNAs (see legend) in the P0 peak of MCF-7 cells. RPM: reads per million mapped reads. I) As in (H), but showing only the reads aligning to rRNAs. The number above each peak denotes the starting position of most reads defining that peak in the corresponding rRNA. J) Amplification by random-primed RT-qPCR of different regions of 28S, 18S and 5.8S rRNAs in different fractions collected after SEC separation of MCF-7 CCM. Gly_GSP: amplification of glycine 5’ halves by using a gene-specific primer during RT. Numbers following rRNA primers (e.g., 28S_310) represent the position of the 5’ end of the expected amplicon.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Size-exclusion Chromatography, Sample Prep, Purification, RNA Sequencing Assay, Amplification, Random Primed, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: A) Schematic representation of the experimental protocol used in panels B-E. S(+): DMEM + 10% FBS. S(-): MEGM media, serum-free. B) RNA analysis by SEC in PBS washes #1 to #4 of MCF-7 cells. Conditions identical to those used in . C) An example of deconvolution analysis (Abs 260/280 ratio-to-RNA-concentration conversion) applied to a representative chromatogram of MCF-7 CCM (ultracentrifugation supernatant). D) Variation of RNA concentration corresponding to the P0 (light red) and P1 (dark red) peaks in PBS washes #1 to #4. The variation in the Abs 280 nm at the BSA peak is plotted in the right Y axis. Initial values correspond to those present in the CCM. E) Denaturing electrophoresis (7M Urea, 6% PAGE) of the concentrated P0 peak from the 4 th PBS wash of HepG2, BJ and MCF-7 cell lines. No RNA purification was performed. “Cells”: MCF-7 RNA lysate. F) U2-OS cells after incubation in serum-free DMEM plus ITS supplement for one hour. Top: monolayer. Bottom: tracking of floating nuclei (yellow line) in three consecutive shots taken one second apart from each other. G) Cluster of floating nuclei. Same conditions as in (F). H) Analysis of eIF2 alpha phosphorylation (Wblot) in nontreated (NT) MCF-7 cells, in cells exposed to four consecutive PBS washes (4 x PBS), in cells cultured after confluency (100%) or exposed to 500 µM sodium arsenite for one hour (ARS 500). Bottom: densitometry analysis in two independent biological replicates of the experiment. I) Denaturing electrophoresis (10% PAGE) of TRIzol-purified total extracellular RNA from U2-OS cells washed for 30 seconds with HBSS, ran alongside 1 µg of purified intracellular RNA from the same cell line. J) Denaturing 10% PAGE exRNA analysis in U2-OS (left) or DU 145 (right) cell-conditioned media (ITS, one hour) obtained in the presence (+) or absence (-) of 200 µM sodium arsenite. K) Denaturing electrophoresis of the purified P0 peak from E.G7-OVA cells, either treated (+) or not treated (-) with recombinant RNase-free DNase I. Sample preparation after SEC was the same as in panel (E). L) Chromatograms of cell-conditioned PBS form E.G7-OVA cells. The sample was separated into two aliquots, one of which was treated with RNase A before SEC (right). M) Proteomic analysis of the RNase-treated or not-treated (NT) P0 peak from E.G7-OVA cells. Blue: histones. Red: ribosomal proteins. NSAF: normalized spectral abundance factor. N) List of the top ten proteins from the large (left) and small (right) ribosomal subunits producing the higher number of spectra in the proteomic analysis of the P0 peak from E.G7-OVA cells.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Concentration Assay, Electrophoresis, Purification, Incubation, Cell Culture, Recombinant, Sample Prep

Journal: bioRxiv

Article Title: Fragmentation of extracellular ribosomes and tRNAs shapes the extracellular RNAome

doi: 10.1101/2020.01.29.923714

Figure Lengend Snippet: A) Cells in culture release tRNAs, ribosomal subunits or ribosomes to the extracellular nonvesicular space. When the CCM is analyzed by SEC, these RNAs define the P0 and P1 peaks, respectively. However, their detection is only possible after addition of RI to the medium. Regarding the mechanism responsible for the release of these RNAs, active secretion (e.g., autophagy-dependent) might contribute, but damaged or dead cells with compromised plasma membrane integrity are probably a main source of extravesicular exRNAs. Other forms of cell death can also release nucleosomes and fragmented DNA (right), although this can also occur actively by autophagy-dependent secretion. In contrast, live cells release EVs in a relatively continue fashion (center). These EVs contain ncRNAs such as tRNAs. Extracellular RNases degrade extravesicular RNAs and generate some stable fragmentation products. These products include tRNA halves, which can assemble into dimers and elute in the chromatographic P1 peak when RI is not added to the medium. We speculate that the P2 peak is composed of rRNA-derived fragments forming tightly bound dsRNAs which are not amenable to standard small RNA sequencing techniques. While full-length tRNAs and YRNAs are not detected in the non-EV fraction in the absence of RI, those which are present inside EVs are protected from degradation. Thus, EVs are probably the only source of full-length ncRNAs in RNase-rich extracellular samples. B) A diagram explaining possible biogenetic routes for extracellular, nonvesicular tRNA Gly GCC 5’ halves.

Article Snippet: The obtained RNA was diluted in 8 μl of ultra-pure RNase-free water, and 7 μl were used as input for NGS library preparation using the NEBNext Small RNA Library Prep Set for Illumina (New England Biolabs).

Techniques: Derivative Assay, RNA Sequencing Assay