Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells bearing the indicated constructs were treated with 25 μM etoposide (Etop; DNA damage agent) for 24 h, 2 μg/mL doxycycline (Dox; viral reactivation) for 24h, or 100 μg/mL cycloheximide (Chx; translational inhibitor) for 6 h, or mock treated with drug diluent and analyzed by western blotting. γH2AX is induced during DNA damage response, KSHV SOX is a viral early protein and marker for infection, and p27 is a short-lived control for translation inhibition. Vinculin is a loading control. Black arrow indicates full length strep-tagged TFIIB and gray arrow indicated full-length endogenous TFIIB. (B) Empty or TFIIB-2xStrep expressing TRExRTA BCBL-1 cells were treated with either 100 ng/mL TRAIL ligand or mock treated with TRAIL dilution buffer for 6 h before western blotting. Caspase-3 cleavage is a control for induction of cell death. (C) Samples in (A) were probed for Caspase-3 and Caspase-8 activation by cleavage. (D) TRExRTA BCBL-1 cells expressing either TFIIB-2xStrep or mutant TFIIB(D207A)-2xStrep were treated with 100 ng/mL TRAIL ligand or mock for 8 h, followed by western blotting. Black arrow indicates cleavage product. (E) TRExRTA BCBL-1 cells expressing TFIIB-2xStrep or mutant TFIIB(D207A)-2xStrep were pretreated with 80 μM of either the caspase-3 inhibitor Z-VAD-DEVD-FMK, the pan-caspase inhibitor Z-VAD-OMe-FMK, or a negative control cysteine protease inhibitor Z-FA-FMK for 2 h. Cells were then treated with either 500 ng/mL TRAIL ligand or mock treated with TRAIL dilution buffer for 7 h followed by western blotting. Boosted strep signal has gamma contrast enhanced for visualization of the cleavage product and GAPDH probed with mouse antibody is a loading control. (F) TFIIB-2xStrep or TFIIB(D207A)-2xStrep expressing TRExRTA BCBL-1 cells were treated with 660 ng/mL TRAIL for 7 h and RNA extracted and sequenced with ribodepletion and ERCC spike-in control RNA for normalization. Volcano plot shows differentially expressed genes in cells containing TFIIB(D207A)-2xStrep relative to cells containing wild-type TFIIB-2xStrep. Genes in red are significantly differentially regulated in TRAIL-treated cells with described links to apoptosis (see Table S1 ), genes in yellow are differentially expressed in both TRAIL-treated and untreated cells, and in blue are genes with unknown function or no known link to apoptosis that are differentially expressed during TRAIL-treatment.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Construct, Western Blot, Marker, Infection, Control, Inhibition, Expressing, Activation Assay, Mutagenesis, Negative Control, Protease Inhibitor

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells treated with 100 μg/mL Chx and endogenous TFIIB protein were analyzed by western blotting. p27 is a control for translational inhibition and GAPDH probed with mouse antibody and vinculin are loading controls. (B) iSLK-KSHV(+) BAC16 cells were reactivated with doxycycline for the given timepoints prior to western blotting. Vinculin serves as a loading control. Note the lack of appreciable procaspase-3 cleavage and activation. These samples come from the same experiment as . (C) iSLK-KSHV(+) BAC16 cells treated with 250 μg/mL cycloheximide (Chx) for given timepoints. Note lack of procaspase-3 activation.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control, Inhibition, Activation Assay

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep protein were pulse-labelled for 18 h with L-HPG, harvested at the indicated timepoints post-chase for strep pulldown, then labelled with picolyl-azide-sulfo-Cy5. For quantification, strep signal was normalized to input signal and the 0 h timepoint and plotted. **** P < 0.0001, * P = 0.0103; one-way ANOVA with Tukey multiple comparison test. (B) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep were treated with 100 μg/mL cycloheximide (Chx) and harvested at indicated timepoints for western analysis. p27 is a control for Chx activity and vinculin is a loading control. (C) iSLK-KSHV(+) BAC16 cells were treated with 250 μg/mL Chx, harvested at the given timepoints and western blotted for the indicated proteins. Vinculin and GAPDH probed with mouse antibody serve as loading controls. (D) TRExRTA BCBL-1 cells or (E) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStep were treated with 100 μg/mL Chx for 6h with or without 5 μM carfilzomib proteasome inhibitor (carf) and endogenous TFIIB and strep-tagged protein analyzed by western blotting. p53 and p27 are positive controls for Chx activity and FK2 marks poly-ubiquitylated protein as a positive control for proteasomal inhibition; GAPDH probed with mouse antibody is a loading control. (F) TRExRTA BCBL-1 cells treated with 100 μg/mL Chx for 8 h with or without 10 μM TAK-243, a UBA1-targetting inhibitor of ubiquitylation, and western blotted with the indicated antibodies. NRF2 is a control for translation and ubiquitylation inhibition, and GAPDH probed with rabbit antibody is a loading control.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Expressing, Comparison, Western Blot, Control, Activity Assay, Positive Control, Inhibition

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) RT-qPCR of TFIIB RNA normalized to 18S rRNA to confirm TFIIB knockdown in TRExRTA BCBL-1 cells nucleofected with TFIIB siRNA (siTFIIB) or non-targeting siRNA (siNTC) followed by 24 h or 48 h of doxycycline (dox)-induced reactivation or mock. ns-nonsignificant, **** P < 0.0001, *** P = 0.0004, * P = 0.0285; two-way ANOVA with Tukey correction. (B) Western blots of samples in (A). SOX is a viral early gene and K8.1 is a viral late gene. GAPDH probed with mouse antibody is a loading control. (C) PCA analysis of RNA-seq samples described in (A) demonstrating triplicate consistency. (D) Violin plot of gene expression changes for differentially expressed cellular (host) and viral genes from RNA-seq analysis of siTFIIB knockdown relative to siNTC control at 48 h post-reactivation. This is the ensemble of all changes unfiltered by P value. ****P < 0.0001; Student’s T-test with Welch correction. (E) Heatmap of KSHV viral gene expression changes in RNA-seq from siTFIIB and siNTC knockdown at the given timepoints. Cells were reactivated in all conditions and normalized to 24 h gene expression in siNTC cells. (F) Heatmap of viral gene expression changes from RNA-seq analysis of TRExRTA BCBL-1 cells expressing TFIIB-2xStrep or TFIIB(D207A)-2xStrep and reactivated for 48 h.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Quantitative RT-PCR, Knockdown, Western Blot, Control, RNA Sequencing, Gene Expression, Expressing

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) RT-qPCR of TFIIB normalized to 18S RNA harvested from reactivated TRExRTA BCBL-1 cells at the given timepoints. **** P < 000.1, *** P = 0.0008; one-way ANOVA with Tukey multiple comparison test. (B) Western analysis of endogenous TFIIB in TRExRTA BCBL-1 cells or (C) iSLK-KSHV(+) BAC16 cells at timepoints post-reactivation or mock. KSHV SOX is an early gene and vinculin, and GAPDH probed with mouse is a loading control. * P = 0.01636; Student’s t-test. (D) As in (A) but in iSLK-KSHV(+) BAC16 cells. (E) Flow analysis of iSLK-KSHV(+) BAC16 cells transduced with Halo or TFIIB-Halo and mock or 48 h reactivation with doxycycline (dox) and sodium butyrate; Halo was labeled with JF646-Halo dye and measured by signal in the APC-A channel. Fluorescence distributions are shown for a representative replicate and mean APC-A quantification for all samples is shown on the right. ns-nonsignificant, ** P = 0.0057; One-way ANOVA with Tukey multiple comparison test. (F) Confocal microscopy of iSLK-KSHV(+) BAC16 cells transduced with Halo protein or TFIIB-Halo, +/-viral reactivation. Hoescht 3342 delineates nuclei. Scale bar = 20 μm.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Quantitative RT-PCR, Comparison, Western Blot, Control, Transduction, Labeling, Fluorescence, Confocal Microscopy

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) Western blotting of iSLK cells containing or lacking the KSHV BAC16. Cells were mock treated or dosed with 5 ug/mL doxycycline (Dox) and 1 mM sodium butyrate (NaBut) for 48 h. KSHV SOX is a marker for lytic viral infection and anti-GAPDH probed with rabbit antibody is a loading control. ( B) Flow analysis of iSLK cells expressing TFIIB-Halo and containing or lacking the KSHV BAC16. Cells were treated with Dox and NaBut and harvested 48h later. Halo was labeled with JF646-Halo dye and was measured by the signal in the APC-A channel and the fold change in fluorescence over mock-treated cells was quantified. *P < 0.0307; Student’s unpaired t-test.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Marker, Infection, Control, Expressing, Labeling, Fluorescence

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) iSLK-KSHV(+) BAC16 cells +/-reactivation for 36 h, treated with 250 μg/mL cycloheximide (Chx) for the indicated timepoints, then western blotted with the indicated antibodies. KSHV SOX is a control for infection, p27 is a control for translation inhibition, and GAPDH probed with rabbit antibody is a loading control. (B) TRExRTA BCBL-1 cells or (C) TRExRTA BCBL-1 cells expressing TFIIB(D207A)-2xStrep were mock-treated or reactivated for 18 h, treated with 100 μg/mL Chx, and western blotted for the indicated proteins. Vinculin is a loading control. (D) Western blots of TRExRTA BCBL-1 cells treated with 18 h of TRIM28 or nontargeting control (siNTC) siRNAs, followed by 100 μg/mL Chx treatment for the indicated times. (E) Western blots of TRExRTA BCBL-1 cells +/-18 h reactivation and treatment with 1mM of the viral DNA replication inhibitor phosphonoacetic acid (PAA) followed by a timecourse with 100 μg/mL Chx. Early viral gene SOX expression is not affected by inhibition of DNA replication, but late gene K8.1 is dependent on DNA replication and is thus not expressed. Black arrow indicates appropriate p27 band under non-specific antibody signal. GAPDH probed with mouse antibody is a loading control.

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control, Infection, Inhibition, Expressing

Journal: bioRxiv

Article Title: The RNA polymerase II general transcription factor TFIIB is a target for transcriptome control during cellular stress and viral infection

doi: 10.1101/2024.01.16.575933

Figure Lengend Snippet: (A) Western blots from of TRExRTA BCBL-1 cells nucleofected with TRIM24 siRNA or non-targeting control (siNTC) for 18 h followed by a timecourse with 100 μg/mL cycloheximide treatment (Chx). p27 is a translation inhibitor control and GAPDH probed with mouse antibody a loading control. (B) Western blots of iSLK-KSHV(+) BAC16 cells +/-reactivation and treated with 1mM of phosphonoacetic acid (PAA). SOX is a viral early gene and K8.1 is a late gene (expressed only after DNA replication).

Article Snippet: Western blotting was performed with Tris-buffered saline and 0.2% Tween-20 (TBS-T) using PageRuler Prestained protein ladder (10-180 kDa formulation; ThermoFisher) and the following primary and secondary antibodies: TFIIB (clone 2F6A3H4, Cell Signaling-4169, 1:1000), GAPDH (anti-mouse, clone 6C5, Thermofisher-AM4300, 1:5000 or anti-rabbit, clone 14C10, Cell Signaling-2118, 1:1000), p27 (ProteinTech-15086-1-AP, 1:1000), TRIM28 (Cell Signaling-4123, 1:1000), TRIM24 (clone E93TN, Cell Signaling-79030, 1:1000), Vinculin (Abcam-ab91459, 1:1000), FK2 (Enzo-BML-PW8810-0100, 1:1000), NRF2 (ProteinTech-16396-1-AP, 1:000), Caspase-3 (Cell Signaling-9662, 1:1000), Caspase-8 (clone E7, Abcam-ab32397, 1:1000), γH2AX (Bethyl-A300-081A-M, 1:1000), p53 (clone 1C12, Cell Signaling-2524, 1:1000), Goat Anti-Mouse IgG(H+L) Human ads-HRP (Southern Biotech, 1:5,000), Goat Anti-Rabbit IgG(H+L) Human ads-HRP (Southern Biotech, 1:5000), Strep-Tag II Antibody HRP Conjugate (Sigma Aldrich-71591-3, 1:2000).

Techniques: Western Blot, Control

Journal: Nucleic Acids Research

Article Title: Loss of the DNA repair protein, polynucleotide kinase/phosphatase, activates the type 1 interferon response independent of ionizing radiation

doi: 10.1093/nar/gkae654

Figure Lengend Snippet: Loss of PNKP promotes IFNβ1 secretion that mediates downstream STAT1 activation. ( A ) Immunoblots showing activation of the T1IFN response as determined by elevated IRF3 and STAT1 phosphorylation following depletion of PNKP in MCF7 cells (first 4 lanes). Secreted interferons/cytokines present in conditioned media (CM) from PNKP-depleted MCF7 cells activate STAT1 independent of radiation (last four lanes). Naïve MCF7 cells were incubated with CM from the corresponding first four lanes) for 24 h prior to cell harvesting. ( B ) CM from PNKP-depleted MCF7 cells stimulate the T1IFN response in other naïve cancer cells. Cells were allowed to attach before 24-hour incubation with CM from PNKP-depleted MCF7 cells. ( C ) ELISA assay to determine levels of secreted IFNβ1 in media supernatants in PNKP-depleted MCF7, T47D and PANC-1 cells relative to the siControl cells 90 h post transfection with siRNA (replicates: n = 5 for MCF7, and n = 4 for both T47D and PANC-1). Data are presented as standard error of the mean of n number of experiments (* P < 0.05, ** P < 0.01 and *** P < 0.001). Statistical significance was determined using unpaired Student's t -test. ( D ) Western blot analysis showing result of neutralizing antibodies (NA) against IFNβ and IFNAR2 in PNKP-depleted MCF7 cells. ( E ) Western blot analysis showing result of neutralizing antibodies against IFNβ, IFNα and IFNAR2 in PNKP-depleted MCF7 cells; time of incubation = 24 h. For the western blots data shown in (A–E) whole cell extracts were used to determine protein expression. ( F ) Subcellular fractionation of MCF7 cells reveals the localization of proteins that mediate T1IFN response following downregulation of PNKP. Fractionation purity was assessed using lamin A/C and nucleolin as nuclear markers, and tubulin and GAPDH as cytoplasmic markers. Whole cell, cytosolic and nuclear extracts were used to determine protein expression.

Article Snippet: Primary antibodies to the following proteins were used: PNKP (1:1000, sc-365724 Santa Cruz, Texas, USA; PA5-82263, Invitrogen, Massachusetts, USA), pSTAT1 (1:1000–1:3000, sc-136229, Santa Cruz; and 9167S, Cell Signaling), total STAT1 (1:1000, sc-462,1:3000, Santa Cruz; MA5-15129, 1:2000, Invitrogen), pIRF3 (1:1000, ab76493, Abcam, Cambridge, UK), total IRF3 (1:1000, 11904S, Cell Signaling, Massachusetts, USA), ISG15 (1:1000, sc-166755, Santa Cruz), STING (1:1000, 13647S, Cell Signaling), pTBK1(1:1000, ab109272, Abcam; and MA5-35869, Invitrogen), total TBK1 (1:1000, MA1-20344, Invitrogen), β-actin (1:2000, sc-47778, Santa Cruz), GAPDH (1:3000, NBP2-27103, Novus, Colorado, USA; and MA5-15738-D680, Invitrogen), β-tubulin (1:3000, 926–42211, Li-Cor, Nebraska, USA; and ab6046, Abcam), MTCO1 (1:1000, 459600, Invitrogen), MAVS (1:1000, sc-166583, Santa Cruz), TFAM (1:2000, PA5-29571, Invitrogen), TREX1 (1:1000, 15107S, Cell Signaling; and MA5-34734, Invitrogen), Lamin A/C (1:1000, MA3-1000, Invitrogen), cGAS (1:1000, PA5-76367, Invitrogen; and 15102S, Cell Signaling), Nucleolin (1:2000, ab22758, Abcam), and anti-dsDNA (1:1000, ab27156, Abcam).

Techniques: Activation Assay, Western Blot, Phospho-proteomics, Incubation, Cell Harvesting, Enzyme-linked Immunosorbent Assay, Transfection, Expressing, Fractionation