Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HIF-1α is a negative regulator of interferon regulatory factors: Implications for interferon production by hypoxic monocytes

doi: 10.1073/pnas.2106017118

Figure Lengend Snippet: Schematic representation of HIF-1α’s role in hypoxic inflammation. HMGB1-exposed primary monocytes release proinflammatory cytokines and type I IFN through activating NF-κB and IRF3 and 5 ( Left ). In hypoxic conditions or following DMOG treatment only NF-κB is activated as HIF-1α represses IRF3 and IRF5 ( Right ). Thus, increased HIF-1α leads to production of proinflammatory cytokines but not IFN.

Article Snippet: Dual-reporter promoter clone for IRF3 promoter was purchased from GeneCopoeia (HPRM38174-pEZX-PG04).

Techniques:

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then treated with TNFα (8 ng/mL) or PMA (10 ng/mL), or untreated (UN) and harvested after 5 minutes (TNFα) or 20 minutes (PMA and UN). Lysates (25 μg) were resolved by SDS-PAGE PAGE and probed for phospho-p65 (S536) and p65. The image shown is representative of at least two independent experiments. B-C , HeLa cells were treated with a mock water control or 0.6 μM of the TBK1/IKKe inhibitor MRT67307 for 1.5 hours, then transfected with 10 μM 2’3’-cGAM(PS) 2 (Rp/Sp) and Trans IT-LT1 at a 1:1 μg/μL ratio, or dsDNA (2 μg/mL) and Trans IT-LT1 at a 1:2 μg/μL ratio, or untreated (UN). Cells were harvested 5 hours after treatment, and lysates were resolved with SDS-PAGE and probed for p-p65 (S536) or p65 as in A (25 μg lysate loaded) or for p-IRF3 (S386) and IRF3 (100 μg lysate loaded). Image shown is representative of at least two independent experiments.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: SDS Page, Transfection

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells were seeded in 12-well plates to achieve ∼ 65% confluency in 24 hours, at which time cells were treated with either 8 ng/mL TNFα or a water vehicle control. Cells were harvested 24 hours after treatment, and RTqPCR was performed. Resulting levels of CCL22 mRNA are shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, two-tail t test; error bars represent standard deviations. B , HeLa cells were seeded and grown as for A and treated with either 10 ng/mL PMA or a 0.0004% DMSO vehicle control. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. C , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were harvested for RTqPCR; levels of RELA/p65 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. D , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. E , HeLa cells described in C and D were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the IRF3 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. F , HeLa cells carrying the shRNAs described above were transfected as in E in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Derivative Assay, Expressing, shRNA, Stable Transfection, SDS Page, Transfection, Plasmid Preparation

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: A , HeLa cells expressing no shRNA (NS) or stably expressing a non-targeting shRNA (NT) or shRNAs against IRF3 (IRF3-1 or IRF3-2) were harvested for RTqPCR; levels of IRF3 RNA are shown relative to untreated (NS) cells. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Tukey’s pairwise comparison; error bars represent standard deviations. B , Lysates (20 μg) from HeLa cells carrying no shRNA (NS), non-targeting shRNA (NT) or shRNAs against RELA/p65 (p65-1 or p65-2) were resolved using SDS-PAGE and probed for RELA/p65 and beta-tubulin. The image shown is representative of at least three independent experiments. C , HeLa cells described in A and B were transfected with a mock control or dsDNA (2 μg/mL) with Trans IT-LT1 at a 1:2 ratio and harvested after 48 hours for RTqPCR. Resulting fold change of CCL22 mRNA is shown relative to the mock control for each individual cell line. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with a one-way ANOVA and Dunnett’s pairwise comparison of each shRNA group to the control non-targeting group; note that this analysis was performed alongside the RELA/p65 shRNA groups from in order that all non-targeting control experiments be included; error bars represent standard deviations. D , HeLa cells carrying the shRNAs described above were transfected as in C in parallel experiments using a GFP expression plasmid (2 μg/mL, Trans IT-LT1, 1:2 ratio) and imaged 48 hours after transfection. Brightfield (BF) shows the confluency of cells in the same field of view as GFP. E-F , HeLa cells ( E ) and MCF7 cells ( F ) were transfected with a mock control or 2 μg/mL of empty plasmid (EV) or the constitutively active IRF3-5D with Trans IT-LT1 (HeLa) at a 1:2 ratio or TransfeX (MCF7) at a 1:4 ratio. Cells were harvested 48 hours after transfection, and RTqPCR was performed. Resulting fold change of CCL22 mRNA relative to the mock control is shown. Each data point represents an independent experiment with values derived from three technical replicates. Significance testing was performed with an unpaired, one-tailed t test; error bars represent standard deviations.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Expressing, shRNA, Stable Transfection, Derivative Assay, SDS Page, Transfection, Plasmid Preparation, One-tailed Test

Journal: bioRxiv

Article Title: Interferon regulatory factor 3 upregulates the Treg recruitment factor CCL22 in response to double-stranded DNA in cancer cells

doi: 10.1101/2022.03.08.483519

Figure Lengend Snippet: Cells were untreated (UN) or transfected with dsDNA (2 μg/mL) using Trans IT-LT1 (JEG-3, 1:3 ratio; HCT 116, 1:4 ratio) or TransfeX (MCF7, 1:4 ratio). Cells were harvested 48 hours after transfection. Lysates (100 μg) were separated with SDS-PAGE and probed for phospho-IRF3 (S386), IRF3, and beta-tubulin. The image shown is representative of at least two independent experiments.

Article Snippet: Primary antibodies to the following human proteins were used: phospho-STING (S366, Cell Signaling Technology, cat. 19781S); RELA/p65 (R&D, cat. AF5078-SP); phospho-RELA/p65 (S536, R&D, cat. MAB72261-SP); IRF3 (R&D, cat. AF4019-SP); phospho-IRF3 (S386, Cell Signaling Technology, cat. 37829T); and beta-tubulin loading control (Abcam, cat. ab6046).

Techniques: Transfection, SDS Page

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Stearic acid cooperative effect with TNF-α for MIP-1α/CCL3 production requires IRF3. ( A ) THP-1 monocytic cells were transfected with either control or IRF3 siRNA and incubated for 36 h. Real time PCR was done to measure IRF3 expression. ( B , C ) IRF3 deficient THP-1 cells were stimulated with stearic acid and TNF-α. MIP-1α/CCL3 expression was determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. ( D ) IRF3 activity reporter monocytic cells were treated with stearic acid (200 µM) or 0.1% BSA (control) or TNF-α (10 ng/mL) or in combination. Culture media were collected after 24 h. Cell culture media were assayed for luciferase activity representing the degree of IRF3/ISRE activation using Quanti-Luc medium. ( E ) Western blot analysis showed that stearic acid induced IRF3 phosphorylation in a time dependent manner in THP-1 monocytes, verifies the role of IRF3 in the cooperative effect of stearic acid in the TNF-α mediated production of MIP-1α/CCL3. ( F ) Expression of phosphorylated IRF3 is shown as determined by densitometry of western blot bands. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Incubation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Cell Culture, Luciferase, Activation Assay, Western Blot

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: Rab6a-mediated Golgi biogenesis after BFA-WO. ( A ) Giantin (top panel) and NMIIA (bottom panel) W-B of complexes pulled down with anti-Rab6a Ab from lysates of LNCaP cells treated with BFA for 60 min and then 30 min WO. Amounts of lysates used for IP were normalized to giantin or NMIIA, as described in . Non-specific rabbit IgG was used for control IP. Red and blue markers indicate BFA and BFA-WO samples, respectively. ( B ) Confocal immunofluorescence images of Rab6a colocalization with giantin and NMIIA in LNCaP BFA-WO cells. ( C ) Quantification of Pearson’s overlap coefficient for indicated pairs of stained proteins in cells after BFA-WO. All confocal images were acquired with the same imaging parameters; bars, 10 μm. Data collected from 90 cells of three independent experiments, results are expressed as a mean ± SD; * p < 0.001. ( D ) Giantin and Rab6a W-B of the postnuclear supernatant (PNS) and Golgi membranes collected from the HeLa cells: control and treated with BFA. ( E ) The representative 3D reconstruction of the SIM imaging of Golgi membranes isolated from HeLa cells: non-treated, and treated with BFA followed by incubation with or without Rab6a protein. The regenerated Golgi membranes were stained with giantin; bars, 2 µm. ( F ) Quantification of the average length of Golgi membranes ( n = 30 from each sample) presented in ( E ). ( G ) Top panel: the reconstructed 3D SIM imaging of Golgi in HeLa cells after 30 min of BFA-WO. Cells were stained with Rab6a (green) and giantin (red). The representative area of Golgi is presented in the bottom panel. Note the Rab6a punctae between giantin-positive emerging Golgi membranes. ( H ) PLA in HeLa cells treated with DMSO, BFA, and BFA-WO. The proximity of giantin and Rab6a was evaluated using mouse anti-giantin and rabbit anti-Rab6a Abs. Red punctae indicate PLA signal, nucleus is in blue, DAPI; bars, 10 μm. ( I ) Quantitation of proximity ligation for Rab6a and giantin is presented as the corrected total fluorescence intensity (a.u.). The results are measured as a mean ± SD; * p < 0.001.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: Immunofluorescence, Staining, Imaging, Isolation, Incubation, Quantitation Assay, Ligation, Fluorescence

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: The overlap of giantin and Rab6a during Golgi biogenesis. ( A ) Confocal immunofluorescence images of Rab6a in HeLa cells after 60 min of BFA-WO, pretreated with scramble, giantin, GM130, or GRASP65 siRNAs. All confocal images acquired with the same imaging parameters; bars, 10 μm. ( B ) Quantification of cells with membranous Rab6a in cells presented in ( A ); n = 90 cells from three independent experiments, results are expressed as a mean ± SD; * p < 0.001. ( C ) Giantin immunostaining in DMSO- and BFA-treated HeLa cells. ( D ) Giantin immunostaining in HeLa cells after 60 min of BFA-WO, transfected with scramble, Rab6a siRNAs, and dominant-negative (GDP-bound) Rab6a(T27N). ( E ) Rab6a W-B of lysates of HeLa cells treated with corresponding siRNAs; β-actin was a loading control. ( F ) Quantifications of cells with perinuclear Golgi in cells from ( C , D ); n = 90 cells from three independent experiments, results expressed as a mean ± SD; *, p < 0.001.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: Immunofluorescence, Imaging, Immunostaining, Transfection, Dominant Negative Mutation

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: Schematic illustrations of Golgi biogenesis. The fusion of Golgi membranes (highlighted below) is presumably initiated by tethering of giantin from the rim of one cisterna to the rim of opposite cisterna via disulfide bond in the luminal domain. Interaction of gaintin and Rab6a via their N-terminus may provide twisting of giantin monomers required for the coiled-coil dimeric structure. Such fusion requires the force presumably created by the action of F-actin based motor protein, non-muscle Myosin IIB.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: