pcdna3 1 neo vector  (Addgene inc)

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: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

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: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

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: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

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: pcDNA3.1(+)-neo was obtained from ThermoFisher (Invitrogen cat. V790-20). pcDNA3.1(+)-neo-IRF3-5D was created using the wild-type template Human V5-IRF3-pcDNA3, a gift from Saumen Sarkar (Addgene plasmid # 32713; http://n2t.net/addgene:32713 ; RRID:Addgene_32713, ( )) and the Q5 site-directed mutagenesis kit (NEB, cat. E0554S) with the forward and reverse primers 5’-CTCGATCTCGACGATGACCAGTACAAGGCCTAC and 5’-TGGGTGGTCGTTGTCAATGTGCAGGTCCACAGT respectively; mutations were confirmed with sequencing. pCpGf-Bsr-GFP was constructed by PCR-amplifying GFP obtained from pSELECT-zeo-GFPBsr (Invivogen, cat. psetz-zgfpbsr) with a Kozak sequence encoded on the forward primer, then inserting it into pCpGfree-vitroBmcs (Invivogen, cat. pcpgvtb-mcsg2) digested with BglII and ApaL1; insertion was confirmed with sequencing. pcDNA3.1(+)puro was previously described ( ). pCpGfree-mcs was obtained from Invivogen (cat. pcpgf-mcs).

Techniques: Transfection, SDS Page