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renilla luciferase plasmid prl-null  (Promega)

 
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    Promega renilla luciferase plasmid prl-null
    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual <t>luciferase</t> assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by <t>Renilla</t> values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.
    Renilla Luciferase Plasmid Prl Null, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/renilla luciferase plasmid prl-null/product/Promega
    Average 90 stars, based on 1 article reviews
    renilla luciferase plasmid prl-null - by Bioz Stars, 2026-03
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    Images

    1) Product Images from "Key role of TLR3 in type I IFN expression and apoptosis induction in IBDV-infected chicken fibroblast cells"

    Article Title: Key role of TLR3 in type I IFN expression and apoptosis induction in IBDV-infected chicken fibroblast cells

    Journal: bioRxiv

    doi: 10.1101/2025.07.16.665101

    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual luciferase assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by Renilla values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.
    Figure Legend Snippet: (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual luciferase assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by Renilla values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.

    Techniques Used: Expressing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay



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    renilla luciferase plasmid prl-null  (Promega)
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    Promega renilla luciferase plasmid prl-null
    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual <t>luciferase</t> assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by <t>Renilla</t> values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.
    Renilla Luciferase Plasmid Prl Null, supplied by Promega, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    plasmid prl-null  (Promega)
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    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual <t>luciferase</t> assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by <t>Renilla</t> values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.
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    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual <t>luciferase</t> assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by <t>Renilla</t> values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.
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    A , B WT and 4E KO MEF ( A ), or shScr and sh4EBP1/2 HEK293 cells ( B ) were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of two independent experiments are shown. C ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 16 h, and ACACA mRNA expression was analyzed by qRT-PCR. D ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 6 h, and translation efficiency (TE) of ACACA mRNA was calculated by measuring the levels of polysomal and total ACACA mRNA by qRT-PCR. n = 4 independent experiments. E , F WT and 4E KO MEF ( E ), or shScr and sh4EBP1/2 HEK293 cells ( F ) were transfected with an ACACA 5’UTR-containing Firefly <t>Luciferase</t> construct and a control <t>Renilla</t> Luciferase vector. Cells were grown in complete medium or glucose (Glc) starved for 6 h, and luminescence was measured. G HEK293 cells were transfected with an HA-tagged ACC1 expressing vector containing or not the ACACA 5’UTR. Cells were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of three independent experiments are shown. H , I 4E KO MEF ( H ) or sh4EBP1/2 HEK293 cells ( I ) were transfected with control siRNA (scr) or siRNAs targeting ACACA and grown in glucose starved medium (Glc strv) for 48 h. Cell death was measured by PI staining and flow cytometry. ACC1 protein levels were analyzed by immunoblotting. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test ( C – F , H , I ); n = 3 independent experiments for ( C , E , F , H , I ). Source data are provided as a Source Data file.
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    renilla luciferase prl null reporter plasmid  (Sartorius AG)
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    A , B WT and 4E KO MEF ( A ), or shScr and sh4EBP1/2 HEK293 cells ( B ) were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of two independent experiments are shown. C ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 16 h, and ACACA mRNA expression was analyzed by qRT-PCR. D ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 6 h, and translation efficiency (TE) of ACACA mRNA was calculated by measuring the levels of polysomal and total ACACA mRNA by qRT-PCR. n = 4 independent experiments. E , F WT and 4E KO MEF ( E ), or shScr and sh4EBP1/2 HEK293 cells ( F ) were transfected with an ACACA 5’UTR-containing Firefly <t>Luciferase</t> construct and a control <t>Renilla</t> Luciferase vector. Cells were grown in complete medium or glucose (Glc) starved for 6 h, and luminescence was measured. G HEK293 cells were transfected with an HA-tagged ACC1 expressing vector containing or not the ACACA 5’UTR. Cells were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of three independent experiments are shown. H , I 4E KO MEF ( H ) or sh4EBP1/2 HEK293 cells ( I ) were transfected with control siRNA (scr) or siRNAs targeting ACACA and grown in glucose starved medium (Glc strv) for 48 h. Cell death was measured by PI staining and flow cytometry. ACC1 protein levels were analyzed by immunoblotting. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test ( C – F , H , I ); n = 3 independent experiments for ( C , E , F , H , I ). Source data are provided as a Source Data file.
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    A , B WT and 4E KO MEF ( A ), or shScr and sh4EBP1/2 HEK293 cells ( B ) were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of two independent experiments are shown. C ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 16 h, and ACACA mRNA expression was analyzed by qRT-PCR. D ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 6 h, and translation efficiency (TE) of ACACA mRNA was calculated by measuring the levels of polysomal and total ACACA mRNA by qRT-PCR. n = 4 independent experiments. E , F WT and 4E KO MEF ( E ), or shScr and sh4EBP1/2 HEK293 cells ( F ) were transfected with an ACACA 5’UTR-containing Firefly <t>Luciferase</t> construct and a control <t>Renilla</t> Luciferase vector. Cells were grown in complete medium or glucose (Glc) starved for 6 h, and luminescence was measured. G HEK293 cells were transfected with an HA-tagged ACC1 expressing vector containing or not the ACACA 5’UTR. Cells were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of three independent experiments are shown. H , I 4E KO MEF ( H ) or sh4EBP1/2 HEK293 cells ( I ) were transfected with control siRNA (scr) or siRNAs targeting ACACA and grown in glucose starved medium (Glc strv) for 48 h. Cell death was measured by PI staining and flow cytometry. ACC1 protein levels were analyzed by immunoblotting. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test ( C – F , H , I ); n = 3 independent experiments for ( C , E , F , H , I ). Source data are provided as a Source Data file.
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    Image Search Results


    (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual luciferase assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by Renilla values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.

    Journal: bioRxiv

    Article Title: Key role of TLR3 in type I IFN expression and apoptosis induction in IBDV-infected chicken fibroblast cells

    doi: 10.1101/2025.07.16.665101

    Figure Lengend Snippet: (A-B) IFN-β and NF-κB promoter activities are downregulated in DF-1 TLR3 KO cells but can be rescued by exogenous expression of TLR3. DF-1 and DF-1 TLR3 KO cells were co-transfected with pLucter (100 ng) and pR-null (30 ng) plasmids (A) , or with pSI-chNFκB-Luc (50 ng) plasmid (B) together with different amounts (100, 200, 400 or 800 ng) of the plasmid expressing chTLR3-His. At 8 h pt the cells were transfected with 250 ng of Poly I:C, and harvested at 24 h after plasmid transfection. A control consisting in cells co-transfected with pLucter, pR-null and the empty pCDNA3, or with the pSI-chNFκB-Luc, and the empty pCDNA3 plasmid (800 ng) was included in each assay. The samples were analyzed by the dual luciferase assay kit. Each determination was carried out in duplicate, and the firefly luciferase expression level of each sample was normalized by Renilla values. Results are expressed as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not transfected with poly I:C. (C-D) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) (C) or with the plasmid pSI-chNFκB-Luc (50 ng) (D). At 8 h pt, cells were either treated with Poly I:C (20 µg) added directly to the culture medium or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase activity quantification. (E) DF-1 and DF-1 TLR3 KO cells were transfected with a siRNA specifically silencing the expression of MDA5 protein, or with a control siRNA. At 24 h pt, the cultures were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng) and 8 h later cells were either treated with poly I:C (20μg) or transfected with increasing amounts (100, 200 and 300 ng) of Poly I:C. The samples were harvested at 24 h after plasmid transfection and used for luciferase assay. (F) DF-1 and DF-1 TLR3 KO cells were transfected with the plasmids pLucter (100 ng) and pR-null (30 ng). At 8 h pt, cells were transfected with 250 ng of Poly I:C and subsequently treated with BFA (50μM) or the vehicle DMSO 1 h later. The samples were harvested at 24 h after plasmid transfection and analyzed using the dual luciferase assay kit. In panels C-F values are presented as the fold induction over the level found in the control DF-1 or DF-1 TLR3 KO cells not treated with Poly I:C. Bars indicate means ± standard deviations based on data of duplicate samples from three independent experiments. *, ** and *** indicate p values of <0.05, <0.01 and <0.001, respectively, as determined by unpaired Student’s test. ns, not significant.

    Article Snippet: The Renilla luciferase plasmid (pRL-null; Promega) was kindly provided for Dr. Pablo Gastaminza.

    Techniques: Expressing, Transfection, Plasmid Preparation, Control, Luciferase, Activity Assay

    A , B WT and 4E KO MEF ( A ), or shScr and sh4EBP1/2 HEK293 cells ( B ) were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of two independent experiments are shown. C ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 16 h, and ACACA mRNA expression was analyzed by qRT-PCR. D ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 6 h, and translation efficiency (TE) of ACACA mRNA was calculated by measuring the levels of polysomal and total ACACA mRNA by qRT-PCR. n = 4 independent experiments. E , F WT and 4E KO MEF ( E ), or shScr and sh4EBP1/2 HEK293 cells ( F ) were transfected with an ACACA 5’UTR-containing Firefly Luciferase construct and a control Renilla Luciferase vector. Cells were grown in complete medium or glucose (Glc) starved for 6 h, and luminescence was measured. G HEK293 cells were transfected with an HA-tagged ACC1 expressing vector containing or not the ACACA 5’UTR. Cells were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of three independent experiments are shown. H , I 4E KO MEF ( H ) or sh4EBP1/2 HEK293 cells ( I ) were transfected with control siRNA (scr) or siRNAs targeting ACACA and grown in glucose starved medium (Glc strv) for 48 h. Cell death was measured by PI staining and flow cytometry. ACC1 protein levels were analyzed by immunoblotting. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test ( C – F , H , I ); n = 3 independent experiments for ( C , E , F , H , I ). Source data are provided as a Source Data file.

    Journal: Nature Communications

    Article Title: mTORC1 regulates cell survival under glucose starvation through 4EBP1/2-mediated translational reprogramming of fatty acid metabolism

    doi: 10.1038/s41467-024-48386-y

    Figure Lengend Snippet: A , B WT and 4E KO MEF ( A ), or shScr and sh4EBP1/2 HEK293 cells ( B ) were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of two independent experiments are shown. C ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 16 h, and ACACA mRNA expression was analyzed by qRT-PCR. D ShScr and sh4EBP1/2 HEK293 cells were grown in complete medium or glucose (Glc) starved for 6 h, and translation efficiency (TE) of ACACA mRNA was calculated by measuring the levels of polysomal and total ACACA mRNA by qRT-PCR. n = 4 independent experiments. E , F WT and 4E KO MEF ( E ), or shScr and sh4EBP1/2 HEK293 cells ( F ) were transfected with an ACACA 5’UTR-containing Firefly Luciferase construct and a control Renilla Luciferase vector. Cells were grown in complete medium or glucose (Glc) starved for 6 h, and luminescence was measured. G HEK293 cells were transfected with an HA-tagged ACC1 expressing vector containing or not the ACACA 5’UTR. Cells were grown in complete medium or glucose starved (Glc strv) for the indicated times, and analyzed by immunoblotting using antibodies against the indicated proteins. Representative results of three independent experiments are shown. H , I 4E KO MEF ( H ) or sh4EBP1/2 HEK293 cells ( I ) were transfected with control siRNA (scr) or siRNAs targeting ACACA and grown in glucose starved medium (Glc strv) for 48 h. Cell death was measured by PI staining and flow cytometry. ACC1 protein levels were analyzed by immunoblotting. Data are shown as the mean ± SD. Statistics: unpaired one-sided Student’s t test ( C – F , H , I ); n = 3 independent experiments for ( C , E , F , H , I ). Source data are provided as a Source Data file.

    Article Snippet: For transfection, HEK293 cells were seeded in 12-well plates and transfected with 250 ng of each 5’UTR Firefly Luciferase reporter and 3 ng Renilla Luciferase expressing pRL null plasmid (Promega), completed to 500 ng DNA with pcDNA3.1 plasmid, using CalFectin transfection reagent (Signagen) according to the manufacturer’s guidelines.

    Techniques: Western Blot, Expressing, Quantitative RT-PCR, Transfection, Luciferase, Construct, Plasmid Preparation, Staining, Flow Cytometry