sirna Search Results


signalsilence sirna ii  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc signalsilence sirna ii
Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δ nef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag -EGFP, EGFP -LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant <t>siRNA</t> (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δ nef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP -LC3B and HIV-1 NL4-3 Δ nef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments
Signalsilence Sirna Ii, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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silence sirna targeting cjun  (Cell Signaling Technology Inc)
92
Cell Signaling Technology Inc silence sirna targeting cjun
Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δ nef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag -EGFP, EGFP -LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant <t>siRNA</t> (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δ nef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP -LC3B and HIV-1 NL4-3 Δ nef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments
Silence Sirna Targeting Cjun, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 92/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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si c jun  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc si c jun
Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA <t>(si-NT)</t> <t>or</t> <t>si-c-Jun</t> (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant
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silencer select negative control sirna  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc silencer select negative control sirna
Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA <t>(si-NT)</t> <t>or</t> <t>si-c-Jun</t> (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant
Silencer Select Negative Control Sirna, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 94/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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igg  (Cell Signaling Technology Inc)
93
Cell Signaling Technology Inc igg
Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA <t>(si-NT)</t> <t>or</t> <t>si-c-Jun</t> (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant
Igg, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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erk1 2  (Cell Signaling Technology Inc)
94
Cell Signaling Technology Inc erk1 2
Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA <t>(si-NT)</t> <t>or</t> <t>si-c-Jun</t> (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant
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foxp3  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology foxp3
The effect of LOC645166/miR-188-5p/NFKBID axis on the protein levels of <t>FOXP3</t> in Jurkat cells. (A) The effect of LOC645166 overexpressed in Jurkat cells on the protein levels FOXP3 ( N = 4). (B) The effect of miR-188-5p electro-transferred in Jurkat cells on the FOXP3 expression ( N = 4). (C) The effect of miR-188-5p co-transfected with its inhibitor in Jurkat cells on the protein levels of FOXP3 ( N = 4). (D) The effect of NFKBID overexpressed in Jurkat cells on protein levels of FOXP3 ( N = 4). (E) The effect of NFKBID knocked down by siRNA on the protein levels of FOXP3 ( N = 4).
Foxp3, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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cb1 sirna  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology cb1 sirna
Pathological changes and <t>CB1</t> expression in livers from mice infected with Schistosoma J. for 6 weeks. (A) Representative images of Masson-stained liver tissue from normal mice with minor fiber deposition around the vein (Ctrl), from infected mice showing the formation of liver fibrosis (Infected), or from infected mice treated with diphenyleneiodonium (DPI) (ip, 1 mg/kg/day). Arrows indicate different pathological changes (100 × magnification). Summarized data from three control mice, six infected mice, or six infected mice with DPI showing the area percentage of positive stain (blue color) for collagen fiber deposition in the liver tissue sections. (B) Western blot gels representing the expression of CB1 in liver homogenates from three control mice, four infected mice, or four infected mice with DPI. The bar summarized densitometric analysis from Western blot gel documents of CB1 and β-actin. *P<0.05 vs control group; # P<0.05 vs infected group.
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claudin  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology claudin
Pathological changes and <t>CB1</t> expression in livers from mice infected with Schistosoma J. for 6 weeks. (A) Representative images of Masson-stained liver tissue from normal mice with minor fiber deposition around the vein (Ctrl), from infected mice showing the formation of liver fibrosis (Infected), or from infected mice treated with diphenyleneiodonium (DPI) (ip, 1 mg/kg/day). Arrows indicate different pathological changes (100 × magnification). Summarized data from three control mice, six infected mice, or six infected mice with DPI showing the area percentage of positive stain (blue color) for collagen fiber deposition in the liver tissue sections. (B) Western blot gels representing the expression of CB1 in liver homogenates from three control mice, four infected mice, or four infected mice with DPI. The bar summarized densitometric analysis from Western blot gel documents of CB1 and β-actin. *P<0.05 vs control group; # P<0.05 vs infected group.
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sirnas for smac  (Santa Cruz Biotechnology)
97
Santa Cruz Biotechnology sirnas for smac
Pathological changes and <t>CB1</t> expression in livers from mice infected with Schistosoma J. for 6 weeks. (A) Representative images of Masson-stained liver tissue from normal mice with minor fiber deposition around the vein (Ctrl), from infected mice showing the formation of liver fibrosis (Infected), or from infected mice treated with diphenyleneiodonium (DPI) (ip, 1 mg/kg/day). Arrows indicate different pathological changes (100 × magnification). Summarized data from three control mice, six infected mice, or six infected mice with DPI showing the area percentage of positive stain (blue color) for collagen fiber deposition in the liver tissue sections. (B) Western blot gels representing the expression of CB1 in liver homogenates from three control mice, four infected mice, or four infected mice with DPI. The bar summarized densitometric analysis from Western blot gel documents of CB1 and β-actin. *P<0.05 vs control group; # P<0.05 vs infected group.
Sirnas For Smac, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 97/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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par2 sirna  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology par2 sirna
SB3 levels modulate SARS-CoV-2 infection of human bronchial cells. A) SB3 mRNA levels in the human bronchial cells (Calu3 cell line) untreated (CTR) or stimulated with the recombinant SB3 (rSB3) for 24 h. The results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. B) Immunofluorescence results of SB3 (green) in Calu-3 cells untreated (CTR) or stimulated with the rSB3 for 24 h. Cell nuclei are counterstained with Dapi (blue) and shown as merge image (lower panels). C) Paracrine effect of rSB3 on SARS-CoV-2 infection in Calu-3 cells. Calu-3 cells were infected with the SARS-CoV-2 Wuhan variant, at a MOI of 0.05 and the production of new infective virus was evaluated 48 h post infection by virus yield reduction assay. Calu-3 cells were untreated (CTR) or stimulated with rSB3 for 24 h prior to infection. D) Effect of <t>siRNA-mediated</t> knockdown of SB3 in Calu-3 cells on viral infection. Calu-3 cells were silenced for SB3 24 h prior to viral infection. TMPRSS2 siRNA-mediated knockdown was used as positive control. Data are reported as mean values ± SD of biological replicates, with each dot representing a single data point. Statistical significance was assessed using a two-tailed Student’s t test, and all p-values are reported.
Par2 Sirna, supplied by Santa Cruz Biotechnology, used in various techniques. Bioz Stars score: 93/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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e2f1  (Santa Cruz Biotechnology)
93
Santa Cruz Biotechnology e2f1
(A) Western blot of <t>E2F1</t> proteins retrieved by in-vitro-transcribed biotinylated ERINA from T47D cell nuclear extracts. Antisense and beads were used as negative controls.
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Image Search Results


Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δ nef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag -EGFP, EGFP -LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant siRNA (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δ nef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP -LC3B and HIV-1 NL4-3 Δ nef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments

Journal: Retrovirology

Article Title: Residues T 48 and A 49 in HIV-1 NL4-3 Nef are responsible for the counteraction of autophagy initiation, which prevents the ubiquitin-dependent degradation of Gag through autophagosomes

doi: 10.1186/s12977-021-00576-y

Figure Lengend Snippet: Autophagy targets HIV Gag for degradation. A HEK293T cells were transfected with HIV-1 NL4-3 Δ nef proviral DNA and treated with rapamycin (4 μM), chloroquine (60 μM) and/or ALLN (25 μM) for 12 h. 48 h later, lysates were analyzed by western blot for gp120, SQSTM1, p55, ACTB, and LC3. Densitometric analyses were performed to determine the relative ratios of gp120, SQSTM1 and p55. B HEK293T cells were co-transfected with Gag -EGFP, EGFP -LC3B or an empty vector. 48 h later, cells were harvested, and Gag was immunoprecipitated. The pulldown fraction was examined for SQSTM1 and LC3. Lysates were also analyzed by western blot for SQSTM1, Gag, LC3 and ACTB. C HEK293T cells were transfected with the HIV-1 NL4-3 provirus or an empty retroviral vector. 48 h later, cells were harvested, and LC3 was immunoprecipitated. The pulldown fraction was examined for LC3, SQSTM1, p55, gp120 and Nef. Lysates were also analyzed by western blot for gp120, SQSTM1, p55, Nef, LC3 and ACTB. D HEK293T cells treated with an irrelevant siRNA (si-ctr) or SQSTM1-specific siRNAs were transfected with HIV-1 NL4-3 Δ nef proviral DNA. 48 h post-transfection, cells were harvested and endogenous LC3 was immunoprecipitated. The pulldown fraction was examined for LC3 and p55. Lysates were analyzed by western blot for SQSTM1, p55, LC3 and ACTB. E HEK293T cells were co-transfected with EGFP -LC3B and HIV-1 NL4-3 Δ nef proviral DNA. Cells were exposed for 4 h to rapamycin (4 μM) or DMSO prior to microscopy visualization for EGFP-LC3 (green), Gag (red) and the nuclei (blue). Scale bar: 10 μm. All images are representative of three independent experiments

Article Snippet: For the SQSTM1 depletion studies, 8 × 10 5 HEK293T cells were transfected with 100 nM of SignalSilence siRNA II specific for SQSTM1 (Cell Signaling, #6399) using Lipofectamine 3000 (ThermoFisher Scientific, #L3000001), following the manufacturer’s instructions.

Techniques: Transfection, Western Blot, Plasmid Preparation, Immunoprecipitation, Retroviral, Microscopy

Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA (si-NT) or si-c-Jun (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant

Journal: Cell Communication and Signaling : CCS

Article Title: Signaling pathways regulating VDAC1 overexpression associated with apoptosis, pyroptosis, and ferroptosis

doi: 10.1186/s12964-025-02647-5

Figure Lengend Snippet: Involvement of the MAPK-p38, JNK, and CaMK-II pathways in cisplatin- and erastin-induced VDAC1 overexpression. A HeLa cells were serum-starved for 5 h, pre-incubated (2 h) with the indicated concentrations of SP203580, SP600125 or KN-62, then incubated with or without cisplatin (15 µM). After 16 h, RNA was isolated and subjected to q-RT-PCR using VDAC1 mRNA specific primers (Table S2). B , C HeLa cells were serum-starved for 5 h, pre-incubated with the indicated inhibitor (10 µM, 2 h) then incubated with or without cisplatin (10 or 15 µM, 48 h) and subjected to VDAC1 oligomerization assayed as described in the Methods section. The immunoblot with the positions of VDAC1 monomers, dimers, trimers and multimers are indicated ( B ) and the levels of VDAC1 dimers were quantified ( C ). D , E HeLa cells were serum-starved for 5 h, and pre-incubated with the JNK inhibitor, SP600125 or with the CaMK-II inhibitor, KN-62 (5 or10 µM, 2 h), then incubated with or without cisplatin (15µM, 48 h) and subjected to immunoblotting using specific antibodies against P-c-Jun, P-ATF-1 or b-actin ( D ) and their levels were quantified ( E ). F , G C6 cells were serum starved for 2 h, pre-incubated with p38-MAPK inhibitor, SB203580 (5 and 10 µM, 2 h), then incubated with or without erastin (10 µM, 24 h). Cells were subjected to immunoblotting using specific antibodies against VDAC1, P-c-Jun, P-c-Fos or P-p38. Ponceau S staining is shown as a loading control ( F ). The protein relative levels were then quantified ( G ). H-K HeLa cells were transfected with non-targeting siRNA (si-NT) or si-c-Jun (100 nM) using JetPrime ( H , I ), or with si-p38 (100 nM) using SilentFect transfection reagent ( J , K ), as described in the Methods section. At 24 h post-transfection, cells were treated with cisplatin (15 µM, 48 h) and subjected to immunoblotting for P-c-Jun, P-p38, P-c-Fos or b-actin expression using specific antibodies (H, J) . Protein expression levels were quantified ( I , K ). Cell death was analyzed by PI standing and FACS analysis and is presented in the bottom of the blots ( H , J ). Results are the means ± SEM (n = 3). ∗p ≤ 0.05, ∗∗p ≤ 0.01, ∗∗∗p ≤ 0.001; NS = non-significant

Article Snippet: The nucleotides in italic were 2′-O-methyl modified. si-c-JUN (CST-6203 S) and si-p38-MAPK (CST-6564 S) were purchased from Cell Signaling Technology (Danvers, MS).

Techniques: Over Expression, Incubation, Isolation, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Control, Transfection, Expressing

The effect of LOC645166/miR-188-5p/NFKBID axis on the protein levels of FOXP3 in Jurkat cells. (A) The effect of LOC645166 overexpressed in Jurkat cells on the protein levels FOXP3 ( N = 4). (B) The effect of miR-188-5p electro-transferred in Jurkat cells on the FOXP3 expression ( N = 4). (C) The effect of miR-188-5p co-transfected with its inhibitor in Jurkat cells on the protein levels of FOXP3 ( N = 4). (D) The effect of NFKBID overexpressed in Jurkat cells on protein levels of FOXP3 ( N = 4). (E) The effect of NFKBID knocked down by siRNA on the protein levels of FOXP3 ( N = 4).

Journal: Mediators of Inflammation

Article Title: The Long Noncoding RNA, LOC645166, in T Cells of Ankylosing Spondylitis (AS) Patients Regulates the FOXP3 Expression via the Axis of LOC645166/miR-188-5p/NFKBID

doi: 10.1155/mi/8574340

Figure Lengend Snippet: The effect of LOC645166/miR-188-5p/NFKBID axis on the protein levels of FOXP3 in Jurkat cells. (A) The effect of LOC645166 overexpressed in Jurkat cells on the protein levels FOXP3 ( N = 4). (B) The effect of miR-188-5p electro-transferred in Jurkat cells on the FOXP3 expression ( N = 4). (C) The effect of miR-188-5p co-transfected with its inhibitor in Jurkat cells on the protein levels of FOXP3 ( N = 4). (D) The effect of NFKBID overexpressed in Jurkat cells on protein levels of FOXP3 ( N = 4). (E) The effect of NFKBID knocked down by siRNA on the protein levels of FOXP3 ( N = 4).

Article Snippet: To examine the effect of NFKBID on the expression of FOXP3, the NFKBID mRNA of Jurkat cells was knocked down by using siRNA (sc-97928, Santa Cruz Biotechnology).

Techniques: Expressing, Transfection

The expression levels of FOXP3 and Treg cell frequencies in CD4 + T cells of AS patients and healthy control. (A) The levels of FOXP3 in CD4 + T cells of AS patients and healthy controls ( N = 5). (B) Flow cytometry analysis of the Treg cell frequencies in CD4 + T cells of AS patients ( N = 14) and healthy controls ( N = 13).

Journal: Mediators of Inflammation

Article Title: The Long Noncoding RNA, LOC645166, in T Cells of Ankylosing Spondylitis (AS) Patients Regulates the FOXP3 Expression via the Axis of LOC645166/miR-188-5p/NFKBID

doi: 10.1155/mi/8574340

Figure Lengend Snippet: The expression levels of FOXP3 and Treg cell frequencies in CD4 + T cells of AS patients and healthy control. (A) The levels of FOXP3 in CD4 + T cells of AS patients and healthy controls ( N = 5). (B) Flow cytometry analysis of the Treg cell frequencies in CD4 + T cells of AS patients ( N = 14) and healthy controls ( N = 13).

Article Snippet: To examine the effect of NFKBID on the expression of FOXP3, the NFKBID mRNA of Jurkat cells was knocked down by using siRNA (sc-97928, Santa Cruz Biotechnology).

Techniques: Expressing, Control, Flow Cytometry

Pathological changes and CB1 expression in livers from mice infected with Schistosoma J. for 6 weeks. (A) Representative images of Masson-stained liver tissue from normal mice with minor fiber deposition around the vein (Ctrl), from infected mice showing the formation of liver fibrosis (Infected), or from infected mice treated with diphenyleneiodonium (DPI) (ip, 1 mg/kg/day). Arrows indicate different pathological changes (100 × magnification). Summarized data from three control mice, six infected mice, or six infected mice with DPI showing the area percentage of positive stain (blue color) for collagen fiber deposition in the liver tissue sections. (B) Western blot gels representing the expression of CB1 in liver homogenates from three control mice, four infected mice, or four infected mice with DPI. The bar summarized densitometric analysis from Western blot gel documents of CB1 and β-actin. *P<0.05 vs control group; # P<0.05 vs infected group.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: Pathological changes and CB1 expression in livers from mice infected with Schistosoma J. for 6 weeks. (A) Representative images of Masson-stained liver tissue from normal mice with minor fiber deposition around the vein (Ctrl), from infected mice showing the formation of liver fibrosis (Infected), or from infected mice treated with diphenyleneiodonium (DPI) (ip, 1 mg/kg/day). Arrows indicate different pathological changes (100 × magnification). Summarized data from three control mice, six infected mice, or six infected mice with DPI showing the area percentage of positive stain (blue color) for collagen fiber deposition in the liver tissue sections. (B) Western blot gels representing the expression of CB1 in liver homogenates from three control mice, four infected mice, or four infected mice with DPI. The bar summarized densitometric analysis from Western blot gel documents of CB1 and β-actin. *P<0.05 vs control group; # P<0.05 vs infected group.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

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

Effects of chronic Schistosoma J. infection or acute Schistosoma J. soluble egg antigen (SEA) treatment on the expression of CB1 and CB2 receptors and of fibrotic markers in HSCs. (A) Relative mRNA levels of CB1 and CB2 in normal control (Ctrl) and infected HSCs (n=4). (B) Western blot gels and summarized densitometric analysis representing the difference in expression of CB1 and CB2 receptors between normal and infected HSCs (n=6). (C) Western blot gels and summarized data showing the expression of collagen I and TIMP-1 expression in normal and infected HSCs (n=6). D. Relative mRNA levels of CB1 and CB2 in SEA-treated HSCs (n=6). E. Western blot gels and summarized data representing the effect of SEA on CB1 and CB2 receptors in normal and SEA-treated HSCs (n=6). (F) Western blot gels and summarized data showing the expression of collagen I and TIMP-1 expression in normal and SEA-treated HSCs (n=6). *P<0.05 vs Ctrl.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: Effects of chronic Schistosoma J. infection or acute Schistosoma J. soluble egg antigen (SEA) treatment on the expression of CB1 and CB2 receptors and of fibrotic markers in HSCs. (A) Relative mRNA levels of CB1 and CB2 in normal control (Ctrl) and infected HSCs (n=4). (B) Western blot gels and summarized densitometric analysis representing the difference in expression of CB1 and CB2 receptors between normal and infected HSCs (n=6). (C) Western blot gels and summarized data showing the expression of collagen I and TIMP-1 expression in normal and infected HSCs (n=6). D. Relative mRNA levels of CB1 and CB2 in SEA-treated HSCs (n=6). E. Western blot gels and summarized data representing the effect of SEA on CB1 and CB2 receptors in normal and SEA-treated HSCs (n=6). (F) Western blot gels and summarized data showing the expression of collagen I and TIMP-1 expression in normal and SEA-treated HSCs (n=6). *P<0.05 vs Ctrl.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Infection, Expressing, Control, Western Blot

CB1 gene silencing blocked fibrotic changes in HSCs induced by SEA. (A) Western blot gels showing the efficiency of CB1 siRNAs to specifically silence corresponding genes in normal HSCs. The following bar summarized densitometric analysis from Western blot gel documents. (B) Western blot gels showing that CB1 siRNA prevented SEA-induced expression of collagen I, α-SMA, and TIMP-1. The following bar summarized densitometric analysis from Western blot gel documents of collagen I, α-SMA, and TIMP-1, respectively (n=6). *P<0.05 vs Scram siRNA, # P<0.05 vs Scram-SEA.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: CB1 gene silencing blocked fibrotic changes in HSCs induced by SEA. (A) Western blot gels showing the efficiency of CB1 siRNAs to specifically silence corresponding genes in normal HSCs. The following bar summarized densitometric analysis from Western blot gel documents. (B) Western blot gels showing that CB1 siRNA prevented SEA-induced expression of collagen I, α-SMA, and TIMP-1. The following bar summarized densitometric analysis from Western blot gel documents of collagen I, α-SMA, and TIMP-1, respectively (n=6). *P<0.05 vs Scram siRNA, # P<0.05 vs Scram-SEA.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Western Blot, Expressing

Superoxide production in Schistosoma J.-infected or SEA-stimulated HSCs. (A) Representative ESR traces of superoxide (O2·−) trapped by CMH using NADPH as a substrate. The bar summarized ESR data showing that SEA stimulated greater O2·− production in HSCs during chronic and acute infection (n=6). *P<0.05 vs Ctrl. (B) Summarized ESR data showing that NADPH oxidase pharmacologic inhibitor DPI (20 μM) blocked its activity. *P<0.05 vs Ctrl; # <0.05 vs SEA. (C–E) Western blot gels and summarized data showing the effects of Nox1 siRNA, Nox4 siRNA, or CB1 siRNA on protein expression of Nox1 or Nox4 in normal HSCs (n=4–6). *P<0.05 vs Scram. (F) Summarized data showing that Nox1 and Nox4 siRNA, but not CB1 siRNA, blocked SEA-induced O2·− production (n=5–6). *P<0.05 vs Scram Ctrl; # P<0.05 vs Scram-SEA. DPI, diphenyleneiodonium; Nox, NADPH oxidase; Scram: Scramble siRNA.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: Superoxide production in Schistosoma J.-infected or SEA-stimulated HSCs. (A) Representative ESR traces of superoxide (O2·−) trapped by CMH using NADPH as a substrate. The bar summarized ESR data showing that SEA stimulated greater O2·− production in HSCs during chronic and acute infection (n=6). *P<0.05 vs Ctrl. (B) Summarized ESR data showing that NADPH oxidase pharmacologic inhibitor DPI (20 μM) blocked its activity. *P<0.05 vs Ctrl; # <0.05 vs SEA. (C–E) Western blot gels and summarized data showing the effects of Nox1 siRNA, Nox4 siRNA, or CB1 siRNA on protein expression of Nox1 or Nox4 in normal HSCs (n=4–6). *P<0.05 vs Scram. (F) Summarized data showing that Nox1 and Nox4 siRNA, but not CB1 siRNA, blocked SEA-induced O2·− production (n=5–6). *P<0.05 vs Scram Ctrl; # P<0.05 vs Scram-SEA. DPI, diphenyleneiodonium; Nox, NADPH oxidase; Scram: Scramble siRNA.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Infection, Activity Assay, Western Blot, Expressing

Effects of NADPH oxidase inhibition and gene silencing on SEA-induced CB1 and CB2 expression in HSCs. (A) Western blot gels demonstrating that DPI (20 μM) blocked SEA-induced increased expression of CB1 in normal HSCs. The following bars separately summarized densitometric analysis from Western blot gel documents of CB1 and CB2 protein expression (n=4). *P<0.05 vs Vehl Ctrl; # P<0.05 vs SEA alone. (B) Representative Western blot gels showing that both Nox1 and Nox4 gene silencing by siRNA blocked SEA-induced increased expression of CB1 in normal HSCs. The below bars summarized densitometric analysis from Western blot gel documents of CB1 and CB2 (n=4). *P<0.05 vs Scram-Vehl; # P<0.05 vs Scram-SEA.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: Effects of NADPH oxidase inhibition and gene silencing on SEA-induced CB1 and CB2 expression in HSCs. (A) Western blot gels demonstrating that DPI (20 μM) blocked SEA-induced increased expression of CB1 in normal HSCs. The following bars separately summarized densitometric analysis from Western blot gel documents of CB1 and CB2 protein expression (n=4). *P<0.05 vs Vehl Ctrl; # P<0.05 vs SEA alone. (B) Representative Western blot gels showing that both Nox1 and Nox4 gene silencing by siRNA blocked SEA-induced increased expression of CB1 in normal HSCs. The below bars summarized densitometric analysis from Western blot gel documents of CB1 and CB2 (n=4). *P<0.05 vs Scram-Vehl; # P<0.05 vs Scram-SEA.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Inhibition, Expressing, Western Blot

NADPH oxidase-mediated CB1 upregulation requires Nrf2 activity in HSCs on SEA stimulation. (A) Immunofluorescence images showing the effect of SEA stimulation on the nuclear translocation of Nrf2 in normal HSCs with scram or Nox1/4 siRNAs (n=6). (B) Nrf2 activity in scram, or Nox1/4 siRNAs-transfected HSCs with or without SEA stimulation (n=6). (C) Western blot gels and summarized data showing the effects of Nrf2 siRNA on the protein expression of CB1 in normal HSCs with or without SEA stimulation (n=4–6). *P<0.05 vs Scram Vehl; # P<0.05 vs Scram SEA.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: NADPH oxidase-mediated CB1 upregulation requires Nrf2 activity in HSCs on SEA stimulation. (A) Immunofluorescence images showing the effect of SEA stimulation on the nuclear translocation of Nrf2 in normal HSCs with scram or Nox1/4 siRNAs (n=6). (B) Nrf2 activity in scram, or Nox1/4 siRNAs-transfected HSCs with or without SEA stimulation (n=6). (C) Western blot gels and summarized data showing the effects of Nrf2 siRNA on the protein expression of CB1 in normal HSCs with or without SEA stimulation (n=4–6). *P<0.05 vs Scram Vehl; # P<0.05 vs Scram SEA.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Activity Assay, Immunofluorescence, Translocation Assay, Transfection, Western Blot, Expressing

Blockade of NADPH oxidase by DPI and Nox1 or Nox4 gene silencing inhibited SEA-induced fibrotic changes in HSCs. (A) Western blot gels and summarized data showing that DPI blocked the effect of SEA-induced increased α-SMA, collagen I, and TIMP-1 protein expression (n=6). *P<0.05 vs Vehl Ctrl; # P<0.05 vs Ctrl SEA. (B) Western blot gels showing that Nox4 and Nox1 siRNA blocked the effect of SEA-induced increases in fibrotic markers α-SMA, collagen I, and TIMP-1 (n=4). *P<0.05 vs Scram Vehl; # P<0.05 vs Scram SEA.

Journal: Free radical biology & medicine

Article Title: Upregulation of cannabinoid receptor-1 and fibrotic activation of mouse hepatic stellate cells during Schistosoma J . infection: Role of NADPH oxidase

doi: 10.1016/j.freeradbiomed.2014.03.015

Figure Lengend Snippet: Blockade of NADPH oxidase by DPI and Nox1 or Nox4 gene silencing inhibited SEA-induced fibrotic changes in HSCs. (A) Western blot gels and summarized data showing that DPI blocked the effect of SEA-induced increased α-SMA, collagen I, and TIMP-1 protein expression (n=6). *P<0.05 vs Vehl Ctrl; # P<0.05 vs Ctrl SEA. (B) Western blot gels showing that Nox4 and Nox1 siRNA blocked the effect of SEA-induced increases in fibrotic markers α-SMA, collagen I, and TIMP-1 (n=4). *P<0.05 vs Scram Vehl; # P<0.05 vs Scram SEA.

Article Snippet: CB1 siRNA and Nrf2 siRNA were purchased from Santa Cruz (CB1 siRNA sc-142443; Nrf2 siRNA sc-37049).

Techniques: Western Blot, Expressing

SB3 levels modulate SARS-CoV-2 infection of human bronchial cells. A) SB3 mRNA levels in the human bronchial cells (Calu3 cell line) untreated (CTR) or stimulated with the recombinant SB3 (rSB3) for 24 h. The results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. B) Immunofluorescence results of SB3 (green) in Calu-3 cells untreated (CTR) or stimulated with the rSB3 for 24 h. Cell nuclei are counterstained with Dapi (blue) and shown as merge image (lower panels). C) Paracrine effect of rSB3 on SARS-CoV-2 infection in Calu-3 cells. Calu-3 cells were infected with the SARS-CoV-2 Wuhan variant, at a MOI of 0.05 and the production of new infective virus was evaluated 48 h post infection by virus yield reduction assay. Calu-3 cells were untreated (CTR) or stimulated with rSB3 for 24 h prior to infection. D) Effect of siRNA-mediated knockdown of SB3 in Calu-3 cells on viral infection. Calu-3 cells were silenced for SB3 24 h prior to viral infection. TMPRSS2 siRNA-mediated knockdown was used as positive control. Data are reported as mean values ± SD of biological replicates, with each dot representing a single data point. Statistical significance was assessed using a two-tailed Student’s t test, and all p-values are reported.

Journal: ACS Infectious Diseases

Article Title: SerpinB3/Protease Activated Receptor‑2 Axis Is Essential for SARS CoV‑2 Infection

doi: 10.1021/acsinfecdis.5c00145

Figure Lengend Snippet: SB3 levels modulate SARS-CoV-2 infection of human bronchial cells. A) SB3 mRNA levels in the human bronchial cells (Calu3 cell line) untreated (CTR) or stimulated with the recombinant SB3 (rSB3) for 24 h. The results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. B) Immunofluorescence results of SB3 (green) in Calu-3 cells untreated (CTR) or stimulated with the rSB3 for 24 h. Cell nuclei are counterstained with Dapi (blue) and shown as merge image (lower panels). C) Paracrine effect of rSB3 on SARS-CoV-2 infection in Calu-3 cells. Calu-3 cells were infected with the SARS-CoV-2 Wuhan variant, at a MOI of 0.05 and the production of new infective virus was evaluated 48 h post infection by virus yield reduction assay. Calu-3 cells were untreated (CTR) or stimulated with rSB3 for 24 h prior to infection. D) Effect of siRNA-mediated knockdown of SB3 in Calu-3 cells on viral infection. Calu-3 cells were silenced for SB3 24 h prior to viral infection. TMPRSS2 siRNA-mediated knockdown was used as positive control. Data are reported as mean values ± SD of biological replicates, with each dot representing a single data point. Statistical significance was assessed using a two-tailed Student’s t test, and all p-values are reported.

Article Snippet: Cell reverse transfections were carried out using Lipofectamine 3000 with 25 nM PAR2 siRNA (Santa Cruz Biotechnology, sc-36188, Dallas, Texas, USA) and negative control siRNA (Qiagen, Hilden, Germany).

Techniques: Infection, Recombinant, Gene Expression, Immunofluorescence, Variant Assay, Virus, Knockdown, Positive Control, Two Tailed Test

Immunofluorescence analysis for Spike and PAR2 in HepG2 cells overexpressing SerpinB3. A) Immunofluorescence analysis of Spike protein (red) in not permeabilized HepG2/SB3 cells untreated or silenced for PAR2 after 2 h incubation with Spike. In the merged images nuclei are counterstained with DAPI (blue). B) Quantification of Spike protein immunofluorescence signal in HepG2/SB3 cells, expressed as mean fluorescence intensity (MFI) normalized to cell count + SD (Mann–Whitney test). C) Immunofluorescence analysis of PAR2 protein (red) in HepG2/SB3 cells after PAR2 silencing. In the merged images nuclei are counterstained with DAPI (blue). D) Quantification of PAR2 protein immunofluorescence expressed as mean fluorescence intensity (MFI) normalized to cell count + SD (Mann–Whitney test). Only significant p values (p < 0.05) were reported.

Journal: ACS Infectious Diseases

Article Title: SerpinB3/Protease Activated Receptor‑2 Axis Is Essential for SARS CoV‑2 Infection

doi: 10.1021/acsinfecdis.5c00145

Figure Lengend Snippet: Immunofluorescence analysis for Spike and PAR2 in HepG2 cells overexpressing SerpinB3. A) Immunofluorescence analysis of Spike protein (red) in not permeabilized HepG2/SB3 cells untreated or silenced for PAR2 after 2 h incubation with Spike. In the merged images nuclei are counterstained with DAPI (blue). B) Quantification of Spike protein immunofluorescence signal in HepG2/SB3 cells, expressed as mean fluorescence intensity (MFI) normalized to cell count + SD (Mann–Whitney test). C) Immunofluorescence analysis of PAR2 protein (red) in HepG2/SB3 cells after PAR2 silencing. In the merged images nuclei are counterstained with DAPI (blue). D) Quantification of PAR2 protein immunofluorescence expressed as mean fluorescence intensity (MFI) normalized to cell count + SD (Mann–Whitney test). Only significant p values (p < 0.05) were reported.

Article Snippet: Cell reverse transfections were carried out using Lipofectamine 3000 with 25 nM PAR2 siRNA (Santa Cruz Biotechnology, sc-36188, Dallas, Texas, USA) and negative control siRNA (Qiagen, Hilden, Germany).

Techniques: Immunofluorescence, Incubation, Fluorescence, Cell Counting, MANN-WHITNEY

Effect of Spike on PAR2 expression and function and role of PAR2 in SARS-CoV-2 infection. A) PAR2 mRNA expression in different cell lines treated or not with recombinant Spike protein. The results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. B) Upper panel: Representative Western Blot analysis of PAR2-induced Erk1/2 phosphorylation in the Calu-3 cell line. Erk1/2 phosphorylation levels were measured after treatment with recombinant Spike protein and the SLIGKV-NH2 activating peptide. Blots display pErk1/2 levels relative to total Erk1/2. GAPDH was used as a loading control. Lower panel: Graph reporting densitometric analysis of Western blots expressed as mean values ± SD of three biological replicates. Data are normalized to GAPDH and expressed as the pErk1/2 to total Erk1/2 ratio vs untreated cells. Statistical significance was assessed by double-tailed t test. C) Efficiency of SARS-CoV-2 infection in Calu-3 cells silenced for PAR2 (siPAR2) or not (CTR) and infected with the Wuhan or Omicron variants, at a MOI of 0.05. The generation of novel infectious virus was evaluated at 48 h post-infection by virus yield reduction assay. Graphs report mean values ± SD of at least two biological replicates. Each condition was tested in triplicate per replicate. Individual replicates are shown as dots. The statistical significance was assessed by double-tailed t test.

Journal: ACS Infectious Diseases

Article Title: SerpinB3/Protease Activated Receptor‑2 Axis Is Essential for SARS CoV‑2 Infection

doi: 10.1021/acsinfecdis.5c00145

Figure Lengend Snippet: Effect of Spike on PAR2 expression and function and role of PAR2 in SARS-CoV-2 infection. A) PAR2 mRNA expression in different cell lines treated or not with recombinant Spike protein. The results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. B) Upper panel: Representative Western Blot analysis of PAR2-induced Erk1/2 phosphorylation in the Calu-3 cell line. Erk1/2 phosphorylation levels were measured after treatment with recombinant Spike protein and the SLIGKV-NH2 activating peptide. Blots display pErk1/2 levels relative to total Erk1/2. GAPDH was used as a loading control. Lower panel: Graph reporting densitometric analysis of Western blots expressed as mean values ± SD of three biological replicates. Data are normalized to GAPDH and expressed as the pErk1/2 to total Erk1/2 ratio vs untreated cells. Statistical significance was assessed by double-tailed t test. C) Efficiency of SARS-CoV-2 infection in Calu-3 cells silenced for PAR2 (siPAR2) or not (CTR) and infected with the Wuhan or Omicron variants, at a MOI of 0.05. The generation of novel infectious virus was evaluated at 48 h post-infection by virus yield reduction assay. Graphs report mean values ± SD of at least two biological replicates. Each condition was tested in triplicate per replicate. Individual replicates are shown as dots. The statistical significance was assessed by double-tailed t test.

Article Snippet: Cell reverse transfections were carried out using Lipofectamine 3000 with 25 nM PAR2 siRNA (Santa Cruz Biotechnology, sc-36188, Dallas, Texas, USA) and negative control siRNA (Qiagen, Hilden, Germany).

Techniques: Expressing, Infection, Recombinant, Gene Expression, Western Blot, Phospho-proteomics, Control, Virus

Effect of silencing for SerpinB3 and PAR2 in Calu-3. A) IFNγ mRNA expression in mock-transfected cells and in cells silenced for SB3 (siSB3) or for unrelated siRNA. B) IFNγ mRNA expression in mock-transfected cells and in cells silenced for PAR2 (siPAR2) or for unrelated siRNA. C) IFNγ mRNA expression in Calu-3 cells after 24 h treatment with the PAR2 inhibitor 1-Piperidine-Propionic Acid (1-PPA). The molecular amplification results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. The statistical significance was assessed by a Mann–Whitney test.

Journal: ACS Infectious Diseases

Article Title: SerpinB3/Protease Activated Receptor‑2 Axis Is Essential for SARS CoV‑2 Infection

doi: 10.1021/acsinfecdis.5c00145

Figure Lengend Snippet: Effect of silencing for SerpinB3 and PAR2 in Calu-3. A) IFNγ mRNA expression in mock-transfected cells and in cells silenced for SB3 (siSB3) or for unrelated siRNA. B) IFNγ mRNA expression in mock-transfected cells and in cells silenced for PAR2 (siPAR2) or for unrelated siRNA. C) IFNγ mRNA expression in Calu-3 cells after 24 h treatment with the PAR2 inhibitor 1-Piperidine-Propionic Acid (1-PPA). The molecular amplification results are expressed as the mean+SEM of gene expression, reported as 2 –Δct relative to basal values. The statistical significance was assessed by a Mann–Whitney test.

Article Snippet: Cell reverse transfections were carried out using Lipofectamine 3000 with 25 nM PAR2 siRNA (Santa Cruz Biotechnology, sc-36188, Dallas, Texas, USA) and negative control siRNA (Qiagen, Hilden, Germany).

Techniques: Expressing, Transfection, Amplification, Gene Expression, MANN-WHITNEY

(A) Western blot of E2F1 proteins retrieved by in-vitro-transcribed biotinylated ERINA from T47D cell nuclear extracts. Antisense and beads were used as negative controls.

Journal: Cancer research

Article Title: ERINA is an estrogen-responsive lncRNA that drives breast cancer through the E2F1/RB1 pathway

doi: 10.1158/0008-5472.CAN-20-1031

Figure Lengend Snippet: (A) Western blot of E2F1 proteins retrieved by in-vitro-transcribed biotinylated ERINA from T47D cell nuclear extracts. Antisense and beads were used as negative controls.

Article Snippet: The siRNAs of E2F1 (sc-29297) and RB (sc-29468) were purchased from Santa Cruz.

Techniques: Western Blot, In Vitro