human hek293t  (ATCC)

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Effect of OAS1 and OAS3 variants on RNase L activation HEK293T-ACE2 cells were transfected with plasmids expressing OAS1 (A) and OAS3 (B) WT and rare variant genes. For OAS1 , we used the rs10774671 (G/G) common genotype. ‘Empty’ denotes transfection with an empty vector. At 24 hpt, the cells were left mock-transfected or transfected with poly(I:C). Total RNA was purified 24 h after poly(I:C) transfection and was analyzed in a Bioanalyzer. The amount of RNA degradation (red rectangle) was quantified by densitometry and normalized to the amount of the 18S rRNA (shown with an arrow on the left) within each sample. Densitometry values were normalized to the values in mock-treated cells transfected with the OAS1 -WT (encoding OAS1-p46) (A) or OAS3 -WT (B) plasmids. two-way ANOVA and Dunnett’s multiple comparisons (n = 2–3 per variant). Red symbols denote significant differences vs. WT (names in bold) (for OAS1 variants: ∗ p = 0.035, p = 0.015, p = 0.014, and p = 0.019 corresponding to T203I, K206Q, R256P and R256W, respectively), (for OAS3 variants: ∗ p = 0.028, p = 0.035, p = 0.032, p = 0.017, and p = 0.034 for R594C, R767C, R932Q, R932W, and A584T + A722G, respectively; & p = 0.059). (C) HEK293T-ACE2 cells were transfected with plasmids expressing the OAS1 WT (as above), OAS3 WT or the indicated variant genes. At 24 hpt, the cells were mock-infected or infected with SARS-CoV-2, and total RNA was purified 24 h after infection. RNA degradation (bands shown in the red rectangle) was quantified by densitometry and normalized to the amount of the 18S rRNA (shown with an arrow on the left). The levels in mock-treated cells, transfected with the OAS1 -WT plasmid were expressed as 1, and the other values normalized to this value. Data are represented as mean ± SD. See and for study controls.

Article Snippet: Human HEK293T , ATCC , CRL-11268.

Techniques: Activation Assay, Transfection, Expressing, Variant Assay, Plasmid Preparation, Purification, Infection

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Activation of RNase L after viral infection and effect of OAS3 on RNase L activation (A) A549-ACE2 cells (rs10774671 A/A genotype) were infected (MOI 1), during 24 and 48 h. Total RNA was purified and analyzed in a Bioanalyzer. (B) Silencing OAS1 or OAS3 with siRNAs reduced the corresponding mRNA expression and attenuated RNA degradation induced by SARS-CoV-2 at 24h. two-way ANOVA and Sidak’s multiple comparison test, ∗∗ p = 0.002 for OAS1 mRNA and ∗ p = 0.010 for OAS3 mRNA (C) Viral load of silenced cells as assessed by measuring the viral titers in the cell culture supernatants using a lysis plaque formation assay. One-way ANOVA and Dunnett’s multiple comparisons test (∗ p = 0.024). (D) Cellular extracts from poly(I:C)-transfected cells were collected and subjected to Western blot analyses using an anti-OAS3 antibody and an anti-actin antibody. Molecular weight markers are indicated (in kDa) on the right. (E and F) Parental A549-ACE2 and two clones of OAS3 KO cells were left mock-treated or treated with poly(I:C) (E), or either left mock-infected or infected with SARS-CoV-2 (MOI 1) (F). RNAs were purified and analyzed in a Bioanalyzer 24 hpt (E) or 24 and 48 hpi (F). (G) Viral titers in the cell culture supernatants of (F) were measured by a lysis plaque formation assay. two-way ANOVA and Tukey’s multiple comparison test, ∗∗ p = 0.002, ∗∗∗ p = 0.0003. (H) Cellular extracts from HEK293T-ACE2 mock-infected cells were collected and subjected to Western blot analyses using an anti-myc antibody (to detect OAS1-p42, OAS-p46 and OAS3, indicated by arrows) and an anti-actin antibody. Molecular weight markers are indicated (in kDa) on the right. (I) HEK293T-ACE2 cells and (J) A549-ACE2 OAS3 KO cells were transfected with pCAGGS plasmids expressing the OAS1 WT, OAS3 WT, or with the empty plasmid, as control. At 24 hpt, the cells were infected with SARS-CoV-2 (MOI 1) for an additional 24 h and viral titers were measured. One-way ANOVA and Sidak’s multiple comparison test, symbols indicate comparison vs. ‘Empty’ (∗) or vs. ‘OAS1-p42’ ( & ). (I) OAS1-p42 ∗∗ p = 0.0065, OAS1-p46 ∗∗∗∗ p < 0.0001, OAS3 ∗∗∗ p = 0.0003; && p = 0.0076. (J) OAS1-p46 ∗∗ p = 0.0028, OAS3 ∗∗ p = 0.0016, & p = 0.0365. Data are represented as mean ± SD.

Article Snippet: Human HEK293T , ATCC , CRL-11268.

Techniques: Activation Assay, Infection, Purification, Expressing, Comparison, Cell Culture, Lysis, Plaque Formation Assay, Transfection, Western Blot, Molecular Weight, Clone Assay, Plasmid Preparation, Control

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Effect of OAS1 and OAS3 expression on the induction of innate immune responses (A) A549-ACE2 cells (expressing OAS1-p42) were transfected twice with siRNAs specific for OAS1 or OAS3 or with a non-targeted (NT) siRNA. At 24 hpt, the cells were transfected with poly(I:C). Total RNA was purified 24h later for RT-qPCR measurement of the indicated inflammatory mediators. One-way ANOVA and Sidak’s multiple comparison test vs. NT, IFNL1: ∗∗ p = 0.0038; TNF: ∗∗ p = 0.0025, ∗∗∗ p < 0.0001; CXCL10: ∗ p = 0.0306). (B) Parental cells and two clones of OAS3 KO cells were transfected with two different concentrations of poly(I:C) (low: 50 ng/mL and high: 250 ng/mL) or none. At 24h, total RNA was purified for RT-qPCR measurement of the indicated inflammatory mediators. two-way ANOVA and Tukey’s multiple comparison test vs. WT, IFNL1: low ∗∗ p = 0.0084, high ∗∗ p = 0.0066, ∗∗∗+ p < 0.0001; TNF: ∗∗ p = 0.0062, ∗∗∗∗ p < 0.0001; CXCL10: ∗∗ p = 0.001; ∗∗∗ p = 0.0007. (C) A549-ACE2 cells were transfected twice with siRNAs specific for OAS1 or OAS3 or with a non-targeted (NT) siRNA and were treated with SARS-CoV-2. Inflammatory cytokine mRNA was determined at 24 hpi. One-way ANOVA and Sidak’s multiple comparison test vs. NT, IFNL1: ∗ p = 0.0463; TNF: ∗ p = 0.0298. (D) OAS3 KO A549-ACE2 cells were transfected with pCAGGS plasmids expressing the OAS1 WT (G/G allele encoding p46) or OAS3 WT forms, or with the empty plasmid, as control. At 24 hpt, the cells were transfected with poly(I:C) and total RNA was purified 24h later. The expression levels of IFNL1, TNF, CCL2, and CXCL10 mRNA were evaluated by RT-qPCR and are expressed as fold change in comparison to cells not exposed to poly(I:C). r.u.: relative units. One-way ANOVA and Sidak’s multiple comparison test vs. Empty, IFNL1: ∗ p = 0.0242; TNF:∗ p = 0.0289; CCL2: ∗∗ p = 0.0019, ∗∗∗ p = 0.0008; CXCL10: ∗ p = 0.0322 for PAS1p46, ∗ p = 0.0403 for OAS3. See also . (E) HEK293T-ACE2 cells were transfected with an empty plasmid, as control, or with plasmids expressing OAS1 rs10774671 (G/G) common genotype either WT or containing rare LOF variant genes. At 24 hpt, the cells were left mock-transfected as controls or transfected with poly(I:C); Total RNA was purified 24 h after poly(I:C) transfection and was analyzed; n = 4 samples obtained in two independent experiments. Values were compared vs. control (∗∗) or vs. WT (&) with one-way ANOVA and Sidak’s multiple comparison tests. IFNL1: ∗∗ p = 0.0026, & p = 0.0215 & p = 0.0215 for K206Q and & p = 0.0296 for R256W. TNF: ∗∗∗∗ p < 0.0001; ∗∗∗ p = 0.0006 for K206Q, ∗∗∗ p = 0.0003 for R256W; & p = 0.0248. Data are represented as mean ± SD.

Article Snippet: Human HEK293T , ATCC , CRL-11268.

Techniques: Expressing, Transfection, Purification, Quantitative RT-PCR, Comparison, Clone Assay, Plasmid Preparation, Control, Variant Assay

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Effect of OAS1 and OAS3 variants on RNase L activation HEK293T-ACE2 cells were transfected with plasmids expressing OAS1 (A) and OAS3 (B) WT and rare variant genes. For OAS1 , we used the rs10774671 (G/G) common genotype. ‘Empty’ denotes transfection with an empty vector. At 24 hpt, the cells were left mock-transfected or transfected with poly(I:C). Total RNA was purified 24 h after poly(I:C) transfection and was analyzed in a Bioanalyzer. The amount of RNA degradation (red rectangle) was quantified by densitometry and normalized to the amount of the 18S rRNA (shown with an arrow on the left) within each sample. Densitometry values were normalized to the values in mock-treated cells transfected with the OAS1 -WT (encoding OAS1-p46) (A) or OAS3 -WT (B) plasmids. two-way ANOVA and Dunnett’s multiple comparisons (n = 2–3 per variant). Red symbols denote significant differences vs. WT (names in bold) (for OAS1 variants: ∗ p = 0.035, p = 0.015, p = 0.014, and p = 0.019 corresponding to T203I, K206Q, R256P and R256W, respectively), (for OAS3 variants: ∗ p = 0.028, p = 0.035, p = 0.032, p = 0.017, and p = 0.034 for R594C, R767C, R932Q, R932W, and A584T + A722G, respectively; & p = 0.059). (C) HEK293T-ACE2 cells were transfected with plasmids expressing the OAS1 WT (as above), OAS3 WT or the indicated variant genes. At 24 hpt, the cells were mock-infected or infected with SARS-CoV-2, and total RNA was purified 24 h after infection. RNA degradation (bands shown in the red rectangle) was quantified by densitometry and normalized to the amount of the 18S rRNA (shown with an arrow on the left). The levels in mock-treated cells, transfected with the OAS1 -WT plasmid were expressed as 1, and the other values normalized to this value. Data are represented as mean ± SD. See and for study controls.

Article Snippet: Human lung epithelial carcinoma A549 (ATCC CCL-185, derived from lung carcinomatous tissue from a 58-year-old Caucassian male), human embryonic kidney HEK293T (ATCC CRL-11268, derived from a fetus), Vero E6 and Vero cells overexpressing the TMPRSS2 (Vero-TMPRSS2, kindly provided by Prof. Luis Enjuanes, CNB-CSIC, derived from the kidney of a normal adult African Green monkey) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc.) containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in a 5% CO 2 incubator.

Techniques: Activation Assay, Transfection, Expressing, Variant Assay, Plasmid Preparation, Purification, Infection

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Activation of RNase L after viral infection and effect of OAS3 on RNase L activation (A) A549-ACE2 cells (rs10774671 A/A genotype) were infected (MOI 1), during 24 and 48 h. Total RNA was purified and analyzed in a Bioanalyzer. (B) Silencing OAS1 or OAS3 with siRNAs reduced the corresponding mRNA expression and attenuated RNA degradation induced by SARS-CoV-2 at 24h. two-way ANOVA and Sidak’s multiple comparison test, ∗∗ p = 0.002 for OAS1 mRNA and ∗ p = 0.010 for OAS3 mRNA (C) Viral load of silenced cells as assessed by measuring the viral titers in the cell culture supernatants using a lysis plaque formation assay. One-way ANOVA and Dunnett’s multiple comparisons test (∗ p = 0.024). (D) Cellular extracts from poly(I:C)-transfected cells were collected and subjected to Western blot analyses using an anti-OAS3 antibody and an anti-actin antibody. Molecular weight markers are indicated (in kDa) on the right. (E and F) Parental A549-ACE2 and two clones of OAS3 KO cells were left mock-treated or treated with poly(I:C) (E), or either left mock-infected or infected with SARS-CoV-2 (MOI 1) (F). RNAs were purified and analyzed in a Bioanalyzer 24 hpt (E) or 24 and 48 hpi (F). (G) Viral titers in the cell culture supernatants of (F) were measured by a lysis plaque formation assay. two-way ANOVA and Tukey’s multiple comparison test, ∗∗ p = 0.002, ∗∗∗ p = 0.0003. (H) Cellular extracts from HEK293T-ACE2 mock-infected cells were collected and subjected to Western blot analyses using an anti-myc antibody (to detect OAS1-p42, OAS-p46 and OAS3, indicated by arrows) and an anti-actin antibody. Molecular weight markers are indicated (in kDa) on the right. (I) HEK293T-ACE2 cells and (J) A549-ACE2 OAS3 KO cells were transfected with pCAGGS plasmids expressing the OAS1 WT, OAS3 WT, or with the empty plasmid, as control. At 24 hpt, the cells were infected with SARS-CoV-2 (MOI 1) for an additional 24 h and viral titers were measured. One-way ANOVA and Sidak’s multiple comparison test, symbols indicate comparison vs. ‘Empty’ (∗) or vs. ‘OAS1-p42’ ( & ). (I) OAS1-p42 ∗∗ p = 0.0065, OAS1-p46 ∗∗∗∗ p < 0.0001, OAS3 ∗∗∗ p = 0.0003; && p = 0.0076. (J) OAS1-p46 ∗∗ p = 0.0028, OAS3 ∗∗ p = 0.0016, & p = 0.0365. Data are represented as mean ± SD.

Article Snippet: Human lung epithelial carcinoma A549 (ATCC CCL-185, derived from lung carcinomatous tissue from a 58-year-old Caucassian male), human embryonic kidney HEK293T (ATCC CRL-11268, derived from a fetus), Vero E6 and Vero cells overexpressing the TMPRSS2 (Vero-TMPRSS2, kindly provided by Prof. Luis Enjuanes, CNB-CSIC, derived from the kidney of a normal adult African Green monkey) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc.) containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in a 5% CO 2 incubator.

Techniques: Activation Assay, Infection, Purification, Expressing, Comparison, Cell Culture, Lysis, Plaque Formation Assay, Transfection, Western Blot, Molecular Weight, Clone Assay, Plasmid Preparation, Control

Journal: iScience

Article Title: OAS1 and OAS3 genetic variants enhance inflammatory responses to SARS-CoV-2

doi: 10.1016/j.isci.2025.113966

Figure Lengend Snippet: Effect of OAS1 and OAS3 expression on the induction of innate immune responses (A) A549-ACE2 cells (expressing OAS1-p42) were transfected twice with siRNAs specific for OAS1 or OAS3 or with a non-targeted (NT) siRNA. At 24 hpt, the cells were transfected with poly(I:C). Total RNA was purified 24h later for RT-qPCR measurement of the indicated inflammatory mediators. One-way ANOVA and Sidak’s multiple comparison test vs. NT, IFNL1: ∗∗ p = 0.0038; TNF: ∗∗ p = 0.0025, ∗∗∗ p < 0.0001; CXCL10: ∗ p = 0.0306). (B) Parental cells and two clones of OAS3 KO cells were transfected with two different concentrations of poly(I:C) (low: 50 ng/mL and high: 250 ng/mL) or none. At 24h, total RNA was purified for RT-qPCR measurement of the indicated inflammatory mediators. two-way ANOVA and Tukey’s multiple comparison test vs. WT, IFNL1: low ∗∗ p = 0.0084, high ∗∗ p = 0.0066, ∗∗∗+ p < 0.0001; TNF: ∗∗ p = 0.0062, ∗∗∗∗ p < 0.0001; CXCL10: ∗∗ p = 0.001; ∗∗∗ p = 0.0007. (C) A549-ACE2 cells were transfected twice with siRNAs specific for OAS1 or OAS3 or with a non-targeted (NT) siRNA and were treated with SARS-CoV-2. Inflammatory cytokine mRNA was determined at 24 hpi. One-way ANOVA and Sidak’s multiple comparison test vs. NT, IFNL1: ∗ p = 0.0463; TNF: ∗ p = 0.0298. (D) OAS3 KO A549-ACE2 cells were transfected with pCAGGS plasmids expressing the OAS1 WT (G/G allele encoding p46) or OAS3 WT forms, or with the empty plasmid, as control. At 24 hpt, the cells were transfected with poly(I:C) and total RNA was purified 24h later. The expression levels of IFNL1, TNF, CCL2, and CXCL10 mRNA were evaluated by RT-qPCR and are expressed as fold change in comparison to cells not exposed to poly(I:C). r.u.: relative units. One-way ANOVA and Sidak’s multiple comparison test vs. Empty, IFNL1: ∗ p = 0.0242; TNF:∗ p = 0.0289; CCL2: ∗∗ p = 0.0019, ∗∗∗ p = 0.0008; CXCL10: ∗ p = 0.0322 for PAS1p46, ∗ p = 0.0403 for OAS3. See also . (E) HEK293T-ACE2 cells were transfected with an empty plasmid, as control, or with plasmids expressing OAS1 rs10774671 (G/G) common genotype either WT or containing rare LOF variant genes. At 24 hpt, the cells were left mock-transfected as controls or transfected with poly(I:C); Total RNA was purified 24 h after poly(I:C) transfection and was analyzed; n = 4 samples obtained in two independent experiments. Values were compared vs. control (∗∗) or vs. WT (&) with one-way ANOVA and Sidak’s multiple comparison tests. IFNL1: ∗∗ p = 0.0026, & p = 0.0215 & p = 0.0215 for K206Q and & p = 0.0296 for R256W. TNF: ∗∗∗∗ p < 0.0001; ∗∗∗ p = 0.0006 for K206Q, ∗∗∗ p = 0.0003 for R256W; & p = 0.0248. Data are represented as mean ± SD.

Article Snippet: Human lung epithelial carcinoma A549 (ATCC CCL-185, derived from lung carcinomatous tissue from a 58-year-old Caucassian male), human embryonic kidney HEK293T (ATCC CRL-11268, derived from a fetus), Vero E6 and Vero cells overexpressing the TMPRSS2 (Vero-TMPRSS2, kindly provided by Prof. Luis Enjuanes, CNB-CSIC, derived from the kidney of a normal adult African Green monkey) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM; Mediatech, Inc.) containing 10% fetal bovine serum (FBS) and 2 mM L-glutamine at 37°C in a 5% CO 2 incubator.

Techniques: Expressing, Transfection, Purification, Quantitative RT-PCR, Comparison, Clone Assay, Plasmid Preparation, Control, Variant Assay

Journal: bioRxiv

Article Title: Enhancing the biocorrosion resistance and biocompatibility of Aluminium substrates using Graphene Oxide-PEDOT:PSS Hybrid Coating

doi: 10.1101/2025.09.29.679258

Figure Lengend Snippet: Comparison of cell viability, assessed by MTT method in the DMEM culture medium, after exposure to ( A ) Al_GO/P sample extracts by indirect contact assay and ( B ) after direct contact with the Al_GO/P samples; at ( i ) 24-hour and ( ii ) 48-hour. Data are expressed as % values of result obtained in comparison to control cells (HEK293T) not exposed to materials or material extracts. Results from indirect contact assay showed Al_GO/P substrates maintained optimal biocompatibility compared to bare Al over 48 hours. While direct contact assay indicated high cell viability for Al_GO50P and Al_GO100P, with Al_GO250P showing minor viability changes at 48 hours, while significant decreases were observed for Al_GO500P and Al_GO1000P over 48 hours. Error bars represent ±1 standard deviation (SD) from mean forthree independent biological replicates (n=3). Statistical significance was assessed using one-way ANOVA; specific p-values of <0.05, <0.01, <0.001, and <0.0001 were indicated as *,**,***, and ****, respectively.

Article Snippet: The Human Embryonic Kidney (HEK293T) cell line [ATCC CRL-11268] was used.

Techniques: Comparison, Control, Standard Deviation