a549 ko cells  (Addgene inc)

Journal: Viruses

Article Title: Mitochondrial Antiviral Signaling (MAVS) Protein Modulates the Transition from Acute to Persistent Parainfluenza Virus Infection and Resistance to Complement-Mediated Cell Lysis

doi: 10.3390/v18040416

Figure Lengend Snippet: Loss of MAVS impairs antiviral gene induction independent of STAT1. ( A ) WT or MAVS KO A549 cells were mock-infected or infected with Le-PIV5 at an MOI of 10 and harvested at 24 or 48 hpi. Total RNA was analyzed by quantitative PCR to assess expression of IFN-β, IFIT1, and OAS2. Gene expression levels are expressed relative to the corresponding mock-infected controls. ( B ) Protein lysates from WT or MAVS KO cells that were mock-infected or infected with Le-PIV5 at a MOI of 10 were collected at 24 and 48 hpi and analyzed by Western blotting for STAT1 protein. β-actin was used as a loading control. ** indicates a p value < 0.01, *** indicates a p value < 0.001, and **** indicates a p value < 0.0001.

Article Snippet: A549 KO-MAVS cells were purchased commercially (A549-DualTM KO-MAVS cells, catalog #a549d-komavs; InvivoGen, San Diego, CA, USA) and grown in DMEM 10% HI FBS, 100 U/mL penicillin, 100 μg/mL streptomycin, 100 μg/mL Normocin.

Techniques: Infection, Real-time Polymerase Chain Reaction, Expressing, Gene Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: S6K1 signaling confers radioresistance to lung cancer cells. ( A ) Cells were radiated at the shown doses, and the surviving fractions were calculated for each cell line as explained in the . Note the dramatic decrease at 4 Gy of the surviving fraction (SF) in the sensitive cells H23 (SF: 0.0001) and H226 (SF: 0.11) compared to the most resistant H661 (SF: 0.4) and A549 cells (SF: 0.52). ( B ) Cells were irradiated at the indicated doses, and cell proliferation was evaluated 4 days after radiation using Alamar blue. Again, H23 and H226 cells showed a lower proliferation rate in our cell models compared to no-irradiated controls, against the most resistant cells, H661 and A549 ( C ) Clonogenic assays showing the colony formation after a 4 Gy dose of radiation. H23 is clearly the most sensitive cell to radiation, followed by H226, H661, and A549. ( D ) Western blot experiments showing higher phosphoactivation of S6 and S6K1 in most radioresistant cells A549 and H661. ( E ) Quantification of Immunoblots using ImageJ software (version 1.54r). The most radioresistance cells H661 and A549 showed an increase in the expression of pS6, the main target of S6K1, with a fold change of 1.7 and 1.8, respectively, compared to the most sensitive H23, used as an internal control. * Denotes a p value < 0.05. *** Denotes a p value < 0.0001. Statistical differences were determined using Tukey’s test as explained in the methods. ( F ) S6K1 expression levels from control patients (non-tumor tissue; n : 104) and lung tumor patients ( n : 986) were downloaded from the Xena TCGA database (University of California).

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Irradiation, Western Blot, Software, Expressing, Control

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: Inhibition of S6K1 increases radiation sensitivity of lung cancer cells. ( A ) Top: Immunoblot assay showing that the pharmacological inhibition of S6K1 with PF-4708671 (5 μM) for 48 h reduces the phosphorylation of S6, a downstream target of S6K1. Bottom: quantitation of p-S6 using ImageJ. Note a reduction of 53% (H661) and 95% (A549) in the expression of pS6 in cells treated with PF-4708671 compared to controls. Statistical differences were determined using a Student’s t -test. ( B , C ) Colony formation in cells pre-treated with DMSO or PF-4708671 plus radiation. Then, cells were treated with low doses of radiation (2 Gy). PF-4708671 was kept until the end of the experiment. Surviving fraction was calculated for each condition compared to non-treated controls. Data showed that PF-4708671 dramatically sensitized the resistant cells H661 (SF: 0.12) and A549 (SF:0.12) to low doses of radiation. ( D ) S6K1 KO cells and control wild types were seeded as before for clonogenic assays, and the surviving colonies were stained and counted. S6K1 genetic deletion decreases the average colony formation (CF) (CF-KO1: 4; CF-KO2: 8) compared to control (CF: 24) after radiation. Statistical differences were determined using Tukey’s test as explained in the methods. Right panel: S6K1 KO was confirmed by Western blot. * Denotes a p value < 0.05. ** Denotes a p value < 0.001. # Denotes the number.

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Inhibition, Western Blot, Phospho-proteomics, Quantitation Assay, Expressing, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: S6K1 Modulates STAT3 Activation to Promote Resistance to Radiotherapy in Lung Cancer

doi: 10.3390/ijms27041915

Figure Lengend Snippet: STAT3 activation increases after radiation to promote radioresistance modulated by S6K1. ( A ) Compared to non-irradiated controls, STAT3 and STAT3 phosphoactivation increases after a single dose of 10 Gy in H661 and A549 cells at 24 and 48 h. ( B ) A549 S6K1-KO cells, transfected with a plasmid expressing a constitutively active form of S6K1 protein, showed an increase in the phospho-activation of STAT3 before and after radiation. ( C ) PF-4708671 (5 µM) antagonizes the phospho-activation of STAT3 and the expression of c-myc in A549 cells after radiation. Protein expressions were studied by Western Blot. ( D ) Expression analysis showing the downregulation of STAT3 activation (p-Ser727) in S6K1 KO cells. S6K1 deletion decreases the p-STAT3 expression after radiation. ( E ) A549 cells treated with the inhibitor Stattic plus radiation showed the lowest number of colonies (CF: 1.3) compared to radiotherapy (CF: 10) or static (CF: 13) alone and control (DMSO; CF: 43) (* p < 0.05, ** p < 0.005, and **** p < 0.0001). ( F ) Transcriptional activity of STAT3 was measured in the presence of vehicle, PF-4708671 (5 μM), radiation (10 Gy) or the combination by using the Dual-Glo ® Luciferase Assay System. PF-4708671 decreased the STAT3 transcriptional activity compared to control, before and after radiation (* p < 0.05, *** p < 0.0005). Statistical differences were determined using Tukey’s test as explained in the methods.

Article Snippet: For S6K1 re-expression in A549 KO cells, we used the pRK7-HA-S6K1-F5A-E389-R3A plasmid [ ] (Addgene # 8991; RRID: Addgene_8991, Watertown, MA, USA).

Techniques: Activation Assay, Irradiation, Transfection, Plasmid Preparation, Expressing, Western Blot, Control, Activity Assay, Luciferase

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intracellular C3 regulates the immune response to infection via NF-κB signaling

doi: 10.1007/s00018-025-05975-4

Figure Lengend Snippet: C3 deficiency alters gene expression in human lung epithelial A549 cell line, which is rescued by cytosolic C3. A ) Three variants of A549 cells expressing C3 (WT), entirely lacking C3 (C3 KO) or expressing only cytosolic C3 (C3 ΔATG1) were used in this study. Western blotting analysis of cell lysates showing the presence of pro-C3 along with mature alpha and beta chains in WT cells ( B ). No C3 was detected in C3 KO cells while C3 ΔATG1 clones expressed only pro-C3 ( C ). D ) While WT and C3 ΔATG1 cells showed similar gene expression, C3 KO cells had strongly altered gene expression compared to WT and C3 ΔATG1 (padj < 0.05). E ) Heat map representation of the expression patterns in WT, C3 KO and C3 ΔATG1 clones (4 clones for each genotype) and F ) Principal component (PC) analysis of RNA-seq data for WT, C3 KO and C3 ΔATG1 clones showed that C3 KO clones cluster separately from WT and C3 ΔATG1 clones. G ) Venn diagram of DEGs across different genotypes. Summary of KEGG enrichment analysis showing pathways with >2 differentially expressed genes between WT and C3 KO ( H ) and between C3 KO and C3 ΔATG1 ( I ). For analysis of differentially regulated genes, we used the following criteria: pair-wise analysis with padj≤ 0.05 & |log2(foldchange)| ≥ 1

Article Snippet: Two individual clones each of WT, C3 ΔATG1 and C3 KO A549 cells were treated with either 1 μg/mL Pam3CSK4 (InvivoGen #tlrl-pms), 1 μg/mL of LPS (InvivoGen #tlrl-eklps), 0.1 ng/mL IL-1β (Mabtech #3415–1 H-6), 10 ng/mL TNF (ImmunoTools #11343015) or 100 ng/mL poly(I: C) (InvivoGen #tlrl-picw) and incubated for 24 h before the supernatants were collected and cells were lysed in lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP40 and 0.5% sodium deoxycholate, supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) (Thermo ScientificTM, #78441).

Techniques: Gene Expression, Expressing, Western Blot, Clone Assay, RNA Sequencing

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intracellular C3 regulates the immune response to infection via NF-κB signaling

doi: 10.1007/s00018-025-05975-4

Figure Lengend Snippet: Cytokine secretion from A549 cells is modulated by the presence of C3. XL cytokine array analysis of supernatants from WT and C3 KO A549 cells infected with M. catarrhalis shows lower secretion from C3 KO cells. Reference spots and negative controls are indicated by + and –, respectively ( A ) with quantification in ( B ), and black arrows indicating IL-8 and MCP-1, as differentially secreted cytokines. Bio-Plex Pro human cytokine 27-plex assay with supernatants from WT, C3 KO and C3 ΔATG1 cells infected with M. catarrhalis ( C ) or Y. enterocolitica ( D ) shows lower secretion from C3 KO cells that is rescued in C3 ΔATG1. Bars represent the average of three biological repetitions indicated with symbols. Two-way ANOVA with Tukey´s multiple comparison test, matched measures within the experiment, * p < 0.05, **** p < 0.0001

Article Snippet: Two individual clones each of WT, C3 ΔATG1 and C3 KO A549 cells were treated with either 1 μg/mL Pam3CSK4 (InvivoGen #tlrl-pms), 1 μg/mL of LPS (InvivoGen #tlrl-eklps), 0.1 ng/mL IL-1β (Mabtech #3415–1 H-6), 10 ng/mL TNF (ImmunoTools #11343015) or 100 ng/mL poly(I: C) (InvivoGen #tlrl-picw) and incubated for 24 h before the supernatants were collected and cells were lysed in lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP40 and 0.5% sodium deoxycholate, supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) (Thermo ScientificTM, #78441).

Techniques: Infection, Plex Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intracellular C3 regulates the immune response to infection via NF-κB signaling

doi: 10.1007/s00018-025-05975-4

Figure Lengend Snippet: Cytosolic C3 contributes to cytokine release after stimulation of different receptors. ( A - F ) Levels of cytokine secretion measured by ELISA in supernatants from WT, C3 KO and C3 ΔATG1 A549 cells after infection with bacteria are reduced in cells lacking C3. IL-8 secretion after infection with M. catarrhalis ( A ), IL-6 after infection Y. enterocolitica ( B ), RANTES secretion after infection with M. catarrhalis ( C ), RANTES secretion after infection with Y. enterocolitica ( D ), MCP-1 secretion after infection with M. catarrhalis ( E ), and MCP-1 secretion after infection with Y. enterocolitica ( F ). ( G - M ) Levels of cytokine secretion in supernatants from WT, C3 KO and C3 ΔATG1 A549 cells are reduced in cells lacking C3 after stimulation of TLR or TNFR, but not after IL-1R stimulation. IL-8 secretion after treatment with Pam3CSK4 ( G ), IL-6 secretion after treatment with TNF ( H ), sICAM-1 release after treatment with Pam3CSK4 ( I ), sICAM-1 release after TNF treatment ( J ), IL-8 secretion after poly(I: C) treatment ( K ), MCP-1 secretion after poly(I: C) treatment ( L ), and IL-8 secretion after IL-1β treatment ( M ). Results are shown as mean ± SD of independent experiments. Each dot represents an average of at least three clones in one biological repeat. The cytokine secretion levels of untreated cells were subtracted from the values of treated cells. One-way ANOVA with Tukey´s multiple comparison test, matched measures within experiment for C-F and I-M. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, ns: not significant

Article Snippet: Two individual clones each of WT, C3 ΔATG1 and C3 KO A549 cells were treated with either 1 μg/mL Pam3CSK4 (InvivoGen #tlrl-pms), 1 μg/mL of LPS (InvivoGen #tlrl-eklps), 0.1 ng/mL IL-1β (Mabtech #3415–1 H-6), 10 ng/mL TNF (ImmunoTools #11343015) or 100 ng/mL poly(I: C) (InvivoGen #tlrl-picw) and incubated for 24 h before the supernatants were collected and cells were lysed in lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP40 and 0.5% sodium deoxycholate, supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) (Thermo ScientificTM, #78441).

Techniques: Enzyme-linked Immunosorbent Assay, Infection, Bacteria, Clone Assay, Comparison

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Intracellular C3 regulates the immune response to infection via NF-κB signaling

doi: 10.1007/s00018-025-05975-4

Figure Lengend Snippet: Cytokine gene transcription and NF-κB pathway activation are dependent on cytosolic C3. mRNA levels for IL-6 are decreased in C3 KO A549 cells compared to WT and C3 ΔATG1 cells after infection with Y. enterocolitica , or treatment with TNF ( A ). Similar is observed for mRNA levels for IL-8 after infection with M. catarrhalis or Y. enterocolitica , however treatment with TNF or Pam3CSK4 does not reach significance ( B ). NF-κB activation assessed using luciferase reporter assay is decreased in C3 KO compared to WT and C3 ΔATG1 cells after treatment with Pam3CSK4 ( C ) or infection with M. catarrhalis ( D ). Representative Western blot showing NF-κB (p65 subunit) translocation to the nucleus ( E ) with quantification in ( F ). C3 KO cells are characterized by diminished translocation of p65 to the nucleus compared to WT and C3 ΔATG1;- and + indicate no treatment and treatment with Pam3CSK4, respectively. A similar result was obtained upon infection with M. catarrhalis , a representative blot of nuclear fractions shown in ( G ) with quantification in ( H ). Representative Western blot analysis showing decreased phosphorylation of IKK in lysates from C3 KO cells compared to WT; - and + indicate no treatment and treatment with Pam3CSK4 ( I ) or M. catarrhalis ( K ) with quantification in ( J ) and ( L ), respectively. Each dot represents one biological repeat for one clone ( A , B ) or an average of two clones in one biological repeat ( C - L ). One-way ( C , D ) or Two-way ( A , B , F , H , J , L ) ANOVA with Tukey´s multiple comparison test, matched measures within the experiment. *p< 0.05, **p < 0.01, ***p < 0.001, ns: not significant

Article Snippet: Two individual clones each of WT, C3 ΔATG1 and C3 KO A549 cells were treated with either 1 μg/mL Pam3CSK4 (InvivoGen #tlrl-pms), 1 μg/mL of LPS (InvivoGen #tlrl-eklps), 0.1 ng/mL IL-1β (Mabtech #3415–1 H-6), 10 ng/mL TNF (ImmunoTools #11343015) or 100 ng/mL poly(I: C) (InvivoGen #tlrl-picw) and incubated for 24 h before the supernatants were collected and cells were lysed in lysis buffer containing 150 mM NaCl, 50 mM Tris, pH 7.5, 1% NP40 and 0.5% sodium deoxycholate, supplemented with HaltTM Protease and Phosphatase Inhibitor Cocktail, EDTA-free (100X) (Thermo ScientificTM, #78441).

Techniques: Activation Assay, Infection, Luciferase, Reporter Assay, Western Blot, Translocation Assay, Phospho-proteomics, Clone Assay, Comparison