a549 ko cells  (Addgene inc)

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: International journal of molecular sciences

Article Title: S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

doi: 10.3390/ijms251910461

Figure Lengend Snippet: Figure 2. Genetic and pharmacological targeting of S6K1 decreases radioresistance in lung cancer cells. (A) Lung cancer cells were seeded in 6-well plates in triplicate and left attached overnight. Then, cells were exposed to PF-4708671 (5µM) or DMSO for 24 h before radiation. Cells were exposed to 2 Gy of radiation and were grown until visible colonies were formed (2 weeks). PF-4708671 was kept for the whole experiment. Colony formation was determined by clonogenic assays. (B) S6K1−/−KO A549 cells were lysated, and the deletion of S6K1 gene was confirmed by Western blot. (C) Colony formation was determined by clonogenic assays in S6K1−/−KO A549 cells after irradiation with a dose of 4 Gy. The S6K1 deletion dramatically sensitizes A549 cells to radiation. * denotes a p value < 0.01, ** and *** <0.001 and **** <0.0001.

Article Snippet: The radiation-sensitive H23 cells were transfected with an empty vector (Addgene #10883, Watertown, MA, USA) or with a vector expressing a modified cDNA expressing a constitutively active form of S6K1 (pRK7-HA-S6K1-F5A-E389-R3A) (Addgene #8991, Watertown, MA, USA) [31,32].

Techniques: Western Blot, Irradiation

Journal: International journal of molecular sciences

Article Title: S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

doi: 10.3390/ijms251910461

Figure Lengend Snippet: Figure 3. S6K1 impairs DNA damage signaling by inhibiting MRN complex activation. (A) Ex- pression levels of MRN complex members were determined in S6K1−/−KO and wild type cells by Immunoblot. The S6K1 genetic deletion increased the activation of p-RAD50, p-ATM and expression of MRE11, compared to wild type controls. (B) Quantitation of expression changes normalized to actin. (C) S6K1−/−KO and wild type A549 cells were irradiated with a dose of 10 Gy. At 4 h after irradiation, cells were lysated and γ-H2AX expression was determined by Immunoblot. ** denotes a p value < 0.05 and *** <0.005.

Article Snippet: The radiation-sensitive H23 cells were transfected with an empty vector (Addgene #10883, Watertown, MA, USA) or with a vector expressing a modified cDNA expressing a constitutively active form of S6K1 (pRK7-HA-S6K1-F5A-E389-R3A) (Addgene #8991, Watertown, MA, USA) [31,32].

Techniques: Activation Assay, Western Blot, Expressing, Quantitation Assay, Irradiation

Journal: International journal of molecular sciences

Article Title: S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

doi: 10.3390/ijms251910461

Figure Lengend Snippet: Figure 4. S6K1 overexpression increases radioresistance by impairing MRN complex signaling. (A) S6K1 overexpression increases radioresistance and proliferation in H23 cells. Cells were trans- fected with a plasmid expressing a constitutively active form of S6K1 (addgene #8991) or an empty vector. At 24 h after transfection, 5000 cells/well were seeded in 96-well plates and left attached overnight. Cells were irradiated with a dose of 3 Gy and incubated for 72 h. Proliferation was performed by Alamar Blue. (B) H23 cells overexpressing an empty vector or an active S6K1 were lysated and the expression levels of MRN members were investigated before radiation and at 4 h after. **** denotes a p value < 0.0001.

Article Snippet: The radiation-sensitive H23 cells were transfected with an empty vector (Addgene #10883, Watertown, MA, USA) or with a vector expressing a modified cDNA expressing a constitutively active form of S6K1 (pRK7-HA-S6K1-F5A-E389-R3A) (Addgene #8991, Watertown, MA, USA) [31,32].

Techniques: Over Expression, Plasmid Preparation, Expressing, Transfection, Irradiation, Incubation

Journal: International journal of molecular sciences

Article Title: S6K1 Controls DNA Damage Signaling Modulated by the MRN Complex to Induce Radioresistance in Lung Cancer.

doi: 10.3390/ijms251910461

Figure Lengend Snippet: Figure 5. Working model showing the regulation of the AKT/mTOR/S6K1 pathway on the MRN complex signaling activation. After radiation, S6K1 phosphorylated and activated directly, or via an intermediate, inhibits MRN complex signaling, leading to defects in DNA repair, mutation accumulation, apoptosis inhibition and radioresistance. Furthermore, the inhibition of S6K1 by pharmacological inhibition or genetic deletion renders the MRN complex active, which promotes the DDR mechanisms, promoting DNA repair or apoptosis if the damage is extensive, making radiotherapy more effective. Diagram created using Biorender.

Article Snippet: The radiation-sensitive H23 cells were transfected with an empty vector (Addgene #10883, Watertown, MA, USA) or with a vector expressing a modified cDNA expressing a constitutively active form of S6K1 (pRK7-HA-S6K1-F5A-E389-R3A) (Addgene #8991, Watertown, MA, USA) [31,32].

Techniques: Activation Assay, Mutagenesis, Inhibition