Journal: Sensors (Basel, Switzerland)

Article Title: Point-of-Care Surface Plasmon Resonance Biosensor for Stroke Biomarkers NT-proBNP and S100β Using a Functionalized Gold Chip with Specific Antibody

doi: 10.3390/s19112533

Figure Lengend Snippet: Stroke Triaging. After stroke occurrence, Time = Brain. Efficacy of care is correlated to a timely diagnosis and treatment. First, a CT/MRI scan is conducted to differentiate between an ischemic and hemorrhagic stroke. This is followed by stroke mechanisms (e.g., using the criteria of Trial of Org 10172 in Acute Stroke Treatment, TOAST classification) to identify a cardioembolic stroke. Patients with cardioembolic stroke harbor a higher risk of poorer stroke outcomes and timely treatment with acute reperfusion treatment is necessary (intravenous tissue plasminogen activator (IV-tPA)) within the time-limited window of 4.5 h. The ability to accurately measure stroke biomarkers (e.g., NT-proBNP and S100β) within 15 min is highly desirable, in order to shorten the classification time.

Article Snippet: S100β protein (CAT#8S9b) and Anti-S100β 8B10 Antibody (CAT#4S37) were purchased from HyTest and used for the development of the biosensor for S100β detection.

Techniques: Biomarker Discovery

Journal: Sensors (Basel, Switzerland)

Article Title: Point-of-Care Surface Plasmon Resonance Biosensor for Stroke Biomarkers NT-proBNP and S100β Using a Functionalized Gold Chip with Specific Antibody

doi: 10.3390/s19112533

Figure Lengend Snippet: Stroke biomarkers measurement using bold SPR chip. ( A ) NT-proBNP; ( B ) S100β.

Article Snippet: S100β protein (CAT#8S9b) and Anti-S100β 8B10 Antibody (CAT#4S37) were purchased from HyTest and used for the development of the biosensor for S100β detection.

Techniques:

Journal: Sensors (Basel, Switzerland)

Article Title: Point-of-Care Surface Plasmon Resonance Biosensor for Stroke Biomarkers NT-proBNP and S100β Using a Functionalized Gold Chip with Specific Antibody

doi: 10.3390/s19112533

Figure Lengend Snippet: Validation in spiked plasma samples. ( A ) NT-proBNP; ( B ) S100β.

Article Snippet: S100β protein (CAT#8S9b) and Anti-S100β 8B10 Antibody (CAT#4S37) were purchased from HyTest and used for the development of the biosensor for S100β detection.

Techniques: Biomarker Discovery, Clinical Proteomics

Journal: Viruses

Article Title: Establishment of a Dual-Reporter Minigenome System for Respiratory Syncytial Virus

doi: 10.3390/v18030304

Figure Lengend Snippet: Mini-NLuc-sfGFP for small-molecule evaluation. ( A ) The experimental protocol involved transfecting the Mini-NLuc-sfGFP minigenome into BSR-T7/5 cells, followed by treatment with a range of small molecules. The inhibitory effects were initially assessed by quantifying the fluorescence intensity or NLuc activity, which led to the selection of promising candidates for further investigation. Created in BioRender. Wu, C. (2026) https://BioRender.com/8qct7m8 . ( B ) AVG-233 reduced the BSR-T7/5 cell viability in a concentration-dependent manner (OD 450 ). ( C ) Similarly, RSV L-protein-IN-4 reduced the BSR-T7/5 cell viability in a concentration-dependent manner (OD 450 ). ( D ) AVG-233 demonstrated a dose-dependent inhibition of Mini-NLuc-sfGFP reporter gene expression, as indicated by the luciferase activity. ( E ) Similarly, RSV L-protein-IN-4 exhibited the dose-dependent inhibition of the Mini-NLuc-sfGFP reporter gene expression, also measured using the luciferase activity. Data are presented as the mean ± SD of three independent experiments, each performed in triplicate.

Article Snippet: Small-molecule inhibitors of RSV L polymerase, namely, AVG-233 (CAS no. 2151937-80-1) and RSV L-protein-IN-4 (CAS no. 851657-60-8), both sourced from Topscience Co. Ltd., China, were diluted in DMSO to create stock solutions at various concentrations.

Techniques: Fluorescence, Activity Assay, Selection, Concentration Assay, Inhibition, Gene Expression, Luciferase

Journal: Viruses

Article Title: Establishment of a Dual-Reporter Minigenome System for Respiratory Syncytial Virus

doi: 10.3390/v18030304

Figure Lengend Snippet: Analysis of the linear relationship between sfGFP and NLuc in Mini-NLuc-sfGFP to evaluate RSV L-protein-IN-4. ( A ) Effects of RSV L-protein-IN-4 on the sfGFP fluorescence at 0, 0.01, 0.05, 0.25, 0.5, 1, 2, and 3 μM. ( B , C ) RSV L-protein-IN-4 demonstrated a dose-dependent inhibition of Mini-NLuc-sfGFP reporter gene expression, as indicated by fluorescence intensity and luciferase activity. ( D ) Linear regression showed a significant correlation between sfGFP and NLuc. Scale bar: 400 μm. Data are presented as the mean ± SD of three independent experiments, each performed in triplicate.

Article Snippet: Small-molecule inhibitors of RSV L polymerase, namely, AVG-233 (CAS no. 2151937-80-1) and RSV L-protein-IN-4 (CAS no. 851657-60-8), both sourced from Topscience Co. Ltd., China, were diluted in DMSO to create stock solutions at various concentrations.

Techniques: Fluorescence, Inhibition, Gene Expression, Luciferase, Activity Assay

Journal: Viruses

Article Title: Establishment of a Dual-Reporter Minigenome System for Respiratory Syncytial Virus

doi: 10.3390/v18030304

Figure Lengend Snippet: Validation of small-molecule inhibitory effects assessed by Mini-NLuc-sfGFP and confirmed in RSV A2. RSV A2 infection was conducted at a multiplicity of infection (MOI) of 0.1, utilizing a primary antibody of mouse anti-RSV F and a secondary antibody of goat anti-mouse FITC conjugate. ( A ) Immunofluorescence imaging showed that AVG-233 inhibited viral infection at 0, 0.01, 0.05, 0.25, 0.5, 1, 2, and 3 μM. ( B ) Viral genome copy numbers via RT-qPCR at equivalent concentrations. ( C ) Immunofluorescence imaging showed that RSV L-protein-IN-4 inhibited viral infection at 0, 0.01, 0.05, 0.25, 0.5, 1, 2, and 3 μM. ( D ) Viral genome copy numbers via RT-qPCR at equivalent concentrations. Scale bar: 400 μm. Data are presented as the mean ± SD of three independent experiments, each performed in triplicate.

Article Snippet: Small-molecule inhibitors of RSV L polymerase, namely, AVG-233 (CAS no. 2151937-80-1) and RSV L-protein-IN-4 (CAS no. 851657-60-8), both sourced from Topscience Co. Ltd., China, were diluted in DMSO to create stock solutions at various concentrations.

Techniques: Biomarker Discovery, Infection, Immunofluorescence, Imaging, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: Dot plot reporting the correlation of miR‐9 and Sp1 expression in primary HNSCC samples evaluated by qRT–PCR. The number of analyzed samples ( n ), the Spearman correlation value ( r ), and its significance ( P ) are reported in the graph. qRT–PCR analyses of SP1 expression in control (shCTR) and anti‐miR‐9 FaDu cells. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. qRT–PCR analyses of Sp1 expression in control (shCTR) and anti‐miR‐9 FaDu cells transiently transduced with PGK‐miR‐9 or control vector as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. qRT–PCR analyses of Sp1 expression in CAL27 cells transfected with pcDNA miR‐9 or control vector. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. Clonogenic assay on FaDu cells silenced for Sp1 (shSP1#3 or #5) or scramble sequence (shCTR). Left graph reports the number of colonies. On the right, WB analyses of the indicated protein expression in FaDu cells. Tubulin was used as loading control. Bottom panel, representative images of clones are shown. In the graph, data represent the mean (± SD) of three independent experiments performed in duplicate and one‐way ANOVA was used to verify the statistical significance. Sphere‐forming assay of the cells described in (E). Left panel reports the mean number of spheres formed in first and second generations. Right graph reports the area of second‐generation spheres. Data represent the mean (± SD) of three independent experiments performed in duplicate, and one‐way ANOVA was used to verify the statistical significance. Each dot represents one analyzed sphere. Bottom panels show representative images of the spheres. WB analyses of the indicated protein expression in control (shCTR) and anti‐miR‐9 FaDu cells, overexpressing or not RSV‐SP1. Actin and histone H3 were used as loading control. Graphs reporting the cell viability of FaDu cells described in G and treated with increasing concentration of gefitinib (GEFI—left panel) or Cetuximab (CTX—right panel) as indicated and evaluated using the MTS assay. Data represent the mean (± SD) of two independent experiments performed in sextuplicate, and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Schematic representation of the pGL3 vectors used to test the potential activity miR‐9 on Sp1 promoter. Vectors are named based on the maximum distance from SP1 ATG. The lines indicate the putative KLF5‐binding sites according to the statistical prediction power according to EPD portal (blue lines P = 0.001, green lines P = 0.0001). Black bars indicate the primer pairs used for the amplification of different region of Sp1 promoter in the ChIP analyses depicted in Fig . Graph reporting the normalized luciferase activity of Sp1 promoter fragments in control (shCTR) or anti‐miR‐9 FaDu cells. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Expressing, Quantitative RT-PCR, Control, Transduction, Plasmid Preparation, Comparison, Transfection, Clonogenic Assay, Sequencing, Clone Assay, Concentration Assay, MTS Assay, Activity Assay, Binding Assay, Amplification, Luciferase

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: qRT–PCR analyses of Sp1 expression in control (shCTR) and anti‐miR‐9 (miR‐9 sponge) FaDu cells. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. qRT–PCR analyses of Sp1 expression in CAL27 cells transiently transduced with and PGK‐miR‐9 (GFP‐miR‐9) or control (GFP control) vector as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. WB analyses of the indicated protein expression in control (shCTR) and miR‐9 CAL27 cells, overexpressing or not RSV‐Sp1 as indicated. Actin was used as loading control. Graphs reporting the cell viability of CAL27 cells described in (C) and treated with increasing concentration of gefitinib (GEFI) and evaluated using the MTS assay. Data represent the mean (± SD) of two independent experiments performed in sextuplicate, and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Graph reporting the normalized luciferase activity of Sp1 promoter fragments in CAL27 cells control or stably overexpressing miR‐9. Data are the mean (± SD) of three independent experiments performed in duplicate, and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units; * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Transduction, Plasmid Preparation, Concentration Assay, MTS Assay, Comparison, Luciferase, Activity Assay, Stable Transfection

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: A Venn Diagram showing the number of miR‐9 potential targets among the genes altered during HNSCC progression as described in Pavón et al . B–D qRT–PCR WB analyses reporting KLF5 mRNA (left graph) and protein (right WB) expression in control (shCTR) or anti‐miR‐9 SCC9 cells (B), in control or miR‐9 overexpressing UMSCC1 cells (C), or in control or miR‐9 overexpressing CAL27 cells (D). Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. In WB analysis, histone H3 was used as loading control. E Graph reporting the normalized luciferase activity of wild type (WT) or mutated KLF5 3’‐UTR described in Fig in control (shCTR), miR‐9 overexpressing, or anti‐miR‐9 CAL27 cells, as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. F Graph reporting the normalized luciferase activity of Sp1 promoter fragments in CAL27 cells transfected with control or KLF5 vectors, as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to calculate the statistical significance. G Chromatin immunoprecipitation (ChIP) assay performed on CAL27 cells. The graph reports the binding of KLF5 to the indicated region of Sp1 promoter expressed as signal relative to input in two independent immunoprecipitations (ChIP1 and ChIP2) using the KLF5 ab. IgG was used as negative control on the same chromatin. H Chromatin immunoprecipitation (ChIP) assay performed on CAL27 cells. The graph reports the binding of KLF5 to the indicated genomic regions used as negative (Neg Ctr 1, Neg Ctr 2, and Neg Ctr 3) or potentially positive controls (EPPK1; LAM2C2; SERPINE1; EPHA2; INPP4B; SOX17) of anti‐KLF5 antibody specificity. The Sp1 ‐673/‐486 fragment was amplified on the same DNA recovered from the IP as internal reference. Data information: In the figure, A.U. = arbitrary units; * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Quantitative RT-PCR, Expressing, Control, Luciferase, Activity Assay, Comparison, Transfection, Chromatin Immunoprecipitation, Binding Assay, Negative Control, Amplification

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: Table reporting the qRT–PCR analyses of gene expression in control (shCTR) and anti‐miR‐9 FaDu cells, as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. On the left, WB analyses of KLF5 and Sp1 expression in control (shCTR) and anti‐miR‐9 FaDu. Histone H3 expression was used as loading control. On the right, protein quantification analyses of KLF5 and SP1 expression normalized on H3 loading control. Data represent the mean (± SD) of three biological replicates, and unpaired t ‐test was used to verify the statistical significance. Schematic design of the KLF5 3’‐UTR. To test the potential miR‐9 binding on KLF5 3’‐UTR, four vectors were generated: WT (wild type containing both the seed sites), mut A or mut B (mutated for one single binding site) and mut A + B (mutated in both seed site). The predicted seed sites for miR‐9‐binding sites are shown in red when present and in blue when mutated. Graph reporting the normalized luciferase activity of wild type (WT) or mutated KLF5 3’‐UTR described in C in control (shCTR) or anti‐miR‐9 FaDu cells, as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. qRT–PCR analyses of KLF5 (left) and Sp1 (right) expression in FaDu cells transfected with control or KLF5 vectors. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. Upper panel shows WB analyses of KLF5 and Sp1 expression in FaDu cells. Actin expression was used as loading control. Lower graph shows the SP1 protein quantification normalized over actin expression. Data represent the mean (± SD) of three independent experiments, and unpaired t ‐test was used to verify the statistical significance. Graph reporting the normalized luciferase activity of Sp1 promoter fragments in FaDu transfected with pcDNA control or KLF5 vectors, as indicated. Data represent the mean (± SD) of three independent experiments performed in duplicate, and unpaired t ‐test was used to verify the statistical significance. Chromatin immunoprecipitation (ChIP) assay performed on FaDu cells. Upper left panel shows WB analysis reporting KLF5 expression in the immunoprecipitation achieved using anti‐KLF5 or control (IgG) antibodies used in the ChIP assay. Vinculin expression was used as loading control. Bottom left panel shows a typical image of amplified PCR fragments using ChIP DNA or genomic DNA, as indicated. Right graph reports the binding of KLF5 to the indicated region of Sp1 promoter expressed as signal relative to input in two independent immunoprecipitations (ChIP1 and ChIP2) using the KLF5 ab. IgG was used as negative control on the same chromatin. Primer pairs used to amplify the different regions of Sp1 promoter are depicted in the schema provided in Fig . Data information: In the figure, A.U. = arbitrary units; * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Quantitative RT-PCR, Gene Expression, Control, Expressing, Binding Assay, Generated, Luciferase, Activity Assay, Transfection, Chromatin Immunoprecipitation, Immunoprecipitation, Amplification, Negative Control

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: Graph reporting the tumor volume of mice ( n = 5 mice/group) injected in both flanks with control (shCTR) or anti‐miR‐9 FaDu cells and treated with vehicle (untreated) or Cetuximab (1 mg/kg, IP injections) every three days for 3 weeks. Data are represented as mean (± SD). WB analyses evaluating the expression of the indicated proteins in tumor explanted from mice described in (A). GAPDH was used as loading control. Graph reporting the normalized pY1068 EGFR expression folded over total EGFR in tumor explanted from mice described in (A). On the left, typical images of Sp1 expression evaluated by immunohistochemistry (IHC) in tumors ( n = 5 mice/group) explanted from mice treated as in (A). On the right, graph reports the Sp1 expression scored from 0 (negative) to 4 (strong) based on its nuclear intensity by a blinded pathologist. Data are represented as mean (± SD), and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Dot plot reporting the qRT–PCR of normalized miR‐9 expression in tumor treated with control (shCTR) and anti‐miR‐9 lentiviruses FaDu cells as described in Fig . Each dot represents a tumor. Data are represented as mean (± SD), and two‐way ANOVA test was used to calculate statistical significance. Graph reporting the percentage of necrotic areas in tumors injected with control and anti‐miR‐9 lentiviruses and treated with radiotherapy alone (IR) or in combination with Cetuximab (IR+CTX) as described in Fig . Data are represented as mean (± SD), and two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Kaplan–Mayer curve evaluating the overall survival of HNSCC patients treated with RT+Cisplatin combination included in the TCGA dataset, segregated on the expression of miR‐9 in the primary tumor (high expression ≥ 75,819 reads n = 34; low expression < 75,819 reads n = 99). Number of total evaluated samples ( n ) and P value are reported in the graph. Statistical significance was calculated with log‐rank test. Data information: In the figure, A.U. = arbitrary units; * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Injection, Control, Expressing, Immunohistochemistry, Comparison, Quantitative RT-PCR

Journal: EMBO Molecular Medicine

Article Title: miR‐9 modulates and predicts the response to radiotherapy and EGFR inhibition in HNSCC

doi: 10.15252/emmm.202012872

Figure Lengend Snippet: Graph reporting cell viability of control (shCTR) and anti‐miR‐9 FaDu cells treated with increasing concentration of the indicated drugs for 72 h and analyzed using the MTS cell viability assay. Data represent the mean (± SD) of three independent experiments each performed in sextuplicate, and unpaired t ‐test was used to calculate the statistical significance per each dose. Clonogenic assay of control (shCTR) and anti‐miR‐9 FaDu cells not irradiated (NIR) or treated with 2 or 5 Gy IR. On the left, typical images of cell clones are shown. On the right, the graph reports the percentage (± SD) of survived cells respect to not irradiate cells in three independent experiments performed in triplicate. Unpaired t ‐test was used to calculate the statistical significance per each dose. Left, WB analyses of the indicated protein expression in control (shCTR) and anti‐miR‐9 FaDu cells not irradiated (NIR) or treated with 5 Gy IR and allowed to repair for the indicated hours (h). Tubulin was used as loading control. On the right, protein quantification analyses of KLF5 and Sp1 expression normalized on tubulin loading control. On the right, graphs report the quantification of the indicated proteins normalized on tubulin expression. Data are expressed as mean (± SD) of three independent experiments, and unpaired t ‐test was used to calculate the statistical significance at time point. Left panel reports typical immunofluorescence images of control (shCTR) or anti‐miR‐9 FaDu cells, not irradiated (NIR) and analyzed 1, 4, or 8 h after 2 Gy IR (γH2AX green, pS10‐H3 red, nuclei in blue). Graphs report the percentage of γH2AX (middle)‐ and pS10‐H3 (right)‐positive cells. Data represent the mean (± SD) of three independent experiments in which at least 10 randomly selected fields were evaluated. Unpaired t ‐test was used to calculate the statistical significance at each time point. Graphs report the qRT–PCR analyses evaluating the expression of miR‐9 (left) or SP1 (right) in cells treated as in (C). Data are the mean (± SD) of three independent experiment performed in duplicate. Two‐way ANOVA with Sidak’s multiple comparison test was used to calculate the statistical significance. Graph reporting cell viability of control (shCTR) and anti‐miR‐9 FaDu cells, overexpressing or not SP1, and treated with increasing concentration of Bleomycin (BLEO) for 72 h and analyzed using the MTS cell viability assay. Data represent the mean (± SD) of three independent experiments each performed in sextuplicate. Two‐way ANOVA with Sidak’s multiple comparison test was used to verify the statistical significance. Data information: In the figure, A.U. = arbitrary units; * P < 0.05; ** P < 0.01; *** P < 0.001. Source data are available online for this figure.

Article Snippet: Briefly, 293T17 cells were transfected with pHIT465 and pHIT60 plus microRNA Precursor (MDH1‐PGK‐GFP‐miR‐9—Addgene #25036), Sp1 (RSV‐Sp1—Addgene #12098), or miR‐9 sponge sequence (pBABE miR‐9 sponge—Addgene #25040) or Control Constructs by standard calcium phosphate transfection protocol.

Techniques: Control, Concentration Assay, Viability Assay, Clonogenic Assay, Irradiation, Clone Assay, Expressing, Immunofluorescence, Quantitative RT-PCR, Comparison