Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A is functionally associated with TP63-binding sites. ( A ) Effect on the IRF6 enhancer activity of the addition of exogenous TP63 in H1299 cells as measured by luciferase activity. All luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05. ( B ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the IRF6 enhancer region in H1299 cells. ( C ) Western blot analysis of exogenous TP63 and TFAP2A expression in H1299 cells. ( D ) Luciferase assay measuring the effect of the addition of increasing amounts of TFAP2A on TP63-mediated activation of the PVRL1 enhancer region in H1299 cells. ( E ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2, PVRL1 and PDGFC associated binding sites, expressed as fold enrichment compared to a negative control region within the same ChIP. Data represent the mean of two independent biological replicates. ( F ) Western blot validation of TP63 and TFAP2A depletion in HFKs used in recruitment ChIPs. ( G ) Quantitative ChIP-PCR comparing TP63 and TFAP2A binding to IRF6, FGFR2, JAG2 and PVRL1 associated binding sites in HFKs depleted for TP63 or TFAP2A compared to scrambled control. Results calculated as fold enrichment compared to a negative control region within the same ChIP and normalized to scrambled control to compare between antibodies and experiments. Data represent the mean of two independent biological replicates. ( H ) Quantitative ChIP-PCR of second negative control binding site (BCL2 upstream) in HFKs depleted for TP63 or TFAP2A expressed as fold change compared to scrambled control. Data represent the mean of two independent biological replicates. ( I ) Quantitative RT-PCR quantification of expression of CL/P associated genes in TFAP2A-depleted human foreskin keratinocytes compared to scrambled control. Graph represents the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Activity Assay, Luciferase, Activation Assay, Western Blot, Expressing, Negative Control, Biomarker Discovery, Control, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: Identification of TFAP2A as a potential TP63 co-factor. ( A ) Table of results from transcription factor motif enrichment analysis of 7574 TP63-binding sites. ( B ) Cumulative frequency distribution plots comparing distance of predicted TFAP2A sites and TP63-binding motifs from the centre of TP63-binding sites/peaks. ( C ) Comparison of localization of TP63-binding sites with or without predicted AP-2 sites. ( D and E ) Quantitative PCR ChIP validation of TP63 (D) and TFAP2A (E) binding to TP63 sites associated with CL/P genes (data represent the mean of three biological replicates expressed as % input ± SEM). ( F ) Semi-quantitative PCR results for sequential re-ChIP assay for TP63 and TFAP2A for the IRF6 , FGFR2, TGFB1 and PVRL1 associated TP63-binding sites, showing both factors co-ChIP.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Comparison, Real-time Polymerase Chain Reaction, Biomarker Discovery

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2C interacts with a subset of TP63-binding sites. ( A ) Quantitative PCR estimation of TFAP2A and TFAP2C mRNA copy number in cycling HFKs. Data represent the mean of three independent biological replicates ± SEM. ( B ) Quantitative PCR ChIP validation comparing TFAP2A (3B5) and TFAP2C (H77) interaction with a subset of TP63-binding regions associated with CL/P genes. Data represent the mean of three biological replicates expressed as % input ± SEM). ( C and D ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C to six TP63 isoforms on activation of the IRF6 (C) or PVRL1 (D) enhancer region in H1299 cells. ( E ) Luciferase assay measuring the effect of the addition of TFAP2A or TFAP2C on TP63-mediated activation of the IRF6 enhancer region in primary human foreskin keratinocytes (HFKs). Luciferase graphs represent the mean fold increase of at least three independent biological ± SEM; Student’s t -test * P < 0.05, ** P < 0.01.

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Binding Assay, Real-time Polymerase Chain Reaction, Biomarker Discovery, Luciferase, Activation Assay

Journal: Nucleic Acids Research

Article Title: Genome-wide analysis of p63 binding sites identifies AP-2 factors as co-regulators of epidermal differentiation

doi: 10.1093/nar/gks389

Figure Lengend Snippet: TFAP2A and TFAP2C are required for efficient differentiation of organotypic raft cultures. ( A ) Western blot analysis of TFAP2A, TFAP2C, TP63 and actin loading control of the HFKs transfected with TFAP2A, TFAP2C, TP63 targeting and scrambled control siRNA. ( B ) Sections of organotypic raft cultures generated from TFAP2A-, TFAP2C- or TP63-depleted HFKs stained for haematoxylin & Eosin (H&E) and indirect immunofluorescent staining of early [keratins 1 (KRT1), intermediate (transglutaminase-1 (TGM1)] and late [Filaggrin (FLG)] markers of differentiation (scale bar = 100 µM). ( C ) Number of bromodeoxyuridine (BrDU) incorporating cells in organotypic raft culture assessed by immunofluorescent staining. Graph represents the average number of BrDU-positive cells in the basal epithelial layer from organotypic raft cultures per 1000 µM, expressed as percentage scrambled control (mean ± SE at least 10 counts each of two independent biological replicates).

Article Snippet: The TFAP2A construct was obtained from Addgene [plasmid SP(RSV); 12100) ( )], and matched TFAP2A and TFAP2C image clones in pCMVsport6 were obtained from MRC Geneservice (Cambridge).

Techniques: Western Blot, Control, Transfection, Generated, Staining

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: 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