Journal: Proteome Science

Article Title: Proteomic analysis of laser capture microscopy purified myotendinous junction regions from muscle sections

doi: 10.1186/1477-5956-12-25

Figure Lengend Snippet: Selection of proteins identified in the MTJ sample

Article Snippet: The primary antibodies used in this work were: goat anti human FHL1 (cat. AHP2070, AbD Serotec), mouse monoclonal anti-syntrophin (cat. SAB4200213, Sigma-Aldrich), mouse monoclonal anti-myomesin (cat. mMaC myomesin B4, DSHB), mouse monoclonal anti-titin (cat. 9 D10, DSHB), mouse monoclonal anti s-laminin (cat. C4, DSHB), mouse monoclonal anti filaminC RR90 (an IgA sub-type [ ]), mouse monoclonal anti-Annexin I (cat. EH17a, DSHB), mouse monoclonal anti-myotilin (RSO34, Novocastra), mouse monoclonal anti-desmin (cat. D76, DSHB), rabbit polyclonal anti-TACP (cat. QC18385, Sigma-Aldrich), mouse monoclonal anti-αB crystallin (cat. CPTC-CRYAB-3, DSHB), mouse monoclonal anti-tubulin (cat. E7, DSHB), mouse monoclonal anti-α actinin (cat EA53, Sigma-Aldrich), mouse monoclonal anti-α-sarcoglycan (cat. IVD3(1)A9, DSHB).

Techniques: Selection, Membrane

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: ( A ) 48 differentially expressed genes were identified by bioinformatics and Venn analysis from four GSE databases (blue cycle indicates GSE2379, red cycle indicates GSE3524, black cycle indicates GSE6631 and gay cycle indicates GSE13601). ( B ) 48 differentially expressed genes were localized in diverse Chromosomes. ( C ) The Heatmap of X-linked FHL1 was shown in four GSE databases. ( D ) Expression level of FHL1 mRNA in 24 representative HNSCCs and the corresponding adjacent tissues by using RT-PCR. ( E ) Expression levels of FHL1 mRNA were decreased in HNSCCs ( n = 105) compared with adjacent tissues ( n = 69) using real-time PCR analysis. For each sample, the relative mRNA level of FHL1 was normalized to β-action. The line within each box represents the median negative Ct value; the upper and lower edges of each box represent the 75th and 25th percentiles, respectively. ( F ) Expression level of FHL1 in 10 representative HNSCCs and the corresponding adjacent tissues tested by Western blotting. ( G ) Kaplan-Meier survival curve indicated overall survival by evaluation of FHL1 mRNA levels in the training cohort. ( H ) Kaplan-Meier survival curve indicated disease-free survival by evaluation of FHL1 mRNA levels in the training cohort.

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Western Blot

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: Associations between FHL1 mRNA levels and clinical parameters in a training cohort (n = 105)

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Expressing, Adjuvant

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: ( A ) Representative images show immunohistochemical staining (IHC) for FHL1 expression in positive control (muscle), well differentiated HNSCC, moderately differentiated HNSCC, and poorly differentiated HNSCC from the left side to the right side. From the first line to the end line indicates H & E staining, ×100 original magnification and ×200 original magnification, respectively. ( B ) Representative images showed FHL1 expression was quantified by using digital image analysis (IOD value). ( C ) Kaplan-Meier survival curve indicated overall survival by evaluation of FHL1 protein levels in the validation cohort through human semiquantitative analysis. ( D ) Kaplan-Meier survival curve indicated disease-free survival by evaluation of FHL1 protein levels in the validation cohort through human semiquantitative analysis.

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Immunohistochemical staining, Staining, Expressing, Positive Control, Biomarker Discovery

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: ( A ) Expression of FHL1 mRNA level was detected in HNSCC cell lines and normal epithelial cells. ( B ) Silencing effect of FHL1 by three siRNAs was evaluated in HN-4 and SCC-25 cells. ( C ) Expression of FHL1 was detected after FHL1 knock down by siRNA-1302 or FHL1 overexpression by Ad-GFP+FHL1 in HN-4, SCC-25 and HN-13 through using Western blotting assay. ( D ) The effect of FHL1 knockdown by siRNA-1302 or FHL1 overexpression by Ad-GFP+FHL1 on the proliferation of SCC-25, HN-4, HN-13 and CAL-27cells analyzed with the CCK-8 cell-counting kit. ( E ) The effect of FHL1 ablation by siRNA-1302 or FHL1 overexpression by Ad-GFP+FHL1 on the colony formation potential of SCC-25, HN-4 and HN-13 cells. ( F ) The distribution of cell cycle after infecting by siRNA-1302 or FHL1 overexpression by Ad-GFP+FHL1 was observed in SCC-25 and HN-13 cells through cell cycle analysis.

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Expressing, Knockdown, Over Expression, Western Blot, CCK-8 Assay, Cell Counting, Cell Cycle Assay

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: ( A ) The apoptosis potential of FHL1 overexpression with Ad-GFP+FHL1 in HN-13 and CAL-27 cells was examined by 7-AAD and Annexin V-PE double staining through flow cytometry. ( B ) Cell cycle-related proteins were assessed by Western blotting after FHL1 overexpression with Ad-GFP+FHL1 in HN-13 and CAL-27 cells. ( C ) Cell cycle-related proteins were assessed by Western blotting after FHL1 ablation with siRNA-1812 and siRNA-1302 in SCC-25 cells. ( D ) Photographs of the tumors that were surgically removed from mice in each group after they were killed at the end of 7 weeks from the HN-13 cells inoculation. ( E ) The left diagram showed that the time course is from growth of HN-13 xenograft tumors that developed into approximately 100 mm 3 in mice to the end of 7 weeks after the cell inoculation. Black arrow indicated intratumoral injection with Ad-GFP+FHL1 or Ad-GFP; the right diagram demonstrated that weight of individual tumors surgically removed from the animals in each group. ( F ) The time course curve and weight diagram for CAL-27 xenograft tumors. ( G ) Representative H & E staining and immunohistochemical staining for Ki67, Cyclin D1 as well as TUNEL assay were conducted in HN-13 cell line xenografts each group (original magnification, ×400).

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Over Expression, Double Staining, Flow Cytometry, Western Blot, Injection, Staining, Immunohistochemical staining, TUNEL Assay

Journal: Oncotarget

Article Title: X-linked FHL1 as a novel therapeutic target for head and neck squamous cell carcinoma

doi: 10.18632/oncotarget.7478

Figure Lengend Snippet: ( A ) Schematic representations of the location of CpG island within the promoter of FHL1 and of the primers designed against the promoter region for Methylation-specific polymerase chain reaction (MSP-PCR) and bisulfite-treated DNA sequencing (BS). Red ticks indicated CpG site in CpG island region. ( B ) Methylation status of FHL1 was investigated in normal oral epithelial cell (NOEC), HNSCC cell lines and eight representative paired HNSCCs by MSP-PCR. ( C ) Methylation status of FHL1 was further revealed in paired adjacent tissues ( n = 34) and HNSCCs ( n = 105) by MSP-PCR. ( D ) The negative association between FHL1 mRNA expression and FHL1 methylation status was illustrated. ( E ) Methylation status of FHL1 was determined in a representative paired adjacent tissue and HNSCC sample by BS. ( F ) Expression restoration of FHL1 was also observed in five HNSCC cells by real-time PCR after the 5-Aza-dC induction. ( G ) Schematic representations of the location of putative binding region within the promoter of FHL1 and of the five primers designed against the promoter region for Chromatin Immunoprecipitation (ChIP)-PCR, and notably occupancy of EZH2 and H3k27me3 were found in both HN-13 and CAL-27 cells.

Article Snippet: Primary antibodies against FHL1 (Santa Cruz Biotechnology, sc-374246, 1:1000 diluted), Cyclin D1, Cyclin E, p53, p27, p21, phosph-ERK1/2, and ERK1/2 (Cell signaling Technology) were used in this study. β-actin antibody was used to normalize protein loading.

Techniques: Methylation, Polymerase Chain Reaction, DNA Sequencing, Expressing, Real-time Polymerase Chain Reaction, Binding Assay, Chromatin Immunoprecipitation