Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A ) Strategy for identification of 4 key genes encoding secreted proteins that are downregulated (fold change > 2) by CLOCK depletion in GSC272 with inducible CLOCK shRNA (ish CLOCK ) versus inducible shRNA control (ishC), and by BMAL1 depletion in T387 and T3565 with BMAL1 shRNA (sh BMAL1 ) versus shRNA control (shC). ( B ) Heat map shows the expression of TFPI2 , COL8A1 , LRRC17, and PLA2R1 in GSC272, T387, and T3565 harboring ishC versus ish CLOCK and shC versus sh BMAL1 . Red and blue indicate higher and lower expression, respectively. ( C and D ) ChIP-seq data analysis shows the heatmap ( C ) and quantification ( D ) of BMAL1-enriched profiles at TFPI2 , COL8A1 , LRRC17, and PLA2R1 promotors in GSCs (T387 and T3565) and NSCs (ENSA and hNP1). n = 4. ( E ) ChIP-seq data analysis shows BMAL1-enriched profiles at the TFPI2 promotor in NSC #1 and #2 (ENSA and hNP1) and GSC #1 and #2 (T387 and T3565). ( F and G ) RT-qPCR for CLOCK and TFPI2 in control, CLOCK-depleted ( F , n = 4), and BMAL1-depleted ( G , n = 3) GSC272. ( H ) RT-qPCR for TFPI2 in GSC20 and GSC272 treated with or without SR9009 (5 μmol/L). n = 3 independent samples. ( I and J ) RT-qPCR for CLOCK and TFPI2 in GSC17 ( I ) or GSC23 ( J ) harboring CLOCK overexpression (OE). n = 3. ( K and L ) Immunoblots for CLOCK, BMAL1, and TFPI2 in GSC272 harboring shC and sh CLOCK ( K ), or shC and sh BMAL1 ( L ). ( M ) Immunoblots for CLOCK and TFPI2 in GSC20 and GSC272 treated with or without SR9009 (1 and 5 μmol/L). ( N and O ) Immunoblots for CLOCK, BMAL1, and TFPI2 in GSC17 ( N ) or GSC23 ( O ) harboring CLOCK OE. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test ( D and F – J ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: shRNA, Control, Expressing, ChIP-sequencing, Quantitative RT-PCR, Over Expression, Western Blot

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A – D ) Immunoblots ( A and B ) and RT-qPCR ( C and D , n = 3) for CD133 ( PROM1 ) and SOX2 in GSC272 harboring shRNA control (shC), CLOCK shRNA (sh CLOCK , A ), or sh BMAL1 ( B ) in the presence or absence of TFPI2 overexpression (OE). n = 3. ( E and F ) In vitro limiting dilution assays in GSC272 harboring shC, sh CLOCK ( E ), or sh BMAL1 ( F ) in the presence or absence of TFPI2 OE. ( G – I ) Representative images ( G ) and quantification of relative tumorsphere number ( H ) and size ( I ) of GSC272 expressing shC, sh CLOCK, or sh BMAL1 in the presence or absence of TFPI2 OE. Scale bar: 200 μm. n = 8. ( J – M ) Representative images and quantification of proliferation in GSC272 harboring shC, sh CLOCK ( J and K ), and sh BMAL1 ( L and M ) with or without TFPI2 OE. n = 3. ( N – Q ) Immunoblots ( N and O ) and RT-qPCR ( P and Q , n = 3) for CD133 ( PROM1 ) and SOX2 in GSC17 and GSC23 harboring Control or CLOCK OE with or without sh TFPI2 . ( R and S ) In vitro limiting dilution assays in GSC17 ( R ) and GSC23 ( S ) expressing Control or CLOCK OE with or without sh TFPI2 . ( T and U ) Representative images ( T ) and quantification of relative tumorsphere number ( U ) of GSC17 or GSC23 harboring CLOCK OE in the presence or absence of sh TFPI2 . Scale bar: 200 μm. n = 8. ( V – Y ) Representative images and quantification of proliferation in GSC17 ( V and W ) and GSC23 ( X and Y ) harboring CLOCK OE in the presence or absence of sh TFPI2 . n = 3. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, 1-way ANOVA test ( C , D , H , I , K , M , P , Q , U , W , and Y ), 2-way ANOVA test ( E , F , R , and S ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: Western Blot, Quantitative RT-PCR, shRNA, Control, Over Expression, In Vitro, Expressing

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A ) Amplification pattern TFPI2 and CLOCK in TCGA GBM patient tumors from indicated datasets. ( B ) High-resolution uniform manifold approximation and projection (UMAP) showing expression of CLOCK and TFPI2 in GSCs (the population of cells are highlighted in circle) of GBM patient tumors based on scRNA-Seq data ( GSE182109 ). ( C ) Relationship between CLOCK and TFPI2 expression in GSCs from GBM patient tumors based on scRNA-Seq data ( GSE182109 ). R and P values are shown. Pearson’s correlation test. ( D ) In vitro limiting dilution assays in GSC272 harboring shRNA control (shC) and TFPI2 shRNA (sh TFPI2 ) treated with or without SR9009 (5 μM). ( E – G ) Representative images ( E ) and quantification of relative tumorsphere number ( F ) and size ( G ) of GSC272 harboring shC and sh TFPI2 and treated with or without SR9009 (5 μM). Scale bar: 200 μm. n = 8. ( H ) Survival curves of nude mice implanted with 2 × 10 5 shC and sh TFPI2 GSC272. Mice were treated with SR9009 (100 mg/kg, i.p., daily) for 10 days beginning at day 7 after orthotopic injection ( n = 8 mice per group). ( I – L ) Representative images and quantification of IHC staining for SOX2 ( I and J ) and CD133 ( K and L ) in shC and sh TFPI2 GSC272 tumors from mice treated with or without SR9009. Scale bar: 100 μm. n = 7. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, 1-way ANOVA test ( F , G , J , and L ), 2-way ANOVA test ( D ), log-rank test ( H ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: Amplification, Expressing, In Vitro, shRNA, Control, Injection, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A – D ) RT-qPCR ( A and B , n = 3) and immunoblots ( C and D ) for TFPI2, CLOCK, and BMAL1 in GSC17 and GSC23 harboring control or TFPI2 overexpression (OE). ( E ) RT-qPCR ( E , n = 3) and immunoblots ( F ) for TFPI2, CLOCK, and BMAL1 in GSC272 harboring shRNA control (shC) or TFPI2 shRNA (sh TFPI2 ). ( G and H ) Immunoblots for CD133 and SOX2 in GSC17 ( G ) and GSC23 ( H ) harboring control or TFPI2 OE and treated with or without SR9009 (5 μM). ( I ) In vitro limiting dilution assays in GSC17 harboring control or TFPI2 OE and treated with or without SR9009 (5 μM). ( J – L ) Representative images ( J ) and quantification of relative tumorsphere number ( K ) and size ( L ) of GSC17 harboring control or TFPI2 OE treated with or without SR9009 (5 μM). Scale bar: 200 μm. n = 8. ( M ) In vitro limiting dilution assays in GSC23 harboring control or TFPI2 OE and treated with or without SR9009 (5 μM). ( N – P ) Representative images ( N ) and quantification of relative tumorsphere number ( O ) and size ( P ) of GSC23 harboring control or TFPI2 OE treated with or without SR9009 (5 μM). Scale bar: 200 μm. n = 8. ( Q ) Survival curves of nude mice implanted with 2 × 10 5 Control and TFPI2 OE GSC23 and treated with SR9009 (100 mg/kg, i.p., daily) for 10 days beginning at day 7 (n = 8 mice per group). ( R – U ) Representative images and quantification of IHC staining for SOX2 ( R and S ) and CD133 ( T and U ) in Control and TFPI2 OE GSC23 tumors from mice treated with or without SR9009. Scale bar: 100 μm. n = 7. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, 1-way ANOVA test ( A , B , E , K , L , O , P , S , and U ), 2-way ANOVA test ( I and M ), log-rank test ( Q ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: Quantitative RT-PCR, Western Blot, Control, Over Expression, shRNA, In Vitro, Immunohistochemistry

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A ) GSEA analysis shows the top 10 enriched hallmark pathways in shRNA control (shC) GSC272 compared with TFPI2 shRNA (sh TFPI2 ) GSC272. Blue bars indicate the top 2 enriched signatures ( FDR < 0.25). ( B and C ) Immunoblots for HIF-1α, P-P65, and P65 in cell lysates of GSC17 ( B ) and GSC23 ( C ) harboring control and TFPI2 overexpression (OE). ( D ) Immunoblots for HIF-1α, P-P6,5 and P65 in cell lysates of shC and sh TFPI2 GSC272. ( E and F ) Immunoblots for HIF-1α, P-P65, and P65 in cell lysates of GSC17 ( E ) and GSC23 ( F ) harboring control and TFPI2 OE treated with or without HIF-1α inhibitor ACF (2 μM). ( G and H ) Quantification of p65 ChIP-PCR in the CLOCK promoter ( G ) and BMAL1 promoter ( H ) of GSC272. IgG was used as Control. n = 3. ( I and J ) RT-qPCR for CLOCK and BMAL1 in GSC17 ( I ) and GSC23 ( J ) harboring TFPI2 OE treated with or without ACF. n = 3. ( K and L ) RT-qPCR for CLOCK and BMAL1 in GSC17 ( K ) and GSC23 ( L ) harboring control or TFPI2 OE treated with or without P65 inhibitor SC75741. n = 3. ( M and N ) Immunoblots for CLOCK and BMAL1 in cell lysates of control and TFPI2 OE GSC17 ( M ) or GSC23 ( N ) treated with or without ACF. ( O and P ) Immunoblots for CLOCK and BMAL1 in cell lysates of control and TFPI2 OE GSC17 ( O ) or GSC23 ( P ) treated with or without SC75741. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, Student’s t test ( G and H ), 1-way ANOVA test ( I – L ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: shRNA, Control, Western Blot, Over Expression, Quantitative RT-PCR

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A and B ) Immunoblots for CD133 and SOX2 in cell lysates of GSC17 ( A ) and GSC23 ( B ) harboring control or TFPI2 overexpression (OE) and treated with or without HIF-1α inhibitor ACF (2 μM). ( C and D ) Immunoblots for CD133 and SOX2 in cell lysates of control and TFPI2 -OE GSC17 ( C ) and GSC23 ( D ) treated with or without P65 inhibitor SC75741 (5 μM). ( E ) In vitro limiting dilution assays in control and TFPI2 -OE GSC17 treated with or without ACF (2 μM) or SC75741 (5 μM). ( F – H ) Representative images ( F ) and quantification of relative tumorsphere number ( G ) and size ( H ) of control and TFPI2 -OE GSC17 treated with or without ACF or SC75741. Scale bar: 200 μm. n = 8. ( I ) In vitro limiting dilution assays in control and TFPI2 -OE GSC23 treated with or without ACF (2 μM) or SC75741 (5 μM). ( J – L ) Representative images ( J ) and quantification of relative tumorsphere number ( K ) and size ( L ) of control and TFPI2 -OE GSC23 treated with or without ACF or SC75741. Scale bar: 200 μm. n = 8. ( M – P ) Representative and quantification of proliferation in GSC17 ( M and N ) and GSC23 ( O and P ) harboring control or TFPI2 OE treated with or without ACF (2 μM) or SC75741 (5 μM). n = 3. Data from multiple replicates are presented as mean ± SD. ** P < 0.01, *** P < 0.001, 1-way ANOVA test ( G , H , K , L , N , and P ), 2-way ANOVA test ( E and I ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: Western Blot, Control, Over Expression, In Vitro

Journal: The Journal of Clinical Investigation

Article Title: Symbiotic exclusivity between CLOCK and TFPI2 drives stemness and immunosuppression in glioblastoma models

doi: 10.1172/JCI199056

Figure Lengend Snippet: ( A ) Schematic diagram depicting the positive feedback loop between TFPI2 and the CLOCK-BMAL1 complex in GSCs and the strategy of dual targeting their downstream signaling pathways to block GSC self renewal. ( B ) Survival curves of nude mice implanted with 2 × 10 5 GSC272 and treated with LDHA inhibitor Stiripentol (150 mg/kg, i.p., every other day), JNK inhibitor JNK-IN-8 (30 mg/kg, i.p., daily), STAT3 inhibitor WP1066 (30 mg/kg, i.p., daily), or Stiripentol in combination with JNK-IN-8 or WP1066 for 2 weeks beginning at day 7. n = 8 mice per group. ( C – F ) Representative images and quantification of IHC staining for SOX2 ( C and D ) and CD133 ( E and F ) in GSC272 tumors from mice with indicated treatments. Scale bar: 100 μm. n = 4. ( G and H ) Representative images ( G ) and quantification ( H ) of flow cytometry for the percentage of CD45 low CD11b + TMEM119 + CD206 + microglia (out of CD45 low CD11b + TMEM119 + microglia) in tumors from 005 GSC-bearing mice with indicated treatments. n = 3. ( I and J ) Representative images ( I ) and quantification ( J ) of flow cytometry for the percentage of CD45 + CD3 + CD8 + IFNγ + T cells (out of CD45 + CD3 + CD8 + T cells) in tumors from 005 GSC-bearing mice with indicated treatments. n = 3. ( K ) Survival curves of C57BL/6J mice implanted with 2 × 10 5 005 GSCs and treated with Stiripentol, JNK-IN-8, or WP1066, and Stiripentol in combination with JNK-IN-8 or WP1066 for 2 weeks beginning at day 7, and with anti-PD1 (10 mg/kg, i.p.) treatment on days 11, 14, and 17. n = 8 mice per group. Data from multiple replicates are presented as mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001, 1-way ANOVA test ( D , F , H , and J ), log-rank test ( B and K ).

Article Snippet: Plasmids of human Tagged ORF Clone of CLOCK (Origene, # RC221408), and TFPI2 (Origene, #RC202760) and mouse Tagged ORF Clone of Clock (Origene, #MR226315), and Tfpi2 (Origene, #MR202782) were used.

Techniques: Protein-Protein interactions, Blocking Assay, Immunohistochemistry, Flow Cytometry

Journal: Journal of thrombosis and haemostasis : JTH

Article Title: Tissue factor Deficiency Increases Alveolar Hemorrhage and Death in Influenza A Infected Mice

doi: 10.1111/jth.13307

Figure Lengend Snippet: We analyzed TF antigen expression in the lungs of uninfected (B, E) and infected (7 dpi) wild-type mice (C, F), and infected TFΔEp mice (G–I) by immunohistochemistry. Tissue sections were incubated with (B, C, E, F–I) or without (A and D) a goat anti-mouse TF polyclonal antibody. The black arrows indicate TF expression in the epithelium of bronchi (Bc). The arrowheads indicate TF staining in adventitial cells of blood vessels (Bv). The white arrow indicates TF expression in adventitial cells surrounding a bronchiole. Original magnification x200. Scale bar is 100 µm.

Article Snippet: Then, slides were incubated overnight at 4°C with 1 μg/mL anti-human TF goat polyclonal antibody (R&D Systems, Minneapolis, MN), rinsed and incubated with 1.5 μg/mL biotinylated rabbit anti-goat secondary antibody (Vector Lab) for 20 min at room temperature.

Techniques: Expressing, Infection, Immunohistochemistry, Incubation, Staining

Journal: Cancer Science

Article Title: Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma

doi: 10.1111/cas.12569

Figure Lengend Snippet: Upregulation of TMPRSS4 and downregulation of TFPI-2 in lung cancer samples. (a) Pseudocolor image showing log10 expression ratios to the average expression level of the control lung region of 90 specimens for each of approximately 1700 probes ( x -axis) across the 90 specimens ( y -axis) tested by microarray. Red indicates upregulation; green indicates downregulation. AC, adenocarcinoma; SCC, squamous cell carcinoma. (b) Average relative mRNA expression levels of TMPRSS4 or TFPI-2 in 90 clinical lung cancer samples. Results are expressed as fold change to the average expression level of non-malignant regions, mean ± SEM. * P < 0.05 compared to non-malignant control. (c) Expression levels of TMPRSS4 /β-actin mRNA in lung cancer cell lines were measured by RT-PCR. Relative TMPRSS4 expression mRNA level to β -actin was calculated by the −Δ C t method. Results are expressed as the mean ± SD. (d) Expression levels of TFPI-2 /β-actin mRNA in lung cancer cell lines were measured by RT-PCR as in (c). Experiments were carried out in technically triplicate (c, d).

Article Snippet: For the overexpression assay, pCMV-Tag2B (Stratagene, La Jolla, CA, USA), FLAG-tagged TMPRSS4 , GFP-tagged TFPI-2 , or pCMV6-AC-GFP (OriGene) mixed with X-tremeGENE 9 DNA Transfection Reagent (Roche) were transfected into the cells according to the manufacturer's protocol.

Techniques: Expressing, Microarray, Reverse Transcription Polymerase Chain Reaction

Journal: Cancer Science

Article Title: Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma

doi: 10.1111/cas.12569

Figure Lengend Snippet: Immunohistochemical TMPRSS4/TFPI-2 staining of non-small-cell lung carcinoma (NSCLC) specimens. (a) Representative images of TMPRSS4 and TFPI-2 staining. (b) Scatter diagram represents the relationship between the expression score of TMPRSS4 ( y -axis) and TMPRSS4 mRNA expression level ( x -axis) in six NSCLC specimens. The expression score was calculated by multiplying the proportion (%) with the intensity score of immunohistochemical analysis. The mRNA expression level was calculated as log10 ratios to average expression level of non-malignant region according to cDNA microarray data. (c) Same scatter diagram for TFPI-2 as for TMPRSS4 in (b). (d) Scatter diagram represents the relationship between the expression score of TMPRSS4 ( y -axis) and TFPI-2 ( x -axis) in six NSCLC specimens.

Article Snippet: For the overexpression assay, pCMV-Tag2B (Stratagene, La Jolla, CA, USA), FLAG-tagged TMPRSS4 , GFP-tagged TFPI-2 , or pCMV6-AC-GFP (OriGene) mixed with X-tremeGENE 9 DNA Transfection Reagent (Roche) were transfected into the cells according to the manufacturer's protocol.

Techniques: Immunohistochemical staining, Staining, Expressing, Microarray

Journal: Cancer Science

Article Title: Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma

doi: 10.1111/cas.12569

Figure Lengend Snippet: TFPI-2 downregulates the expression of TMRPSS4 in lung cancer cells. (a) NCI-H358, NCI-H520, NCI-H1975, A549, and NCI-H2228 cells were transfected with GFP plasmid alone (lane 1) or with GFP-TFPI-2 (lane 2) for 48 h, and protein levels of TFPI-2 were detected with α-GFP antibody. (b) mRNA expression levels of TMPRSS4/GAPDH in the same experiments as (a) were detected with RT-PCR. Cells treated with GFP plasmid were calculated as 1 in (b). (c) Five cell lines were co-transfected with a GFP or GFP-TFPI-2 expression plasmid and a Luc or TMPRSS4 -Luc reporter plasmid for 24 h together with an internal control RL-TK plasmid, and the effect on the reporter activity of TMPRSS4 by TFPI-2 was evaluated by the luciferase assay. (d, e) NCI-H358, NCI-H520, and NCI-H1975 cells were transfected with negative control or TFPI-2 siRNAs for 48 h, and mRNA expression levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were evaluated by RT-PCR. Each value from cells treated with negative siRNA was calculated as 1. * P < 0.05 compared to GFP alone or to negative control siRNA. All experiments were carried out three times independently. Representative data are shown for Western blotting (a).

Article Snippet: For the overexpression assay, pCMV-Tag2B (Stratagene, La Jolla, CA, USA), FLAG-tagged TMPRSS4 , GFP-tagged TFPI-2 , or pCMV6-AC-GFP (OriGene) mixed with X-tremeGENE 9 DNA Transfection Reagent (Roche) were transfected into the cells according to the manufacturer's protocol.

Techniques: Expressing, Transfection, Plasmid Preparation, Reverse Transcription Polymerase Chain Reaction, Activity Assay, Luciferase, Negative Control, Western Blot

Journal: Cancer Science

Article Title: Methylation-induced downregulation of TFPI-2 causes TMPRSS4 overexpression and contributes to oncogenesis in a subset of non-small-cell lung carcinoma

doi: 10.1111/cas.12569

Figure Lengend Snippet: TFPI-2 is methylated in lung cancer specimens and cell lines. (a) Methylation levels of TFPI-2 were measured using MethyLight analysis in 87 clinical lung cancer samples. Methylation levels of β -actin were used as a control. TFPI-2 /β -actin methylation levels in H1975 cells (which had the most TFPI-2 methylation of the lung cancer cell lines tested) were calculated as 100. We dichotomized the lung cancer patients by their TMPRSS4 expression levels, and we calculated the average expression levels of TMPRSS4 and methylation levels of TFPI-2 in each group. (b) Methylation levels of TFPI-2 in lung cancer cell lines were measured using MethyLight analysis. The experiment was carried out in technically triplicate. (c–e) NCI-H358, NCI-H520, and NCI-H1975 cells were treated with 5 μM 5-aza-2′-deoxycytidine for 72 h or 0.5 μM trichostatin A (TSA) for 24 h. Changes in the methylation level induced by these treatments were measured using MethyLight analysis (c) and changes in the mRNA levels of TFPI-2/GAPDH (d) and TMPRSS4/GAPDH (e) were measured by RT-PCR. * P < 0.05 compared to DMSO alone. Experiments were carried out in triplicate.

Article Snippet: For the overexpression assay, pCMV-Tag2B (Stratagene, La Jolla, CA, USA), FLAG-tagged TMPRSS4 , GFP-tagged TFPI-2 , or pCMV6-AC-GFP (OriGene) mixed with X-tremeGENE 9 DNA Transfection Reagent (Roche) were transfected into the cells according to the manufacturer's protocol.

Techniques: Methylation, Expressing, Reverse Transcription Polymerase Chain Reaction