Journal: Cell reports

Article Title: Post-translational Regulation of FNIP1 Creates a Rheostat for the Molecular Chaperone Hsp90

doi: 10.1016/j.celrep.2019.01.018

Figure Lengend Snippet:

Article Snippet: Rabbit anti-PP5 , Cell Signaling Technology , Cat# 2289; RRID:AB_2168757.

Techniques: Virus, Recombinant, Mass Spectrometry, Software

Journal: Molecular oncology

Article Title: Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6-independent manner.

doi: 10.1002/1878-0261.12072

Figure Lengend Snippet: Fig. 3. PP5 mediates palbociclib-induced AMPK activation, autophagy, and apoptosis. (A) PP5 overexpression suppresses the effect of palbociclib on pAMPKa, autophagy, and apoptosis. Hep3B cells were transfected with vector or DDK-PP5 for 24 h and treated with palbociclib (15 lM) for another 24 h. (B) Pretreatment with arachidonic acid (AA), a PP5 activator, reversed the effect of palbociclib on pAMPKa, autophagy, and apoptosis. Hep3B cells were pretreated with AA (100 lM) for 4 h and then treated with palbociclib for 24 h. Autophagy was determined by LC3 immunoblotting. Apoptotic cells were measured by flow cytometry. *P < 0.05. (C) PP5 activity in Hep3B and PLC5 cells. (D) Palbociclib inhibits PP5 activity in PP5-containing Hep3B lysate. (E) Palbociclib suppresses activity of GST-PP5. Cantharidin, a known Ser/Thr phosphatase inhibitor, served as a positive control. *P < 0.05

Article Snippet: PP5 antibody (C-20) (sc-32588) was used for immunoblotting, and the PP5 antibody (H-7) (sc271816) used for immunoprecipitation was obtained from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: Activation Assay, Over Expression, Transfection, Plasmid Preparation, Western Blot, Cytometry, Activity Assay, Positive Control

Journal: Molecular oncology

Article Title: Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6-independent manner.

doi: 10.1002/1878-0261.12072

Figure Lengend Snippet: Fig. 5. In vivo effects of palbociclib in Huh7 xenograft nude mice. (A,B) Palbociclib suppresses Huh7 xenograft tumor growth and weight. Nude mice were subcutaneously implanted with 5 9 106 Huh7 cells. The mice received vehicle or palbociclib (150 mgkg1) orally every three days. The tumor area was measured twice a week. At the end of treatment, the tumors were harvested and the tumor weights were determined before lysis. Points/bars, mean (n = 5); bars, SD. **P < 0.01. (C) Western blot analysis of pAMPK, AMPK, and LC3 in Huh7 tumors. (D) PP5 activity in Huh7 tumors. *P < 0.05.

Article Snippet: PP5 antibody (C-20) (sc-32588) was used for immunoblotting, and the PP5 antibody (H-7) (sc271816) used for immunoprecipitation was obtained from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: In Vivo, Lysis, Western Blot, Activity Assay

Journal: Molecular oncology

Article Title: Palbociclib induces activation of AMPK and inhibits hepatocellular carcinoma in a CDK4/6-independent manner.

doi: 10.1002/1878-0261.12072

Figure Lengend Snippet: Fig. 6. Expression of PP5 in human HCC tissues. (A) Expression of PP5 in HCC tumors and normal liver tissue. The protein expressions of PP5 in the HCC tumors and its adjacent normal part were analyzed in 153 patients with HCC by IHC. Representative images were shown here. (B) PP5 expression in HCC tumors is significantly higher in HCC tumors than in normal liver tissue. The expressions of PP5 were quantified by H-score and compared by Student’s t-test. P < 0.001. Bar, mean; error bar, SE. ***P < 0.001. (C) PP5 expression in paired tumor and normal liver tissue. Each dot represented the H-score obtained from indicated tissues, and each line linked an individual patient.

Article Snippet: PP5 antibody (C-20) (sc-32588) was used for immunoblotting, and the PP5 antibody (H-7) (sc271816) used for immunoprecipitation was obtained from Santa Cruz Biotechnology (San Diego, CA, USA).

Techniques: 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 ) 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: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: High expression of PPP5C is associated with T stage, malignant degree and prognosis in BCa as analyzed by ONCOMINE microarray database. (A) Meta-analysis of the PPP5C expression between the BCa and the normal urothelial tissue from 3 microarray datasets containing 7 groups (P<0.001). (B) Each expression difference of the bladder cancer and normal tissue in the Sanchez-Carbayo Bladder 2 dataset (P<0.0001), Lee Bladder dataset (P=0.028), Dyrskjot Bladder 3 (P<0.0001). (C) The Kaplan-Meier analysis on the correlation between the PPP5C and the survival using the Als dataset (P=0.0007). (D and E) The two microarray datasets (Blaveri bladder 2 and Dyrskjot Bladder) show that the expression of PPP5C in T stage ≥T1 was higher than that in Ta. (F) The Blaveri bladder 2 dataset indicated that the expression of PPP5C in BCa of high-grade malignancy was higher than that in BCa of low-grade malignancy (P=0.0385).

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Expressing, Microarray

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: PPP5C expression and patient characteristics of ALS Bladder dataset.

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Expressing

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: Lentivirus-mediated interference downregulates the expression of PPP5C in urinary bladder cancer cells. (A) T24 and BT5637 cells were examined by fluorescence microscopy after lentivirus infection for 72 h. (B and C) Knock-down efficiency of PPP5C mRNA level in T24 and BT5637 infected by Lv-shPPP5C was determined by real-time qPCR ( *** P<0.001). (D and E) The protein level of PPP5C was downregulated in T24 and BT5637 cells. Lv-shCon, lentivirus-mediated non-silencing shRNA; Lv-shPPP5C, lentivirus-mediated non-silencing shPPP5C. Data were presented as mean ± SD from at least three experiments.

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Expressing, Fluorescence, Microscopy, Infection, Knockdown, shRNA

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: Lentivirus-mediated knockdown of PPP5C suppresses cell viability of urinary bladder cancer cells. (A and B) After infection with Lv-shCon or Lv-shPPP5C, the growth curves of T24 and BT5637 cells were determined by MTT assay. Data are presented as mean ± SD from at least three experiments ( *** P<0.001). (C and D) Representative images of T24 and BT5637 cells colony formation under micro- and macro-views after infection with Lv-shCon or Lv-shPPP5C. (E and F) The number of colonies formed in T24 and BT5637 cells treated as in (C and D). Data are presented as mean ± SD from at least three experiments ( * P<0.05, *** P<0.001).

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Knockdown, Infection, MTT Assay

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: Knock-down of the PPP5C level inhbits the growth of BCa in vivo . (A and C) The subcutaneous xenograft nude mice and the tumor sample were collected and imaged. (B and D) The tumor size and weight were significantly decreased in nude mice infected with lv-shPPP5C-T24 cell (n=6) as compared with those in nude mice infected with lv-shCon-T24 (n=6) ( * P<0.05, ** P<0.01, *** P<0.001).

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Knockdown, In Vivo, Infection

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: Disruption of PPP5C expression causes cell cycle arrest in bladder cancer cells. (A) Flow cytometric analysis showing cell cycle distribution of T24 and BT5637 cells infected by Lv-shCon or Lv-shPPP5C. (B) Cell percentages in G0/G1, S and G2/M of T24 and BT5637 cells treated as in (A) were analyzed statistically ( * P<0.05, ** P<0.01, *** P<0.001).

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Disruption, Expressing, Infection

Journal: International Journal of Oncology

Article Title: Disruption of serine/threonine protein phosphatase 5 inhibits tumorigenesis of urinary bladder cancer cells

doi: 10.3892/ijo.2017.3997

Figure Lengend Snippet: Apoptotic cells are increased after PPP5C silencing. (A) Apoptosis of T24 and BT5637 was analyzed in lv-shCon and lv-shPPP5C groups using flow cytometry and Annexin V/PI staining. (B) Early apoptosis (V + /PI − ) and late apoptosis (V + /PI + ) of the T24 and BT5637 cells were statistically analyzed ( * P<0.05, **P<0.01, *** P<0.001).

Article Snippet: Then, the membranes were incubated with the following primary antibodies: PPP5C (#:11715-1-AP; Proteintech Group, Chicago, IL, USA), CDK4 (#:11026-1-AP; Proteintech Group), c-Myc (#:sc-40; Santa Cruz Biotechnology, Santa Cruz, CA, USA), p27 (#:3686; Cell Signaling Technology, Danvers, MA, USA), BAD (#:10435-1-AP; Proteintech Group), Beclin1 (#:3495; Cell Signaling Technology) and GAPDH (#:10494-1-AP; Proteintech Group) for 12 h at 4°C.

Techniques: Flow Cytometry, 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

Journal: Allergy

Article Title: Protein phosphatase 5 mediates corticosteroid insensitivity in airway smooth muscle in patients with severe asthma

doi: 10.1111/all.13003

Figure Lengend Snippet: A, Total RNA was extracted from growth-arrested ASM cells in n = 5 healthy controls and n = 5 patients with severe asthma and levels of PP5 and GAPDH were measured by real-time PCR. Results are expressed as means ± SEM. ***P<0.001 versus healthy controls. B, Representative histogram showing PP5 expression assessed by flow cytometry in one healthy control and one patient with severe asthma. B, Quantitative analysis of PP5 levels assessed in n=5 healthy controls and n=5 subjectss with severe asthma. Data are expressed as means ± SEM, **P<0.01 versus healthy controls.

Article Snippet: PP5 primers (considered as proprietary information, cat# sc-44602-PR, 540 bp) were purchased from Santa Cruz Biotech.

Techniques: Real-time Polymerase Chain Reaction, Expressing, Flow Cytometry, Control

Journal: Allergy

Article Title: Protein phosphatase 5 mediates corticosteroid insensitivity in airway smooth muscle in patients with severe asthma

doi: 10.1111/all.13003

Figure Lengend Snippet: A, Growth-arrested ASM cells from n = 3 severe asthmatics were transfected with Silencer predesigned siRNA PP5 or non-silencing control scrambled siRNA (100 nM). After transfection, cells were lysed and assayed for PP5 by real time PCR. Results are expressed as means ± SEM. ***P<0.001 versus cells transfected with scrambled siRNA. Growth arrested ASM cells from severe asthmatics transfected with scrambled or PP5 siRNA (100 nM) were treated with 100 nM fluticasone for 4 hr. Cells were used for C, assessing GILZ expression by real-time PCR. Each experiment was performed in duplicate and repeated in cells from n = 4 patients with severe asthma. The results are expressed as means ± SEM. *P<0.05 versus scrambled siRNA controls; D, GRα nuclear translocation was determined by assessing the staining intensity of GRα in the nucleus of cells from n = 4 patients with severe asthma. The results are expressed as means ± SEM. *P<0.05 versus Fp stimulated cells transfected with scrambled siRNA; **P<0.01 versus basal cells transfected with PP5 siRNA. B-C, CCL11 (B) and CCL5 (C) in the supernatants of transfected cells with either PP5 siRNA or scramble siRNA and stimulated with TNFα (10 ng/ml, 24 hr) in the presence or the absence of fluticasone (100 nM) were determined by ELISA. Results are shown as means ±SEM of experiments performed in triplicate in n = 3 patients with severe asthma. *P<0.05, **P<0.01 versus respective control siRNA.

Article Snippet: PP5 primers (considered as proprietary information, cat# sc-44602-PR, 540 bp) were purchased from Santa Cruz Biotech.

Techniques: Transfection, Control, Real-time Polymerase Chain Reaction, Expressing, Translocation Assay, Staining, Enzyme-linked Immunosorbent Assay

Journal: Allergy

Article Title: Protein phosphatase 5 mediates corticosteroid insensitivity in airway smooth muscle in patients with severe asthma

doi: 10.1111/all.13003

Figure Lengend Snippet: Representative immunostaining in bronchial biopsies for PP5, α-smooth muscle actin or isotype control antibody in one healthy control (A, left panels) or one patient with severe asthma (A, right panels) at magnification X200. Positive staining was visualised with DAB detection (with hematoxylin counter stain) and quantified using the thresholding technique with Image J software. B, % ASM area per biopsy in healthy controls (n=8) and patients with severe asthma (n=20). NS, no statistical difference. C, PP5 staining within ASM bundle expressed as % staining per total area of ASM in n = 8 healthy controls and n= 20 patients with severe asthma. Data represented as means ± SEM, P value versus healthy controls.

Article Snippet: PP5 primers (considered as proprietary information, cat# sc-44602-PR, 540 bp) were purchased from Santa Cruz Biotech.

Techniques: Immunostaining, Control, Staining, Software