Journal: Nature communications

Article Title: miR-508 sustains phosphoinositide signalling and promotes aggressive phenotype of oesophageal squamous cell carcinoma.

doi: 10.1038/ncomms5620

Figure Lengend Snippet: Figure 1 | Reduced INPP5J levels correlate with ESCC progression. (a,b) Western blotting analysis of INPP5J expression in eight pairs of ESCC samples (T) and adjacent normal tissues (ANT; a); and in two NEECs and 12 cultured ESCC cell lines (b). a-Tubulin served as the loading control. (c) IHC staining indicating that INPP5J protein expression is downregulated in human ESCC compared with normal oesophageal tissues. Clinical staging and clinicopathological TNM classification were determined according to the criteria proposed by International Union Against Cancer criteria. Immunohistochemical staining of INPP5J in representative samples of ESCC, and normal oesophageal tissues were shown. Scale bars, 50 mm (insets 20 mm). (d) Kaplan–Meier analysis of 5-year overall survival (left panel) or disease-free survival (right panel) curves for ESCC patients with low versus high expression of INPP5J (n ¼ 207; Po0.001). (e) Reconstitution of INPP5J inhibits ESCC tumorigenicity, as demonstrated by an ESCC tumour xenograft model. Left panel: representative images of tumour-bearing mice. Right panel: images of tumours from all mice in each group. (f) IHC staining detecting Ki67 and p-Akt (Ser 473) expression in the indicated tumours. Scale bars, 20 mm.

Article Snippet: Western blotting was performed using the primary antibodies, anti-p-Akt (1:500; Ser 473), anti-p-Akt (1:500; Thr 308), anti-Akt (1:1,000), anti-p-GSK3b (1:500), anti-GSK3b (1:1,000), anti-CyclinD1 (1:1,000), anti-Bim (1:1,000), anti-p21 (1:500), anti-pRb (1:1,000), anti-Rb (1:1,000), antiPTEN (1:1,000) antibodies (Cell Signaling), anti-INPP4A antibody (1:500; Abcam) and anti-INPP5J antibody (1:500; Proteintech).

Techniques: Western Blot, Expressing, Cell Culture, Control, Immunohistochemistry, Immunohistochemical staining, Staining

Journal: Nature communications

Article Title: miR-508 sustains phosphoinositide signalling and promotes aggressive phenotype of oesophageal squamous cell carcinoma.

doi: 10.1038/ncomms5620

Figure Lengend Snippet: Figure 2 | miR-508 directly targets INPP5J and activates PI3K/Akt signalling. (a) Predicted miR-508 target sequences in the 30UTR of INPP5J (INPP5J-30UTR) and mutant containing three altered nucleotides in the seed sequence of miR-508 (miR-508-mut). (b) miR-508 was significantly overexpressed in ESCC analysed using published miaroarray data (NCBI/GEO/GSE6188, n ¼ 257, including 104 non-tumour and 153 tumour samples; boundaries of the boxes represent the lower and upper quartile. Lines within boxes and whiskers denote median and extremum, respectively. Po0.001; w2- test). (c) Western blotting analysis of INPP5J expression in vector- or miR-508-transduced cells, or in cells transfected with negative control or miR-508 antagomir. a-Tubulin served as the loading control. (d) Luciferase assay of indicated cells transfected with the pGL3-INPP5J-30UTR reporter with increasing amounts (10 and 50 nM) of negative control (NC), miR-508 mimic, control antagomir, miR-508 antagomir or miR-508-mut mimic. (e) Western blotting analysis of expression of p-Akt (Ser 473), p-Akt (Thr 308), Akt, p-GSK3b, GSK3b, p21, CyclinD1, Bim and pRb and p-pRb in the indicated cells. a-Tubulin served as the loading control. Each bar represents the mean±s.d. of three independent experiments. *Po0.05.

Article Snippet: Western blotting was performed using the primary antibodies, anti-p-Akt (1:500; Ser 473), anti-p-Akt (1:500; Thr 308), anti-Akt (1:1,000), anti-p-GSK3b (1:500), anti-GSK3b (1:1,000), anti-CyclinD1 (1:1,000), anti-Bim (1:1,000), anti-p21 (1:500), anti-pRb (1:1,000), anti-Rb (1:1,000), antiPTEN (1:1,000) antibodies (Cell Signaling), anti-INPP4A antibody (1:500; Abcam) and anti-INPP5J antibody (1:500; Proteintech).

Techniques: Mutagenesis, Sequencing, Western Blot, Expressing, Plasmid Preparation, Transfection, Negative Control, Control, Luciferase

Journal: Nature communications

Article Title: miR-508 sustains phosphoinositide signalling and promotes aggressive phenotype of oesophageal squamous cell carcinoma.

doi: 10.1038/ncomms5620

Figure Lengend Snippet: Figure 3 | miR-508 directly targets phosphoinositide phosphatase PTEN and INPP4A, and sustains phosphoinositide signalling. (a) Relative Akt activity (left panel) and INPP5J expression (right panel) in indicated cells. Recovery of INPP5J expression in miR-508-transduced cells comparable to that in control cells, via transfection with INPP5J ORF (without 30UTR), only partially inhibited miR-508-induced Akt activity. (b) Upper panel: predicted miR-508 target sequence in PTEN-30UTR and INPP4A-30UTR. Lower panel: western blotting analysis of the expression of PTEN and INPP4A in the indicated cells. a-Tubulin served as the loading control. (c) Luciferase assay of indicated cells transfected with the pGL3-PTEN-30UTR, pGL3-INPP4A-30UTR reporter with miR-508 mimic, miR-508 antagomir or miR-508-mut mimic. (d) miRNP immunoprecipitation assay showed associations of miR-508 with INPP5J, PTEN and INPP4A. GAPDH served as a negative control. (e) Detection of PI(3,4,5)P3 level (left panel) or PI(3,4)P2 (right panel) in miR-508- transduced cells or miR-508-inhibited cells compared with controls. (f) Relative Akt activity in miR-508-overexpressing or miR-508-inhibited ESCC cells compared with controls. (g) Relative Akt activity in the indicated cells, which were serum-starved and subsequently stimulated with EGF (10 ng ml 1, upper panel) or insulin (100 nM, lower panel) for indicated times. Each bar represents the mean±s.d. of three independent experiments. *Po0.05.

Article Snippet: Western blotting was performed using the primary antibodies, anti-p-Akt (1:500; Ser 473), anti-p-Akt (1:500; Thr 308), anti-Akt (1:1,000), anti-p-GSK3b (1:500), anti-GSK3b (1:1,000), anti-CyclinD1 (1:1,000), anti-Bim (1:1,000), anti-p21 (1:500), anti-pRb (1:1,000), anti-Rb (1:1,000), antiPTEN (1:1,000) antibodies (Cell Signaling), anti-INPP4A antibody (1:500; Abcam) and anti-INPP5J antibody (1:500; Proteintech).

Techniques: Activity Assay, Expressing, Control, Transfection, Sequencing, Western Blot, Luciferase, Immunoprecipitation, Negative Control

Journal: Nature communications

Article Title: miR-508 sustains phosphoinositide signalling and promotes aggressive phenotype of oesophageal squamous cell carcinoma.

doi: 10.1038/ncomms5620

Figure Lengend Snippet: Figure 7 | Clinical relevance of miR-508 and expression of its targets in ESCC. (a) The Kaplan–Meier analysis of 5-year overall survival curves for ESCC patients with low versus high expression of PTEN (upper panel; n ¼ 207; Po0.001) or with low versus high expression of INPP4A (lower panel; n ¼ 207; Po0.001). (b) miR-508 levels were significantly associated with expression of INPP5J, PTEN, INPP4A and p-Akt (Ser 473) in 207 primary human ESCC specimens. Upper panel: two representative cases are shown. Scale bars, 50 mm. Lower panel: percentages of specimens showing low or high miR- 508 expression relative to levels of INPP5J, PTEN, INPP4A and p-Akt (Ser 473). (c) Analysis of expression of miR-508 with the expression levels of INPP5J, PTEN, INPP4A and p-Akt (Ser 473) in 10 freshly collected human ESCC samples, including five clinical stage I-II and five clinical stage III-IV samples. (d) Proposed model. Upregulation of miR-508 represses multiple phosphoinositide phosphatases to sustain phosphoinositide signalling, leading to constitutive PI3K/Akt activation and ESCC aggressiveness.

Article Snippet: Western blotting was performed using the primary antibodies, anti-p-Akt (1:500; Ser 473), anti-p-Akt (1:500; Thr 308), anti-Akt (1:1,000), anti-p-GSK3b (1:500), anti-GSK3b (1:1,000), anti-CyclinD1 (1:1,000), anti-Bim (1:1,000), anti-p21 (1:500), anti-pRb (1:1,000), anti-Rb (1:1,000), antiPTEN (1:1,000) antibodies (Cell Signaling), anti-INPP4A antibody (1:500; Abcam) and anti-INPP5J antibody (1:500; Proteintech).

Techniques: Expressing, Activation Assay

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Representative microphotographs of immunohistochemistry staining of PIB5PA and PTEN (brown) on melanocytic tumour tissue sections. Scale bar, 100 μm. ( b ) Quantification of PIB5PA expression levels in melanocytic tumours (see also , and Supplementary Fig. S1). Data shown are mean immunoreactive score (IRS)±s.e.m.; Student’s t -test; * P <0.01. ( c ) Whole-cell lysates from fresh melanoma isolates were subjected to western blot analysis. Data shown are representative of three individual western blot analyses. ( d ) Representative regression analysis of relationship between PIB5PA and PTEN expression as shown in c . Levels of PIB5PA and PTEN were normalized to those of GAPDH. Quantification of each band was determined using NIH Image J. ( e ) Comparison of pSer473-Akt levels between fresh melanoma isolates negative for both PIB5PA and PTEN, and those expressing at least one of the proteins. Levels of pSer473-Akt were normalized to those of total Akt. Quantification of each band was determined using NIH Image J ( n =3, mean±s.e.m.; Student’s t -test; * P <0.05). ( f ) Whole-cell lysates from pooled melanocytes of three different lines (HEMa-LP, HEMa-DP and HEMn-LP) and melanoma cell were subjected to western blot analysis. Data shown are representative of three individual experiments. BRAF and NRAS mutational status of the cell lines was also indicated. W, wild-type; M, BRAF V600E or NRAS Q61R mutation. ( g ) Quantitative reverse transcription–PCR analysis of total RNA extracted from melanocytes and melanoma cells showing that PIB5PA mRNA levels are commonly downregulated in melanoma cells compared with melanocytes. The relative abundance of PIB5PA mRNA expression in melanocytes was arbitrarily designated as 1 ( n =3, mean±s.e.m.).

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Immunohistochemistry, Staining, Expressing, Western Blot, Comparison, Mutagenesis, Reverse Transcription

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Whole-cell lysates from ME1007 and Mel-FH cells, either non-transfected or transfected with empty vector-HA or PIB5PA-HA constructs, were subjected to western blot analysis. Data shown are representative of three individual experiments. ( b – e ) ME1007 and Mel-FH cells were transiently transfected with empty HA, PIB5PA-HA, or mutant PIB5PA (PIB5PA SKICH )-HA constructs. ( b ) Seventy-two hours later, apoptotic cells were quantitated by measurement of sub-G1 DNA content ( n =3, mean±s.e.m.). ( c ) Forty-eight hours later, whole-cell lysates were subjected to western blot analysis. Data shown are representative of three individual experiments. ( d ) Seventy-two hours later, cells were subjected to proliferation assays using the BrdU incorporation method ( n =3, mean±s.e.m.). ( e ) Twenty-four hours later, viable cells (2,000 cells per well in six-well plates) were allowed to grow for 12 days before being fixed with methanol and stained with crystal violet. Data shown are representative of three individual experiments. Scale bar, 1 cm.

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Transfection, Plasmid Preparation, Construct, Western Blot, Mutagenesis, BrdU Incorporation Assay, Staining

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Mel-FH.PIB5PA and ME1007.PIB5PA cells that carried a lentiviral-based 4-OHT-responsive inducible gene expression system and their parental counterparts were exposed to 4-OHT at indicated concentrations for 24 h. Whole-cell lysates were subjected to western blot analysis. ( b ) Whole-cell lysates from Mel-FH.PIB5PA and ME1007.PIB5PA cells treated with 4-OHT (10 nM) for 24 h with or without withdrawal of 4-OHT for another 24 h were subjected to western blot analysis. ( c ) Mel-FH.PIB5PA and ME1007.PIB5PA cells treated with 4-OHT (10 nM) for 24 h with or without withdrawal of 4-OHT for another 48 h were subjected to proliferation assays using the BrdU incorporation method ( n =3, mean±s.e.m.). ( d ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were seeded onto six-well plates (2,000 cells per well) for 24 h before the addition of 4-OHT (10 nM). Seventy-two hours later, 4-OHT was withdrawn from representative wells. Cells were allowed to grow for another 9 days before being fixed with methanol and stained with crystal violet. Scale bar, 1 cm. ( e ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transiently transfected with vector alone or a myr-Akt construct. Twenty-four hours later, cells were treated with 4-OHT (10 nM) for a further 24 h. Whole-cell lysates were subjected to western blot analysis. ( f ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transiently transfected with vector alone or a myr-Akt construct. Twenty-four hours later, cells were treated with 4-OHT (10 nM) for a further 48 h before cell proliferation was measured by the BrdU incorporation method ( n =3, mean±s.e.m.). ( g ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transfected with vector alone or a myr-Akt construct. Twenty-four hours later, viable cells (2,000 cells per well in six-well plates) were allowed to grow for a further 12 days before being fixed with methanol and stained with crystal violet. Scale bar, 1 cm.

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Gene Expression, Western Blot, BrdU Incorporation Assay, Staining, Transfection, Plasmid Preparation, Construct

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) The PIB5PA mRNA expression levels in HEMa-LP melanocytes transduced with the control or PIB5PA shRNA by quantitative PCR ( n =3, mean±s.e.m.). ( b ) Whole-cell lysates from HEMa-LP melanocytes transduced with the control or PIB5PA shRNA were subjected to western blot analysis. ( c ) Anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PIB5PA or PTEN shRNA. Scale bar, 0.5 mm. ( d ) Quantification of anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PIB5PA or PTEN shRNA as shown in c ( n =3, mean±s.e.m.; Student’s t -test; * P <0.05). ( e ) HEMa-LP melanocytes transduced with the control, PIB5PA or PTEN shRNA were counted on days 3, 6 and 9, using an automated cell counter ( n =3, mean±s.e.m.). ( f ) HEMa-LP melanocytes transduced with the PIB5PA shRNA were cointroduced with the control or Akt shRNA. Twenty-four hours later, whole-cell lysates were subjected to western blot analysis. ( g ) HEMa-LP melanocytes transduced with the PIB5PA shRNA were cointroduced with the control or Akt shRNA. Ninety-six hours later, viable cells were counted in an automated cell counter ( n =3, mean±s.e.m.).

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Expressing, Transduction, Control, shRNA, Real-time Polymerase Chain Reaction, Western Blot, Growth Assay

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Viable ME1007.PIB5PA cells (1 × 10 7 ), with or without pretreatment with 4-OHT (10 nM) for 36 h, were subcutaneously injected into flanks of nu/nu mice. Two days later, mice were administered with either vehicle (DMSO; n =6) or 4-OHT (10 nM g −1 ; n =6) via intraperitoneal injections every 3 days. Mice were killed and tumours were collected at 36 days after melanoma cell injection. Data shown are representative of three individual experiments. Scale bar, 5 mm. ( b ) Comparison of weight of tumours from animals treated with 4-OHT with those treated with vehicle control ( n =6, mean±s.e.m.; Student’s t -test; * P <0.05). ( c ) Whole-cell lysates of crude tumour tissues were subjected to western blot analysis. Data shown are representative of three individual western blot analyses of randomly selected tumour samples from mice with or without treatment with 4-OHT. ( d ) Representative tumours generated by ME1007.PIB5PA cells with or without cotransduction with myr-Akt in nu/nu mice with or without treatment with 4-OHT as stated in a . Scale bar, 5 mm. ( e , f ) Comparison of volume ( e ) and weight ( f ) of tumours generated by ME1007.PIB5PA cells with or without cotransduced myr-Akt in nu/nu mice with or without treatment with 4-OHT as stated in a ( n =6, mean±s.e.m.; Student’s t -test; * P <0.05). ( g ) Whole-cell lysates of crude tumour tissues from tumours generated by ME1007.PIB5PA cells with or without cotransduced with myr-Akt in nu/nu mice with or without treatment with 4-OHT as stated in a . Data shown are representative of three individual experiments.

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Injection, Comparison, Control, Western Blot, Generated

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were serum-starved for 16 h in the presence or absence of 4-OHT (10 nM) before stimulation with EGF (100 nM) for indicated periods. Whole-cell lysates were subjected to western blot analysis. ( b ) Quantification of levels of pSer473-Akt in Mel-FH.PIB5PA cells as shown in a , which were normalized to those of total Akt ( n =3, mean±s.e.m.). ( c ) Mel-FH and ME1007 cells were transiently transfected with a PTEN construct. Twenty-four hours later, cells were serum-starved for 16 h, followed by stimulation with EGF (100 nM) for indicated periods. Whole-cell lysates were subjected to western blot analysis. ( d ) Quantification of levels of pSer473-Akt in Mel-FH cells as shown in c , which were normalized to those of total Akt ( n =3, mean±s.e.m.). ( e ) Mel-FH.PIB5PA and ME1007.PIB5PA cells were transiently transfected with a PTEN construct. Twenty-four hours later, cells were switched into serum-free medium with or without 4-OHT (10 nM) for a further 16 h before the addition of EGF (100 nM) for 15 min. Whole-cell lysates were subjected to western blot analysis. ( f ) Mel-AT and Mel-JD were transfected with the control, PIB5PA, PTEN or PIB5PA plus PTEN siRNA. Twenty-four hours later, whole-cell lysates were subjected to western blot analysis. ( g ) HEMa-LP melanocytes were transfected with the control, PIB5PA, PTEN or PIB5PA plus PTEN siRNA. Twenty-four hours later, whole-cell lysates were subjected to western blot analysis. ( h ) Anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PIB5PA, PTEN or PIB5PA plus PTEN shRNA. Data shown are representative of three individual experiments. Scale bar, 0.5 mm. ( i ) Quantification of anchorage-independent growth assay of HEMa-LP melanocytes transduced with the control, PIB5PA, PTEN or PIB5PA plus PTEN shRNA as shown in h ( n =3, mean±s.e.m.). ( j ) HEMa-LP melanocytes transduced with the control, PIB5PA, PTEN or PIB5PA plus PTEN shRNA as shown in h were counted on days 3, 6 and 9, using an automated cell counter ( n =3, mean±s.e.m.).

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Western Blot, Transfection, Construct, Control, Growth Assay, Transduction, shRNA

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) Total RNA from ME1007 and Mel-FH cells treated with 5-aza (5-aza-2′-deoxycytidine; 10 μM) for 96 h were subjected to quantitative PCR (qPCR) analysis ( n =3, mean±s.e.m.). ( b ) Whole-cell lysates from HEMa-LP melanocytes, and ME1007 and Mel-FH melanoma cells treated with 5-aza (10 μM) for 96 h were subjected to western blot analysis. ( c ) Whole-cell lysates from ME1007 and Mel-FH cells treated with SAHA (4 μM) for indicated periods were subjected to western blot analysis. ( d ) Total RNA from ME1007 and Mel-FH cells treated with SAHA (4 μM) for indicated periods were subjected to qPCR analysis ( n =3, mean±s.e.m.). ( e ) ME1007 and Mel-FH cells were transiently transfected with the pGL3-basic-based reporter constructs, pGL3-vector and pGL3- INPP5J core promoter (pGL3- INPP5J −2,016/+114), respectively. Twenty-four hours later, cells were treated with SAHA (4 μM) for a further 24 h, followed by measurement of the luciferase activity ( n =3, mean±s.e.m.). ( f ) ME1007 and Mel-FH cells were transfected with the control or PIB5PA siRNA. Twenty-four hours later, total RNA was subjected qPCR analysis of PIB5PA mRNA expression ( n =3, mean±s.e.m.). ( g ) ME1007 and Mel-FH cells were transfected with the control or PIB5PA siRNA. Twenty-four hours later, cells were treated with SAHA (4 μM) for a further 24 h. Whole-cell lysates were subjected to western blot analysis. ( h ) Quantification of pSer473-Akt normalized to total Akt levels as shown in g ( n =3, mean±s.e.m.). ( i ) Total RNA from ME1007 and Mel-FH cells treated with MC1568 (5 μM), MS275 (5 μM) and SAHA (4 μM), respectively, for 24 h was subjected to qPCR analysis of PIB5PA mRNA. The relative abundance of PIB5PA mRNA in cells treated with MC1568, MS275 or SAHA was normalized to that in cells without treatment ( n =3, mean±s.e.m.). ( j ) Whole-cell lysates from ME1007 and Mel-FH cells treated with MC1568 (5 μM), MS275 (5 μM) and SAHA (4 μM), respectively, for 24 h were subjected to western blot analysis. ( k ) Whole-cell lysates from ME1007 and Mel-FH cells transfected with the control, HDAC1, HDAC2 or HDAC3 siRNA were subjected to western blot analysis. ( l ) ME1007 cells were cotransfected with the control, HDAC2 or HDAC3 siRNA, and pGL3- INPP5J -(−2,016/+114). Luciferase activity was measured 24 h after transfection ( n =3, mean±s.e.m.).

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Construct, Plasmid Preparation, Luciferase, Activity Assay, Control, Expressing

Journal: Nature Communications

Article Title: PI(4,5)P2 5-phosphatase A regulates PI3K/Akt signalling and has a tumour suppressive role in human melanoma

doi: 10.1038/ncomms2489

Figure Lengend Snippet: ( a ) A schematic illustration of construction of a luciferase reporter constructs. ( b ) ME1007 cells were transiently transfected with indicated pGL3-basic-based reporter constructs. Twenty-four hours later, cells were treated with MS275 (5 μM) for a further 24 h, followed by measurement of the luciferase activity ( n =3, mean±s.e.m.). ( c ) ME1007 cells were cotransfected with the control, HDAC2 or HDAC3 siRNA, and indicated pGL3-basic-based reporter constructs. Luciferase activity was measured 24 h after transfection ( n =3, mean±s.e.m.). ( d , e ) Coimmunoprecipitation assays showing physical association of HDAC2 and HDAC3 with Sp1 in ME1007 cells. ( f ) Formaldehyde-crosslinked chromatin of ME1007 and Mel-FH cells was subjected to immunoprecipitation with antibodies against HDAC2, HDAC3 or Sp1. The precipitates were subjected to PCR amplification using primers directed to the −516/−383 (133 bp) fragment of the INPP5J promoter. Data shown are representative of three individual experiments. ( g ) Western blotting showing siRNA knockdown of Sp1 in ME1007 cells. ( h ) ChIP assays showing knockdown of Sp1 abolished the association of HDAC2 and HDAC3 with the INPP5J promoter. ( i ) ME1007 cells were cotransfected with the control or Sp1 siRNA and pGL3-vector or pGL3- INPP5J -(−2,016/+144) (pGL3- INPP5J ). Twenty-four hours later, cells were treated with MS275 (5 μM) for a further 24 h, followed by measurement of the luciferase activity ( n =3, mean±s.e.m.). ( j ) quantitative PCR analysis showing that siRNA knockdown of Sp1 elevated the levels of PIB5PA mRNA, which could not be further increased by MS275 ( n =3, mean±s.e.m.). ( k ) ME1007 and Mel-FH cells were transiently transfected with the pGL3-basic-based reporter constructs, pGL3-vector, pGL3- INPP5J -(−1,516/+144) (pGL3- INPP5J-WT ) and a reporter with the Sp1 binding site-enriched fragment deleted as shown in a (pGL3- INPP5J -mutant), respectively. Twenty-four hours later, cells were treated with MS275 (5 μM) for a further 24 h, followed by measurement of the luciferase activity ( n =3, mean±s.e.m.). ( l ) ChIP assays showing increased acetylation of the H3 and H4 histone subunits associated with INPP5J promoter (−516/−383) when HDAC2 or HDAC3 was knocked down.

Article Snippet: Assay-on-demand for PIB5PA (assay ID: Hs00300155_m1), PTEN (assay ID: Hs02621230_s1), HDAC1 (assay ID: Hs02621185_s1), HDAC2 (assay ID: Hs00231032_m1), HDAC3 (assay ID: Hs00187320_m1) and GAPDH (assay ID: Hs99999905_m1) were used according to the manufacturer’s protocol (Applied Biosystem).

Techniques: Luciferase, Construct, Transfection, Activity Assay, Control, Immunoprecipitation, Amplification, Western Blot, Knockdown, Plasmid Preparation, Real-time Polymerase Chain Reaction, Binding Assay, Mutagenesis