Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: Journal of medicinal chemistry

Article Title: Eradication of Therapy-Resistant Cancer Stem Cells by Novel Telmisartan Derivatives.

doi: 10.1021/acs.jmedchem.4c01865

Figure Lengend Snippet: Figure 3. TKI resistance correlates with CSC features in K562-R cells. (A) Jess Simple Western immunoassays to determine the protein expression of STAT5 (∼90 kDa), pSTAT5-tyr694/699 (∼90 kDa), and ABCB1 (∼180 kDa) in K562 and K562-R cells. GAPDH (∼40 kDa) served as loading control. (B) Flow cytometry analysis of CD44 expression in K562 and K562-R cells. Shown is the proportion of CD44+ cells [%]. Data represent the mean ∓SEM of 3 independent experiments. Statistical analysis was done with the Student’s unpaired t-test; ***p < 0.001. (C) K562 and K562-R cells were stained using DyeCycle Violet (DCV) and analyzed by flow cytometry. SP subsets are indicated by polygonal gates and the percentage of cells within these gates is given. Quantification of SP and non-side population (NSP) subsets in K562 and K562-R cells. Data represent the mean + SEM of ≥3 independent experiments. Statistical analysis was done with the Student’s paired t-test; **p < 0.01 (D) Concentration-dependent impact of verapamil, valspodar, and elacridar on the metabolic activity of K562-R cells, cotreated with imatinib (1 μM) for 72 h. Statistical analysis was done by one-way ANOVA; *p < 0.05, #p < 0.0001, compared to the vehicle-treated control (DMSO + imatinib (1 μM)). Data represent the mean + SEM of ≥3 independent MTT experiments with three replicates each. (E) Metabolic activity of K562 cells after incubation with verapamil, valspodar, and elacridar at 10 μM for 72 h. The vehicle-treated control (DMSO) served as a reference (100%). Data represent the mean + SEM of ≥3 independent MTT experiments with three replicates each.

Article Snippet: Antibodies specific to STAT5 (R&D Systems #AF2168; RRID/AB_355174), ABCB1 (Cell Signaling #12683; RRID/AB_2715689), ABCC1 (Cell Signaling #72202; RRID/AB_2799816), ABCG2 (Cell Signaling #42078; RRID/ AB_2799211), or GAPDH (Cell Signaling #2118; RRID/ AB_561053) each combined with 0.2 mg/mL protein lysate, and the pSTAT5 antibody (Millipore #05−886R; RRID/AB_1977514) in combination with 2 mg/mL protein lysate were used.

Techniques: Simple Western, Expressing, Control, Flow Cytometry, Staining, Concentration Assay, Activity Assay, Incubation

Journal: Journal of medicinal chemistry

Article Title: Eradication of Therapy-Resistant Cancer Stem Cells by Novel Telmisartan Derivatives.

doi: 10.1021/acs.jmedchem.4c01865

Figure Lengend Snippet: Figure 4. Telmisartan derivatives inhibit the SP phenotype and repress the phosphorylation status of STAT5 in K562-R cells. (A) K562-R cells were stained with DCV at 10 μM and concurrently incubated with 10 μM of 1b, 2b, 3b, 4b, 5c, 6b, 6c, 7a−c, valspodar, elacridar, or 160 μM of verapamil for 90 min. The subsequent analysis was carried out by flow cytometry. SP subsets are indicated by polygonal gates, and the percentage of cells within these gates is given. (B) Quantification of SP and NSP subsets of ≥3 independent experiments, shown as the mean + SEM. Statistical analysis was done with the Student’s paired t-test; **p < 0.01, #p < 0.001, compared to the proportion of the SP in vehicle-treated cells (CTR, DMSO) (C) K562-R cells were stained with DCV and concurrently incubated with indicated concentrations of 7b (telmi-ester), verapamil, valspodar, and elacridar. SP subsets were subsequently analyzed by flow cytometry. Shown is the mean + SEM of ≥3 independent experiments. Statistical analysis was done by one-way ANOVA; *p < 0.05, **p < 0.01, #p < 0.0001, compared to the proportion of the SP in vehicle-treated cells

Article Snippet: Antibodies specific to STAT5 (R&D Systems #AF2168; RRID/AB_355174), ABCB1 (Cell Signaling #12683; RRID/AB_2715689), ABCC1 (Cell Signaling #72202; RRID/AB_2799816), ABCG2 (Cell Signaling #42078; RRID/ AB_2799211), or GAPDH (Cell Signaling #2118; RRID/ AB_561053) each combined with 0.2 mg/mL protein lysate, and the pSTAT5 antibody (Millipore #05−886R; RRID/AB_1977514) in combination with 2 mg/mL protein lysate were used.

Techniques: Phospho-proteomics, Staining, Incubation, Flow Cytometry

Journal: Journal of medicinal chemistry

Article Title: Eradication of Therapy-Resistant Cancer Stem Cells by Novel Telmisartan Derivatives.

doi: 10.1021/acs.jmedchem.4c01865

Figure Lengend Snippet: Figure 6. The developed telmisartan derivatives target the CSC pool of solid cancers. (A) Quantification of the SP subsets detected in indicated ovarian cancer, prostate cancer, breast cancer, and lung cancer cell lines. Shown is the mean + SEM of ≥3 independent experiments. Statistical analysis was done with the Student’s paired t-test; **p < 0.01, #p < 0.0001, compared to the proportion of the SP in vehicle-treated (CTR) cells. (B) Jess Simple Western immunoassays determining the expression of STAT5 (∼90 kDa) and pSTAT5 (∼90 kDa). A2780 V SP, IGROV-1 SP, MCF7, and A549 cells were treated with vehicle (CTR, DMSO) or 10 μM of the compounds 5c, 6c, 7a, 7b, and 7c for 72 h, respectively. GAPDH (∼40 kDa) served as loading control. (C) A2780 V SP and IGROV-1 SP cells were treated with 10 μM of 5c, 6c, 7a, 7b, or 7c in combination with 10 nM of paclitaxel (PTX). PC3-DR and DU145-DR cells were treated with 10 μM of 5c, 6c, 7a, 7b, or 7c in combination with 12.5 nM of docetaxel (DTX). CTR represents the vehicle-treated (DMSO) control. After 72 h, the metabolic activity of the cells was determined by a modified MTT assay. Data represent the mean + SEM of n = 3 independent experiments with three replicates each. Statistical analysis was done with the Student’s paired t-test; *p < 0.05, **p < 0.01, ***p < 0.001, compared to cells treated with PTX or DTX alone (CTR). (D) A2780 V SP cells were treated with 10 μM of 5c, 6c, 7a, 7b, or 7c in combination with indicated concentrations of paclitaxel (PTX; 0−300 nM). IC50 values of PTX were calculated by curve fitting to normalized response. Data represent n = 3 independent experiments with three replicates each.

Article Snippet: Antibodies specific to STAT5 (R&D Systems #AF2168; RRID/AB_355174), ABCB1 (Cell Signaling #12683; RRID/AB_2715689), ABCC1 (Cell Signaling #72202; RRID/AB_2799816), ABCG2 (Cell Signaling #42078; RRID/ AB_2799211), or GAPDH (Cell Signaling #2118; RRID/ AB_561053) each combined with 0.2 mg/mL protein lysate, and the pSTAT5 antibody (Millipore #05−886R; RRID/AB_1977514) in combination with 2 mg/mL protein lysate were used.

Techniques: Simple Western, Expressing, Control, Activity Assay, Modification, MTT Assay

Journal: Cancer cell

Article Title: Integrative multi-omic cancer profiling reveals DNA methylation patterns associated with therapeutic vulnerability and cell-of-origin.

doi: 10.1016/j.ccell.2023.07.013

Figure Lengend Snippet: Figure 5. STAT5A hypermethylation associated with pervasive STAT5A regulon changes (A) Unsupervised clustering of STAT5A regulon genes using Pearson correlation of scaled RNA sequencing data. Annotations denote STAT5A expression and methylation levels. Mean activity indicates the overall sum of regulon activity. The color scale is proportional to expression (red: upregulation; blue: down- regulation). (B) Unsupervised clustering of STAT5A regulon genes using Pearson correlation of scaled global proteome data. (C) Violin plot comparing regulon activity in hypermethylated STAT5A (yellow) and normally methylated STAT5A (gray) samples. Median values are shown as solid black lines, and first and third quartiles are represented by dashed lines. Statistical significance was determined using a Wilcoxon rank-sum test. (D) Pathway members and interactions in the STAT5A regulon. The mean expression differences between STAT5A hypermethylated samples and normally methylated samples are indicated by shading of the filled squares. See also Figure S5 and Table S3.

Article Snippet: Staining employed the Dako Autostainer Link 48 with EnVision FLEX visualizing kit (K800221-2; Dako, Agilent Technologies Inc.) and rabbit polyclonal antibody against human STAT5A (HPA042128, Atlas Antibodies, 1:150 dilution).

Techniques: RNA Sequencing, Expressing, Methylation, Activity Assay