Journal: Asian Pacific Journal of Cancer Prevention : APJCP

Article Title: Unexpected Lower Expression of Oncoprotein Gankyrin in Drug Resistant ABCG2 Overexpressing Breast Cancer Cell Lines

doi: 10.22034/APJCP.2017.18.12.3413

Figure Lengend Snippet: The Expression of ABCG2 at mRNA Level in MCF-7 Derived Breast Cancer Cell Lines. ABCG2 mRNA level is compared between drug resistance cell line MCF-7/MX and its parental non-resistance MCF-7 cells. Real-time RT-PCR analysis was performed on total RNA extracted from cells. Values were normalized to the β-actin content of samples and expressed as mean ±SEM (n = 3). Statistical significance was calculated by one-way Analysis of variance (ANOVA) and Tukey’s post-hoc; ***p < 0.001 compared with the non-resistance parental cell line.

Article Snippet: ABCG2 overexpressing cell line MCF-7/MX and MDR1 overexpressing cell line MCF-7/ADR were kindly provided by Professor Herman Lage (Molecular pathology department, Charite Campus Mitte, Berlin, Germany).

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) Western blot detecting TLK2 protein ectopically expressed in T47D cells after induction with different doses of Dox. ( b ) Cell survival following induction of TLK2 expression in T47D cells was measured by clonogenic assay. Error bars represent the s.d. of three replicate measurements per condition. ( c ) TLK2 overexpression significantly enhanced anchorage-independent growth of T47D cells. TLK2 was overexpressed in T47D by treating with 100 ng ml −1 Dox before the soft-agar assays were performed. Error bars represent the s.d. of three replicate measurements per condition. ( d ) Transwell migration and matrigel invasion assays. Following TLK2 induction for 2 weeks, Dox was either continued or withdrawn to test if the increased cell migration and invasion is dependent on TLK2 overexpression. Error bars represent the s.d. of two replicate measurements per condition. ( e ) Alterations of key signalling molecules in breast cancer were examined by Western blot following TLK2 overexpression in T47D. TLK2 was induced by 200 ng ml −1 Dox for 2 weeks. Dox, doxycyclin. ( f ) Transwell migration assays following SRC, EGFR or FAK knockdown in T47D cells overexpressing TLK2. The indicated concentration of siRNAs against SRC or 20 nM of siRNAs against EGFR or FAK were transfected for 3 days following induction of TLK2 expression in T47D cells for two weeks (200 ng ml −1 Dox). Western blot validation of SRC, EGFR or FAK silencing in T47D cells overexpressing TLK2 was shown in . Error bars represent the s.d. of two replicate measurements per condition. ( g ) Co-immunoprecipitation of TLK2 with SRC in engineered MCF7 cells inducibly expressing Flag-tagged TLK2. Engineered MCF7 cells was treated with 200 ng ml −1 Dox for 48 h and nuclear or cytoplasmic proteins were purified following subcellular fractionation. IP was performed using an established monoclonal antibody against SRC after conjugation with agarose beads. Western blot was performed using an anti-Flag antibody to detect the presence of TLK2 in SRC complex. Dox, Doxycyclin.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Western Blot, Expressing, Clonogenic Assay, Over Expression, Migration, Knockdown, Concentration Assay, Transfection, Biomarker Discovery, Immunoprecipitation, Purification, Fractionation, Conjugation Assay

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a , b ) TLK2 knockdown (KD) by esiRNA and siRNA transfection. ( a ) Cell proliferation was assessed by MTT assay in TLK2-high or low breast cancer cell lines or benign breast epithelial cells after TLK2 knockdown. Unt, untreated. Ctrl, control. Error bars represent the s.d. of three replicate measurements per condition. ( b ) Cell migration assessed by Boyden chamber transwell assay following TLK2 KD for 48 h in MCF7 and MDAMB361 cells using TLK2 esiRNA or siRNA. NIH 3T3 cells are used as chemo-attractant for MCF7 and MDAMB361 cells. Error bars represent the s.d. of three replicates measurements per condition. ( c ) Clonogenic assay was performed following Dox-inducible shRNA silencing of TLK2 in breast cancer cells. 0.5 mg ml −1 of Dox was used for this assay. Dox, doxycycline. Error bars represent the s.d. of three replicate measurements per condition. ( d ) TLK2 inhibition suppresses anchorage-independent growth of MCF7 and MDAMB361 cells. Dox (0.5 μg ml −1 ) was administered for 2 days to induce TLK2 shRNA and then soft-agar colony formation assay was performed. Error bars represent the s.d. of three replicate measurements per condition. ( e ) The cell growth inhibition after TLK2 KD using a siRNA#2 (with the same targeting sequence as the shTLK2) can be rescued by inducible TLK2 overexpression. Multiple silent mutations at the shTLK2 targeting region are introduced into the TLK2 ORF without affecting the amino acid sequence, to reduce the inhibition of ectopically expressed TLK2 by siRNA#2. TLK2 expression was induced by treating MCF7 cells with 100 or 2,000 ng ml −1 Dox; siTLK2 was then transfected and incubated for 2 weeks for clonogenic assay. Error bars represent the s.d. of two replicate measurements per condition. P values were calculated based on t- test. * P <0.05; ** P <0.01; *** P <0.001. Dox, doxycycline.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Knockdown, esiRNA, Transfection, MTT Assay, Control, Migration, Boyden Chamber Transwell Assay, Clonogenic Assay, shRNA, Inhibition, Soft Agar Assay, Sequencing, Over Expression, Expressing, Incubation

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) The effect of TLK2 inhibition in the MCF7 xenograft tumours inducibly expressing a TLK2 shRNA, in the presence or absence of concomitant tamoxifen treatment. The average tumour growth in each treatment group (8 mice per group). Error bars represent the s.d. of tumour volumes of 8 mice measurements per condition. P values were calculated based on ANOVA to compare the tumour volumes. ( b ) Kaplan–Meier survival plot comparing the progression-free survival of different treatment groups (based on tumour-doubling time). Generalized Wilcoxon test was used to calculate the P values for comparing progression-free survival between different treatment groups. ( c ) Quantitative western blot analysis of TLK2 protein expression in the tumours harvested after 15 days of treatment (5 mice/group), or at the end point (8 mice per group). Error bars represent the s.d. of relative TLK2 levels of 5 or 8 mice measurements per condition. * P <0.05; ** P <0.01; *** P <0.001. Dox, doxycycline. Corresponding western blot images are shown in .

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Inhibition, Expressing, shRNA, Western Blot

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) The heat-map of consistently altered signalling proteins revealed by RPPA analyses of MCF7 cells ±TLK2 KD and MCF7 xenograft tumours harvested 2 weeks after induction of TLK2 inhibition. Proteins that showed a consistent trend of changes ( P <0.1) in both in vitro and in vivo models are shown in the heat-map, and are sorted by the mean P values of different comparisons. For RPPA profiling, each knockdown experiment was repeated three times biologically. P values were calculated based on t- test and are shown in grey scale. ( b ) Boxplots of normalized fluorescent intensities of Bcl2 or ERα after TLK2 knockdown in MCF7 cells (3 repeats for each group) or MCF7 xenograft tumours (5 tumours in each group). The whiskers indicate the max and min values and horizontal lines represent the 1st, 2nd and 3rd quartiles. ( c ) Western blot validation of Bcl2 and ERα protein changes after TLK2 knockdown in MCF7 cells. ( d ) Q-PCR results quantifying relative mRNA level of TLK2, ERα, or Bcl2 after TLK2 silencing by transfecting MCF7 cells with 10 nM of siCtrl, esiTLK2, or siTLK2 #1. Error bars represent the s.d. of three replicate measurements per condition.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Inhibition, In Vitro, In Vivo, Knockdown, Western Blot, Biomarker Discovery

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) Flow cytometry results showing cell cycle changes after TLK2 knockdown in asynchronized MCF7 and MDAMB361 cells. 10 nM siRNAs (for MCF7 cells) or indicated concentration of siRNAs (for MDAMB361 cells) were transfected for 72 h. Ctrl, Control. ( b ) Cell apoptosis assessed by Annexin V assay in asynchronized MCF7 and MDAMB361 cells following TLK2 knockdown via 20 nM esiTLK2 or siTLK2#1 transfection for 72 h. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t- test. * P <0.05; ** P <0.01; *** P <0.001. ( c ) Cell cycle profile of synchronized MCF7 cells (by double thymidine block) after TLK2 inhibition. After TLK2 knockdown for 24 h, MCF7 cells were synchronized at the G1/S border using 2.5 mM double thymidine (DT), and then released. Cells were collected every 2 h after cell cycle release for up to 12 h, and analysed for DNA content using flow cytometry. ( d ) Western blot was done to examine the changes of key signalling molecules involved in G1/S cell cycle regulation using the cell lysates obtained from the same experiment as in 7c. ‘DT' indicates synchronized MCF7 cells by DT block. ( e ) A schematic of normal G1/S cell cycle signalling and alternations following TLK2 inhibition (black arrows). In normal cell cycle, the cyclin E level starts to increase in late G1 phase, and then collapses as the cells enter S phase , followed by increased cyclin A expression . Rb regulates G1/S transition by repressing the E2F transcription factors that control the expression of cyclin A. Once Rb is phosphorylated (that is, at S807/S811), it releases E2Fs, which will allow cells to enter S phase . p27 inhibits the two G1 cyclin/cdk complexes, cyclin D/Cdk4 and cyclin E/Cdk2 (refs , ), both of which are the key upstream kinases of Rb (ref. ). During normal G1/S progression, the p27 proteins complexed with G1 cyclin/cdks were phosphorylated by the p27-free cyclin E/Cdk2 complexes at T187, which were then targeted for SKP2-mediated proteasome degradation . D, Cyclin D. E, Cyclin E. A, Cyclin A. R, restriction point.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Flow Cytometry, Knockdown, Concentration Assay, Transfection, Control, Annexin V Assay, Blocking Assay, Inhibition, Western Blot, Expressing

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) Cell cycle profile of MCF7 cells synchronized by nocodazole block after TLK2 or TLK1 knockdown. After TLK2 or TLK1 silencing by transfecting 10 nM of esiTLK2 or siTLK1 for 24 h, MCF7 cells were synchronized at mitosis using 200 nM nocodazole for 15 h, and then released. Cells were collected at the indicated time after cell cycle release. To precisely determine S-phase cell population, 10 μM of BrdU was added for 1.5 h before cell collection. The cell cycle distributions were determined based on DNA content and BrdU incorporation . ( b ) Western blot was done to examine the changes of key signalling molecules involved in G1/S cell cycle regulation and apoptosis using the cell lysates obtained from same experiment as in Fig. 8a. ‘Noc' indicates the MCF7 cells synchronized at mitosis by nocodazole block (before cell cycle release). ( c ) Cell apoptosis assessed by Annexin V assay in asynchronized MCF7 and MDAMB361 cells following 20 nM of esiTLK2, siTLK1, or siCtrl treatment for 72 h. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t- test. ** P <0.01.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Blocking Assay, Knockdown, BrdU Incorporation Assay, Western Blot, Annexin V Assay

Journal: Nature Communications

Article Title: Comprehensive functional analysis of the tousled-like kinase 2 frequently amplified in aggressive luminal breast cancers

doi: 10.1038/ncomms12991

Figure Lengend Snippet: ( a ) Nominating potential TLK2 inhibitors based on analysis of a public kinase profiling data set . Two potential TLK2 inhibitors were selected based on relatively strong TLK2 inhibition (shown in bar chart) and low number of off-targets (shown in line chart). ( b ) The activity of the potential TLK2 kinase inhibitors Go6983 and GF109203X against TLK2 kinase were measured by in vitro kinase assay using recombinant active TLK2 protein. Myelin Basic Protein (MBP) was used as a substrate. ( c ) Clonogenic assays following Go6983 or GF109203X treatment of the MCF7 cells inducibly expressing TLK2. Go6983 (4 μM) or GF109203X and 0.2 or 1 μg ml −1 of Dox were administered. Error bars represent the s.d. of two replicate measurements per condition. P values are calculated based on t- test. * P <0.05; ** P <0.01.

Article Snippet: MCF7 cells (7.5 × 10 6 ) with Dox-dependent expression of shTLK2 were injected bilaterally to 4–6 week old female athymic nude mice (Harlan Sprague-Dawley) supplemented with 17β-estradiol pellets.

Techniques: Inhibition, Activity Assay, In Vitro, Kinase Assay, Recombinant, Expressing

Journal: Scientific Reports

Article Title: Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis

doi: 10.1038/srep24730

Figure Lengend Snippet: ( a ) Single MCF-7 cells were extracted with different time, and the vertical axis represents the MS intensity of ADP (m/z 426). ( b ) The mass spectrum of unicellular metabolites.

Article Snippet: MCF-7 (human breast cancer cell) cell lines were purchased from ABGENT.

Techniques:

Journal: Scientific Reports

Article Title: Integrated Droplet-Based Microextraction with ESI-MS for Removal of Matrix Interference in Single-Cell Analysis

doi: 10.1038/srep24730

Figure Lengend Snippet: ( A – C ) ATP, ADP and AMP in MCF-7 cells. ( a – c ) Standard solutions of ATP, ADP and AMP with the concentration of 10 μM. ( D – F ) GSH, GSSG and UDP-Glc-NAc in MCF-7 cells. ( d , e ) Standard solutions of GSH and GSSG with the concentration of 10 μM. ( f ) MS/MS spectrum of UDP-Glc-NAc from MassBank database ( http://www.massbank.jp ).

Article Snippet: MCF-7 (human breast cancer cell) cell lines were purchased from ABGENT.

Techniques: Concentration Assay, Tandem Mass Spectroscopy