Journal: Scientific Reports

Article Title: The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G 1

doi: 10.1038/s41598-018-34199-9

Figure Lengend Snippet: shRNA–mediated knock-down of JunB in ALK+ ALCL cell lines results in reduced proliferation. Western blots showing JunB levels ( A – C ) and growth curves ( D – F ) of the indicated cell lines expressing either control or JunB shRNA. Growth curves represent five experiments from three infections ( D ), five experiments from five infections ( E ), and three experiments from one infection ( F ). Note: JunB#6 and JunB#1 shRNAs overlap in their seed sequence and are not distinct. BrdU/7-AAD double staining of Karpas 299 ( G ) or SUP-M2 ( H ) cells expressing control or JunB shRNA. Results represent the average and standard deviation of three independent experiments from three infections for Karpas 299 cells, and eight independent experiments from six infections for SUP-M2 cells. ( I ) Bar graph showing the percentage of cells in S phase (% BrdU positive cells) observed when control shRNA or JunB shRNA–expressing Karpas 299 cells were transfected with plasmids expressing either EGFP-P2A or EGFP-P2A-FLAG-JunB. The results represent the average and standard deviation of four independent experiments. ( J ) Approximate doubling times and time spent in each stage of the cell cycle was determined for Karpas 299 and SUP-M2 cells expressing control or JunB shRNA. ( K ) Representative flow cytometry data and summary of Ki-67 expression within the G 0 /G 1 population of SUP-M2 cells expressing control or JunB shRNA. The summary represents the average and standard deviation of three independent experiments from two separate infections. P values were obtained by performing independent, two-tailed t tests comparing the JunB knock-down to control shRNA–expressing cells ( D – F and K ) or between JunB knock-down cells with or without JunB cDNA ( I ). ANOVA with Tukey’s post hoc test was performed in ( I ). * P < 0.05, ** P < 0.01, *** P < 0.001. Molecular mass markers (in kDa) are indicated to the left of western blots.

Article Snippet: The UCONN-L2 ALK+ ALCL cell line was obtained from Dr. Raymond Lai (University of Alberta; Edmonton, AB, Canada).

Techniques: shRNA, Knockdown, Western Blot, Expressing, Control, Infection, Sequencing, Double Staining, Standard Deviation, Transfection, Flow Cytometry, Two Tailed Test

Journal: Scientific Reports

Article Title: The c-Jun and JunB transcription factors facilitate the transit of classical Hodgkin lymphoma tumour cells through G 1

doi: 10.1038/s41598-018-34199-9

Figure Lengend Snippet: shRNA–mediated knock-down of c-Jun in ALK+ ALCL cell lines does not impair proliferation. Western blots showing c-Jun levels ( A , C , and E ) and growth curves ( B , D , and F ) of the indicated cell lines expressing either control or c-Jun shRNA. Growth curves represent four experiments from four infections (c-Jun#1) and five experiments from three experiments (c-Jun#5) ( B ), three experiments from three infections ( D ), and three experiments from one infection ( F ). Although, not included in the growth curve shown, c-Jun knock-down in UCONN-L2 had no effect on proliferation in other infections. ( G ) The percentage of cells in each stage of the cell cycle was measured by BrdU/7-AAD double staining for SUP-M2 cells expressing either control or c-Jun shRNA. The results represent three experiments from three infections. No statistically significant differences in growth rate or cell cycle distribution were observed between any c-Jun knock-down cells and respective control shRNA–expressing cells. Molecular mass markers (in kDa) are indicated to the left of western blots.

Article Snippet: The UCONN-L2 ALK+ ALCL cell line was obtained from Dr. Raymond Lai (University of Alberta; Edmonton, AB, Canada).

Techniques: shRNA, Knockdown, Western Blot, Expressing, Control, Infection, Double Staining

Journal: Nature medicine

Article Title: Wiskott–Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma

doi: 10.1038/s41591-018-0262-9

Figure Lengend Snippet: (a) Western Blot analysis of ALK+ ALCL cell lines (SU-DHL1, JB6, Karpas-299 and DEL) transduced with doxycycline-inducible lentivirus co-expressing WASP and WIP (W&W) or a control reporter GFP (Ctrl). Black arrows: endogenous WASP and WIP; red arrows: Flag-tagged WASP and WIP. MAC-1 cell line and normal T cells were used as controls. The blot is representative of at least two independent experiments with similar results. Actin was used as a loading control. Uncropped blots are available in Supplementary Figure 11.

Article Snippet: Human ALK+ ALCL cell lines (TS, SU-DHL1, JB6, Karpas-299, DEL, SUP-M2 and L82) and ALK- cell lines (MAC-1, FePD and Jurkat) ALCL cell lines were obtained from DSMZ (German collection of Microorganisms and Cell Cultures).

Techniques: Western Blot, Transduction, Expressing, Control

Journal: Nature medicine

Article Title: Wiskott–Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma

doi: 10.1038/s41591-018-0262-9

Figure Lengend Snippet: (a) Western Blot performed on human ALK+ ALCL cells lines and ALK- T lymphoma lines or normal T cells blotted with the indicated antibodies. The blot is representative of two independent experiments with similar results. Actin was used as a loading control. Uncropped blots are available in Supplementary Figure 11.

Article Snippet: Human ALK+ ALCL cell lines (TS, SU-DHL1, JB6, Karpas-299, DEL, SUP-M2 and L82) and ALK- cell lines (MAC-1, FePD and Jurkat) ALCL cell lines were obtained from DSMZ (German collection of Microorganisms and Cell Cultures).

Techniques: Western Blot, Control

Journal: Nature medicine

Article Title: Wiskott–Aldrich syndrome protein (WASP) is a tumor suppressor in T cell lymphoma

doi: 10.1038/s41591-018-0262-9

Figure Lengend Snippet: (a) Gene-expression profiling analysis of ALK, WASP, WIP, N-WASP, CDC42 and TNFRS8 (CD30) on different cases of human T cell lymphomas: AITL, n = 40; PTCL-NOS, n = 74; ALK- ALCL, n = 24, ALK+ ALCL, n = 30. The boxes represent the first and third quartiles, and the line represents the median. The whiskers represent the upper and lower limits of the range (ALK+ ALCL vs AITL, ****P = 5.85 X 10−6; ALK+ ALCL vs PTCL-NOS, ****P = 6.08 X 10−12; ALK+ ALCL vs ALK- ALCL, **P = 0.0075; significance was determined by unpaired, two-tailed Student’s t-test). TNFRS8 (CD30) is strongly expressed in ALK- or ALK+ ALCL but not in other T cell lymphoma (TCL) subtypes.

Article Snippet: Human ALK+ ALCL cell lines (TS, SU-DHL1, JB6, Karpas-299, DEL, SUP-M2 and L82) and ALK- cell lines (MAC-1, FePD and Jurkat) ALCL cell lines were obtained from DSMZ (German collection of Microorganisms and Cell Cultures).

Techniques: Gene Expression, Two Tailed Test

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: Quantification of GzB levels in ALK+ ALCL cell lines expressing GzB shRNA

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Expressing, shRNA, Construct, Control

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: Knock-down of GzB in ALK+ ALCL cell lines does not affect growth rate. The growth rate of Karpas 299 (A) , SUP-M2 (B) , or SR (C) cells expressing the indicated GzB shRNA was compared to control shRNA-expressing cells. The results represent the average and standard deviation of 3 (SUP-M2) or 4 (Karpas 299 and SR) independent experiments. The only time point where we observed a statistically significant difference between the GzB and control shRNA-expressing cell lines was at the 48 h time point where the GzB shRNA #1-expressing SR cell line was modestly higher than the control shRNA-expressing cells ( p = 0.042).

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Knockdown, Expressing, shRNA, Control, Standard Deviation

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: ALK+ ALCL cell lines with GzB knock-down show significant reduction in GzB activity. A . Cleavage of the synthetic GzB substrate, Ac-IEPD-pNA, was examined using lysates from the indicated ALK+ ALCL cell lines expressing either control or GzB shRNA. The results represent the average and standard deviation of 3 independent experiments. p values comparing cells expressing GzB shRNA to cells expressing control shRNA were obtained by performing paired, one-tailed t-tests. * p < 0.05, ** p < 0.01, *** p < 0.005. B . Lysates from Karpas 299 cells (above) or SUP-M2 cells (below) expressing the indicated shRNAs were incubated with purified vitronectin for the indicated times and vitronectin cleavage was examined by western blotting. Arrows indicate the major observed vitronectin cleavage product. Cleavage of vitronectin was also examined when incubated with lysates from the GzB-negative, Hodgkin lymphoma cell line, KM-H2, which served as a negative control. Anti-β-actin blots were performed to demonstrate that equivalent amounts of lysate were added to the reactions. The percentage of cleaved vitronectin (% cleaved vitronectin) was determined by densitometry, and represents the percentage of cleaved vitronectin in each lane relative to cleaved vitronectin in the lane where vitronectin was incubated with control shRNA lysate for 6 h. Molecular mass standards are indicated to the left of the western blots.

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Knockdown, Activity Assay, Expressing, Control, shRNA, Standard Deviation, One-tailed Test, Incubation, Purification, Western Blot, Negative Control

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: Knock-down of GzB in ALK+ ALCL cell lines. Western blots (A) or flow cytometry plots (B) comparing the expression of GzB in ALK+ ALCL cell lines stably expressing either a non-targeting (control) shRNA or the indicated GzB shRNAs. Molecular mass standards are indicated to the left of the western blots.

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Knockdown, Western Blot, Flow Cytometry, Expressing, Stable Transfection, Control, shRNA

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: GzB knock-down reduces the sensitivity of ALK+ ALCL cell lines to staurosporine-induced apoptosis. Karpas 299 (A) , SR (B) , or SUP-M2 (C) cells expressing either control or GzB shRNA were left untreated (DMSO) or were treated with the indicated concentrations of staurosporine (STS) for 6 h at 37°C. Lysates were then prepared and western blotted with an anti-PARP antibody. The arrow indicates cleaved PARP. The anti-β-actin blot demonstrates equivalent protein loading. The percentage of cleaved PARP (% cleaved PARP) was determined by densitometry and represents the percentage of cleaved PARP as a fraction of total PARP. Molecular mass standards are indicated to the left of the western blots. Karpas 299 (D) or SR (E) cells expressing either control or GzB shRNA were left untreated (DMSO) or were treated with the indicated concentrations of staurosporine (STS) for 6 h at 37°C. DNA fragmentation was then examined by TUNEL staining and results were expressed as the percentage of TUNEL-positive cells. The results shown represent the mean and standard deviation of at least 4 (Karpas 299) or 5 (SR) independent experiments. p values comparing cells expressing GzB shRNA to cells expressing control shRNA were obtained by performing paired, one-tailed t-tests. * p < 0.05, § p < 0.01, # p < 0.005, δ p < 0.001.

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Knockdown, Expressing, Control, shRNA, Western Blot, TUNEL Assay, Staining, Standard Deviation, One-tailed Test

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: GzB knock-down reduces the sensitivity of ALK+ ALCL cell lines to doxorubicin-induced apoptosis. SUP-M2 (A) or SR (B) cells expressing either control or GzB targeting shRNA were left untreated (untr.) or were treated with indicated concentrations of doxorubicin (Doxo) for 12 h at 37°C. Cells were then lysed and lysates were probed with an anti-PARP antibody. The arrow indicates cleaved PARP. The anti-β-actin blot demonstrates equivalent protein loading. The percent cleaved PARP (% cleaved PARP) was determined by densitometry and represents the percentage of cleaved PARP as a fraction of total PARP. Molecular mass standards are indicated to the left of the western blots. SUP-M2 (C) or SR (D) cells expressing either control or GzB shRNA were left untreated (DMSO) or were treated with the indicated concentrations of doxorubicin (doxo) for 12 h at 37°C. DNA fragmentation was then examined by TUNEL staining and results were expressed as the percentage of TUNEL-positive cells. The results shown represent the mean and standard deviation of 4 independent experiments. p values comparing cells expressing GzB shRNA to cells expressing control shRNA were obtained by performing paired, one-tailed t-tests. * p < 0.05, § p < 0.01, # p < 0.005, δ p < 0.001.

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Knockdown, Expressing, Control, shRNA, Western Blot, TUNEL Assay, Staining, Standard Deviation, One-tailed Test

Journal: Molecular Cancer

Article Title: Expression of granzyme B sensitizes ALK+ ALCL tumour cells to apoptosis-inducing drugs

doi: 10.1186/1476-4598-13-199

Figure Lengend Snippet: Expression of granzymes in ALK+ ALCL cell lines. A . RT-PCR was performed on RNA isolated from the indicated ALK+ ALCL cell lines to examine the expression of the indicated genes. RNA isolated from the NKL (human NK leukemia) and NK-92 (human NK lymphoma) cells served as positive controls for granzyme expression, whereas RNA isolated from HCT116 (human colon carcinoma) cells served as a negative control. In the “no template” sample, no template cDNA was used in the PCR reaction. Base pair standards are indicated to the left of images. B . Western blot analysis examining GzA and GzB expression in the lysates of ALK+ ALCL cell lines. The KM-H2 cell line (Hodgkin lymphoma) serves as a negative control, while the NKL cell line serves as a positive control for GzA protein expression. Molecular mass standards are indicated to the left of the western blots. C . Quantitative RT-PCR was performed to compare GzA mRNA expression levels between the ALK+ ALCL cell lines and the NKL cell line. The results were normalized to either β-tubulin (left) or GAPDH (right) levels. The results represent the average and standard deviation of three independent experiments. ND = not detected.

Article Snippet: The Karpas 299 and SUP-M2 ALK+ ALCL cell lines were purchased from the Leibniz Institute DSMZ-German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Isolation, Negative Control, Western Blot, Positive Control, Quantitative RT-PCR, Standard Deviation