Journal: Molecular Cancer

Article Title: Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

doi: 10.1186/1476-4598-6-33

Figure Lengend Snippet: Rapid IR-induced protein modulation of Flt3, Hdm2 and Bcl-2 family members in AML cell lines . A . Cells were exposed to 25 Gy and fixed after the indicated time (minutes). The percentage of normal nuclei in a total of 200 cells was determined in Hoechst stained cells for each time point. The results shown represent the mean of three separate experiments and the error bars show standard error of mean (SEM = Standard Deviation/√n). The star denotes statistical significance relative to the control, this is determined by a Students two-tailed t -test, p < 0.05. B . mRNA level of FLT3 and HDM2 was determined by Real-time PCR in one typical experiment. GAPDH was used as endogenous control. C . IR down regulated Hdm2 protein and up regulated Flt3 protein in these AML cell lines. The diagram shows measured intensity on three separate Western blots (normalized to Actin). The error bars show standard error of mean SEM and the stars represents significance as in A . D . Visualization of the protein modulations of Flt2 and Hdm2 shown in C . in addition to modulation of proteins in the Bcl-2 family. Mean intensity on the Western blots written below the corresponding panel were measured and normalized to Actin and to the control. The values shown are arbitrary units and represent one typical experiment.

Article Snippet: Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported [ ], and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. ).

Techniques: Staining, Standard Deviation, Control, Two Tailed Test, Real-time Polymerase Chain Reaction, Western Blot

Journal: Molecular Cancer

Article Title: Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

doi: 10.1186/1476-4598-6-33

Figure Lengend Snippet: IR decrease Hdm2 protein in HL-60 (p53 -/-) and in primary AML cells . A . HL-60 cells treated with 25 Gy and harvested at indicated time (minutes) demonstrated a rapid decrease in Hdm2 protein after IR. There was no detectable Flt3 protein at 130 kDa. B . Primary AML cells also treated with IR and harvested at indicated time, demonstrated the rapid Flt3 increase and Hdm2 attenuation as previously observed. This was followed by an increase in Hdm2, reflecting functional and elevated p53 protein. Mean intensity on the Western blots were measured and normalized to Actin and to the control. Values shown are arbitrary units and represent one typical experiment.

Article Snippet: Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported [ ], and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. ).

Techniques: Functional Assay, Western Blot, Control

Journal: Molecular Cancer

Article Title: Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

doi: 10.1186/1476-4598-6-33

Figure Lengend Snippet: Daunorubicin therapy of primary AML cells and cell lines increased Flt3 protein and attenuated Hdm2/Mcl-1 . A . Treatment of NB4 and MV4-11 cells with DNR resulted in an increase in Flt3 and a decrease in Hdm2. The mean intensity on one representative Western blot was calculated and normalized to Actin and to the control. The numbers shown are in arbitrary units and represent one typical experiment. The percentage of living cells was determined by flow cytometry. The living cells distinct forward and side scatter properties were used to separate viable cells from dead cells. B . Increasing doses of DNR induce Flt3 and down regulate Hdm2 protein in primary AML cells in vitro . Note that Hdm2 is down regulated in HL-60 cells, a cell line with lack of full length Flt3 protein and with deleted alleles for p53. All wells on the SDS-PAGE gel was loaded with equal amounts of protein, and Coomassie staining of the gel after blotting confirmed this equal loading.

Article Snippet: Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported [ ], and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. ).

Techniques: Western Blot, Control, Flow Cytometry, In Vitro, SDS Page, Staining

Journal: Molecular Cancer

Article Title: Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

doi: 10.1186/1476-4598-6-33

Figure Lengend Snippet: Induction therapy of AML reciprocally regulates Flt3 and Hdm2 proteins in vivo . AML cells sampled from a patient undergoing induction chemotherapy with an anthracycline and cytarabin were subjected to Western blotting and analyzed for Flt3, Hdm2 and p53 expression. The mean intensity on one representative Western blot was calculated and normalized to Actin. The numbers shown are in arbitrary units.

Article Snippet: Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported [ ], and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. ).

Techniques: In Vivo, Western Blot, Expressing

Journal: Molecular Cancer

Article Title: Pre-apoptotic response to therapeutic DNA damage involves protein modulation of Mcl-1, Hdm2 and Flt3 in acute myeloid leukemia cells

doi: 10.1186/1476-4598-6-33

Figure Lengend Snippet: Summary of the results . A . FLT3 and TP53 mutational status of the cell lines used in this study. B . A summary of the ability of IR and DNR to induce apoptosis in the three different cell lines studied (n.d; not determined). C . Overview of the concerted protein modulations elicited by DNA-damaging therapy found in this study (n.d; not determined).

Article Snippet: Interestingly, lack of full length Flt3 protein in HL-60 has previously been reported [ ], and we were not able to detect full length Flt3 in different batches of HL-60 cells from ATCC and DMZS (Fig. ).

Techniques:

Journal: Protein Science : A Publication of the Protein Society

Article Title: Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

doi: 10.1002/pro.3695

Figure Lengend Snippet: Ribbon and cartoon structures of LacI/GalR homologs. (a) The homodimer of the lactose repressor protein (LacI) (PDB ID 1EFA 16) is shown with one subunit as a gray ribbon and the other in green. On the “green” monomer, the linker region is shown in magenta. The DNA‐binding and regulatory domains used to construct the chimeras are indicated on the structure. Note that the regulatory domains mediate the dimerization needed to create a high affinity binding site for one DNA operator (blue ladder),16, 17, 18 as well as binding the allosteric effector (black spheres); below the effector, the position analogous to GalR E230 is indicated with blue spheres. The figure was rendered using UCSF Chimera.19 (b) The LacI protein structure has been rotated and zoomed to show positions 48, 52, 55, 58, and 61 in the linker region. Amino acids nearest the plane of the viewer are shown in magenta ball‐and‐stick; those facing towards the rear of the structure (on the partner linker region) are in green wireframe. (c) The domain structure of the wild‐type LacI homodimer is represented as a green cartoon; the PurR homodimer is represented in purple; and the GalR homodimer is represented in teal. These color schemes are used to indicate the source of the DNA binding domains (small ovals; LacI positions 1–44), linkers (bars; LacI positions 45–61), and regulatory domains (large ovals; PurR positions 60–340 or GalR positions 60–343) in the chimeric repressors “LLhP” and “LLhG.” All variants of LLhG in this manuscript contain the E62K mutation (“+K”), indicated by the yellow asterisk, as well as the “E230K” mutation (which is discussed in Section 3 and Section 4 and indicated on panel a)

Article Snippet: Plasmids expressing the coding regions of full‐length LacI (plasmid numbers #31490 and #90058), LLhP (#90038), and LLhG (#90051) are available from addgene ( https://www.addgene.org/ ).

Techniques: Binding Assay, Construct, Mutagenesis

Journal: Protein Science : A Publication of the Protein Society

Article Title: Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

doi: 10.1002/pro.3695

Figure Lengend Snippet: Comparison of LLhP and LLhG+K variants binding operator lacO 1. in vitro binding to lacO 1 versus values from in vivo repression assays for variants of LLhP (magenta squares) and LLhG+K (green circles); data were compiled from References 11, 12, 13, 14, 15. The data for wild‐type, tetrameric LacI (compiled from References 15, 20) are shown with a black triangle. The arrows outside the axes indicate that repression was enhanced as DNA binding affinity became tighter. In addition to altered affinity from amino acid changes, LLhP had enhanced binding in the presence of 0.4 mM corepressor hypoxanthine12, 14; values were determined +/− this effector; “plus” data are indicated with open squares. Solid lines represent the best fit to the data, and correlation coefficients are consistent with the linear relationship expected for the known in vivo concentrations.11, 21 The dashed black line is to aid visual extrapolation of the LLhG+K data to that of wild‐type LacI. Both X and Y error bars represent the standard deviations (std) of averages (avg) determined from at least three separate experiments; to generate this plot from the non‐logarithmic values reported in the cited references, errors were propagated as 0.434*(std)/(avg). Repression data were taken from Reference 15; note that this publication used a different normalization scale than the separate normalizations previously used for LLhP and LLhG+K in References 13, 14 so that data for the different chimeras could be directly compared to each other

Article Snippet: Plasmids expressing the coding regions of full‐length LacI (plasmid numbers #31490 and #90058), LLhP (#90038), and LLhG (#90051) are available from addgene ( https://www.addgene.org/ ).

Techniques: Comparison, Binding Assay, In Vitro, In Vivo

Journal: Protein Science : A Publication of the Protein Society

Article Title: Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

doi: 10.1002/pro.3695

Figure Lengend Snippet: Looping in the lac operon. (a) When dimeric repressor is bound to the lacO 1 DNA operator, transcription of the downstream lacZYA genes is repressed. (b) Dimeric repressor protein is capable of binding other sites in the Escherichia coli genome such as lacO 2, lacO 3 and nonspecific genomic DNA. (c) Tetrameric LacI can simultaneously bind two operator sites, leading to DNA looping. The regulatory domains of two wild‐type GalR dimers also have the capability to form protein–protein interactions via its regulatory domains, which provides another means to facilitate tetramerization and DNA looping. (d) Prior experiments indicated that LLhG+K has looping capabilities, similar to its parent protein GalR.10 Since DNA looping depends highly on interoperator spacing (x‐axis), in vivo repression can be altered by changing this distance. In the experiments shown, repression of the reporter gene was assessed using four strains of E. coli, each containing a lacZ gene under control of the lacO sym and lacO 2 operators with varied interoperator spacing (x axis). Values were normalized to a “no repressor” control and higher values represent increased repression. Note that LLhG+K repression was sensitive to changes in operator spacing, whereas LLhP was not

Article Snippet: Plasmids expressing the coding regions of full‐length LacI (plasmid numbers #31490 and #90058), LLhP (#90038), and LLhG (#90051) are available from addgene ( https://www.addgene.org/ ).

Techniques: Binding Assay, Protein-Protein interactions, In Vivo, Control

Journal: Protein Science : A Publication of the Protein Society

Article Title: Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

doi: 10.1002/pro.3695

Figure Lengend Snippet: Comparison of in vivo repression and in vitro binding for LLhG+K (top), LacI (middle), and LLhP (bottom) variants binding to various operators. For variant proteins, the K d values for binding to operators lacO sym (black circles), lacO disC (black squares), and lacO 2 (gray diamonds) are plotted against K d values for binding to operator lacO 1 (x axis values). Error bars on both the X and Y parameters represent one standard deviation of the average values. The lines are from linear regression of the data and slopes are shown on the plots. (Top) K d values for nine LLhG+K variants at five linker positions binding to operator lacO 1 are from Reference 11. K d values for lacO sym and lacO 2 are summarized in Table ​Table2.2. For lacO disC, most K d values were out of range for the binding assay and a lower limit is shown. The large black square (behind a gray diamond) highlights the V52P variant that had tighter lacO disC binding than the other variants. (Middle) K d values for 12 LacI variants at position 52 are from Reference 20. Note that the lacO sym data may be better described by a nonlinear curve and may reflect either a physical or technical limit to tightest possible binding. (Bottom) K d values for seven LLhP variants at three linker positions were determined in the absence (filled) and presence (white middle) of corepressor hypoxanthine (“HX”).12 Two positions had weak binding to lacO sym (“outliers,” gray diamonds”)

Article Snippet: Plasmids expressing the coding regions of full‐length LacI (plasmid numbers #31490 and #90058), LLhP (#90038), and LLhG (#90051) are available from addgene ( https://www.addgene.org/ ).

Techniques: Comparison, In Vivo, In Vitro, Binding Assay, Variant Assay, Standard Deviation

Journal: Protein Science : A Publication of the Protein Society

Article Title: Homolog comparisons further reconcile in vitro and in vivo correlations of protein activities by revealing over‐looked physiological factors

doi: 10.1002/pro.3695

Figure Lengend Snippet: Differences between measured and effective in vivo repressor concentrations. Ideal LLhP+HX binding curves were calculated from the lacO 1 K d values reported in Reference 12. The measured in vivo repressor protein concentration10, 15 is shown with the right‐most dashed line. The left‐most dashed lines shows the repressor protein concentration corresponding to the fractional operator occupancy estimated from the LLhP+HX in vivo repression data reported in Reference 15 and shown in Figure ​Figure22 of this manuscript. These calculations were performed as in Reference 11, using reporter gene levels in the absence of repressor protein to determine zero operator binding and repression by wild‐type tetrameric LacI to estimate 100% operator occupancy. (LacI looping does not alter this estimation; using zero reporter gene activity to estimate 100% occupancy yields similar results.) The center dashed line indicates the prior estimation of effective repressor concentration based on LLhG+K repression values11; this calculation failed to account for looping‐enhanced repression of LLhG+K variants. Binding to nonspecific genomic DNA accounts for some of the difference between measured and effective repressor concentrations11

Article Snippet: Plasmids expressing the coding regions of full‐length LacI (plasmid numbers #31490 and #90058), LLhP (#90038), and LLhG (#90051) are available from addgene ( https://www.addgene.org/ ).

Techniques: In Vivo, Binding Assay, Protein Concentration, Activity Assay, Concentration Assay

Journal: The Journal of Cell Biology

Article Title: Fusion Competence of Myoblasts Rendered Genetically Null for N-Cadherin in Culture

doi:

Figure Lengend Snippet: Fusion of N-cadherin homozygous null, heterozygous, and wild-type myoblasts with multinucleate myofibers in vivo. N-cadherin −/−, +/−, and +/+ primary myoblasts were each transduced with retroviral constructs containing the full-length lacZ gene and then injected into the tibialis anterior muscles of nude mice. At 2 d, myoblasts were present in unfused clusters (shown here for +/+, upper left ). By 12 d after injection for all phenotypes (+/+, top right , +/−, bottom left , and −/−, bottom right ) lacZ expression was also similar but appeared in fields of hundreds of large-diameter muscle fibers indicative of fusion with pre-existing host fibers. Bar, 60 μm.

Article Snippet: Primary myoblasts were transduced with a retroviral construct, MFG KB5 containing the full length lacZ gene , exposed to the substrate fluorescein di-β- d -galactopyranoside (Molecular Probes, Eugene, OR) and cells expressing β-galactosidase isolated with the fluorescence-activated cell sorter as described ( ).

Techniques: In Vivo, Transduction, Retroviral, Construct, Injection, Muscles, Expressing

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 2 (a) Dose-dependent down-regulation by curcumin of the expression of cyclin D1 protein in prostate cancer cells. 56105 cells were plated in 1 ml medium overnight in 6-well plates and treated with the indicated concentrations of curcumin. After 24 h, whole cell extracts were prepared, resolved on 10% SDS – PAGE, electrotransferred, and probed with anti-CyclinD1 antibo- dies (1 : 1000 dilution) for 2 h as described in Materials and meth- ods. (b) Time-dependent down-regulation by curcumin of the expression of cyclin D1 protein in prostate cancer cells. 56105

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, SDS Page

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 3 Expression of cyclin D1 protein in various human breast cancer cell lines. (a) 16106 cells were plated in 1 ml med- ium overnight in 6-well plates. After 3 h, whole-cell extracts were prepared, resolved in 10% SDS – PAGE, electrotransferred and probed with anti-Cyclin D1 antibodies (1 : 1000 dilution) for 2 h as described in Materials and methods. (b – d) Curcumin down- regulates the expression of cyclin D1 protein in various human breast cancer (b and c) and in SCC (d) cell lines. 16106 cells were plated in 1 ml medium overnight in 6-well plates and then treated with 50 mM curcumin. After 3 h, whole-cell extracts were pre- pared, resolved on 10% SDS – PAGE, electrotransferred and probed with anti-Cyclin D1 antibodies (1 : 1000 dilution) for 2 h as described in Materials and methods

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, SDS Page

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 4 Effect of curcumin on cyclin D2 and cyclin D3 protein in various human cancer cell lines. (a) 16106 cells were plated in 1 ml medium overnight in 6-well plates. After 3 h, whole-cell ex- tracts were prepared, 60 mg protein was resolved on 12% SDS – PAGE, electrotransferred and probed with anti-Cyclin D2 antibo- dies (1 : 1000 dilution) for 2 h as described in Materials and meth- ods. (b – e) Curcumin down-regulates the expression of cyclin D2 and cyclin D3 protein in human colon cancer (b), prostate and breast (c), in breast (d) and SCC (e ) cell lines. 16106 cells were plated in 1 ml medium overnight in 6-well plates and then treated with 50 mM curcumin. After 3 h, whole-cell extracts were pre- pared, 60 mg protein was resolved on 12% SDS – PAGE, electro- transferred and probed with either anti-Cyclin D2 antibodies or anti-Cyclin D3 antibodies (1 : 1000 dilution) for 2 h as described in Materials and methods

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: SDS Page, Expressing

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 6 Lactacystin blocks curcumin-induced down-regulation of the cyclin D1 protein in prostate cancer LNCaP (a) and in breast cancer MCF-7 (b) cells. 16106 cells were plated in 1 ml medium overnight in 6-well plates. After 24 h, cells were pre- treated with 10 mM lactacystin for 2 h and then exposed to 50 mM curcumin for 3 h. Thereafter, whole cell extracts were pre- pared, resolved in 10% SDS – PAGE, electrotransferred, and probed with anti-Cyclin D1 antibodies (1 : 1000 dilution) for 2 h as described in Materials and methods

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: SDS Page

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 7 Curcumin down-regulates the expression of cyclin D1 mRNA in prostate cancer LNCaP (a) and breast cancer MCF-7 (b) cells. 56105 cells were plated in 1 ml medium overnight in 6-well plates and treated with 50 mM curcumin for indicated times. Thereafter, mRNA was isolated and transcribed to cDNA. The cDNA was then used for PCR reactions. The PCR product was resolved on 2% agarose gels and probed with Cyclin D1 cDNA for 2 h as described in Materials and methods. Actin was used as an internal control for equal amount of template. The experi- ments were performed at least three times and similar results were obtained each time

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Expressing, Isolation, Control

Journal: Oncogene

Article Title: Curcumin-induced suppression of cell proliferation correlates with down-regulation of cyclin D1 expression and CDK4-mediated retinoblastoma protein phosphorylation.

doi: 10.1038/sj.onc.1206048

Figure Lengend Snippet: Figure 8 Curcumin down-regulates the cyclin D1 promoter ac- tivity in prostate cancer LNCaP cells. LNCaP cells were transi- ently transfected with 1 mg of cyclin D1 luciferase reporter. Cells were then incubated with 10 mM curcumin at the indicated times. Cells were lysed and measured for luciferase activity. The values represent the means+s.e. from three different experiments

Article Snippet: The polyclonal antibodies anti-cyclin D1 against amino acids 1 – 295, which represents full-length cyclin D1 of human origin were obtained from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA).

Techniques: Transfection, Luciferase, Incubation, Activity Assay

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 1. Schematic presentation of BRCA1 and location of the investigated missense variants. RING = Really Interesting New Gene, NES = Nuclear Export Signal, NLS = Nuclear Localisation Signal, BRCT = BRCA1 C-terminal. Figure adapted from [8].

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques:

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 2. Protein expression levels of BRCA1 variants determined by Western blot analysis: HEK293FT cells were transiently transfected with BRCA1 WT, known benign and pathogenic control variants and 14 missense BRCA1 VUSs. The black dots represent individual normalised band intensi- ties. Each column represents the mean of three to six independent replicates (n = 3–6). The benign (green) and pathogenic (orange) control variants are grouped to the left. Variants marked with a red * indicate p < 0.05. Error bars represent standard deviation.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Expressing, Western Blot, Transfection, Control, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 3. mRNA levels of BRCA1 variants in HEK293FT cells determined by qPCR: HEK293FT cells were transfected with plasmids encoding BRCA1 WT and the four variants found to be expressed at protein levels lower or similar to the included pathogenic controls, as shown in Figure 2. Each column represents the mean of three or four independent replicates (n = 3–4), and the black dots represent individual values after normalisation using actin. Error bars represent standard deviation.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Transfection, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 5. Assessment of BRCA1 protein variant stability after 8 h by cycloheximide chase assay: HEK293FT cells were transiently transfected with BRCA1 WT, known benign and pathogenic control variants and 11 missense BRCA1 VUSs. The columns show normalised mean protein levels of three to five independent replicates (n = 3–5) after 8 h of treatment with cycloheximide relative to the levels at 0 h of treatment (100%) for each individual variant. The black dots represent individual normalised band intensities. Error bars represent standard deviation. The benign and pathogenic control variants are coloured green and orange, respectively. Variants marked with a red * indicate a significant reduction in protein stability compared with WT protein (p < 0.05).

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Variant Assay, Transfection, Control, Standard Deviation

Journal: Genes

Article Title: Functional Analyses of Rare Germline Missense BRCA1 Variants Located within and outside Protein Domains with Known Functions.

doi: 10.3390/genes14020262

Figure Lengend Snippet: Figure 6. Assessment of protein interactions between BRCA1 and BARD1 or PALB2 by Co-IP assay: (A) HEK293FT cells were transiently co-transfected with EV or BRCA1 construct together with Flag- PALB2. Cells were harvested 48 h post transfection and co-immunoprecipitation (Co-IP) was performed. Input = input cell lysates, Co-IP = eluates from the Flag-column. BRCA1 (220 kDa) was detected with anti-BRCA1. PALB2-Flag (130 kDa) was detected with anti-Flag. Representative results from one of in total three experiments are shown. (B) Identical experiment to (A), with BARD1-V5 and V5 antibody coupled to the magnetic beads. BARD1-V5 (100 kDa) was detected with anti-V5. (C) Quantified results from BRCA1-PALB2 Co-IP. Western blot bands from three biological replicates were quantified by Image Lab software (n = 3). Black dots represent individual normalised band intensities. Graphs represent mean % compared to the WT. Error bars represent standard deviation. The benign (green) and pathogenic (orange) control variants are grouped to the left. In the initial analysis, the variant p.Lys503Arg appeared to have a reduced binding to PALB2, but this interaction was shown to be similar to the WT/benign controls when quantifying against the amount of the variant input samplemarked by a red ∧). (D) Identical experiment to (C), but with BRCA1-BARD1 Co-IP.

Article Snippet: The plasmid pDEST-mCherry-LacR-BRCA1 encoding mCherry-tagged wild type (WT) full-length human BRCA1 protein was a gift from Daniel Durocher (Addgene plasmid #71115; http://n2t.net/addgene:71115 (accessed on 2 January 2020); RRID:Addgene_71115) [34].

Techniques: Co-Immunoprecipitation Assay, Transfection, Construct, Immunoprecipitation, Magnetic Beads, Western Blot, Software, Standard Deviation, Control, Variant Assay, Binding Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis.

doi: 10.4161/cc.27523

Figure Lengend Snippet: Figure 4. Tenovin-1 enhances p53 pathway signaling in wtp53 MyLa cells. (A) Immunoblot shows baseline expression levels of p53 using anti- body (FL393) that detects full-length p53 of human origin. Densitometric analysis of protein bands was performed relative to GAPDH loading con- trol. (B) Immunoblot using another antibody (7F5; 2527) specific for the N terminus of p53 shows that p53 is upregulated only in the cell line bear- ing wtp53 (MyLa). (C) Tenovin-1 treatment upregulates activated (acety- lated) p53 expression only in wtp53 MyLa nuclear lysates. Densitometric analysis of protein bands was performed relative to TATA-binding pro- tein (TATA) loading control. (D) Tenovin-1 treatment upregulates p53 promoter activity in wtp53 MyLa cells. Data represented as mean ± stan- dard deviation of 3 experiments (6 replicates per experiment) with simi- lar results (*P < 0.05). (E) Immunoblot shows that the upregulated p53 in MyLa cells is associated with upregulation of p21, a downstream target of p53. Densitometric analysis of protein bands was performed relative to GAPDH loading control.

Article Snippet: Blots were exposed to SIRT1 and p53 (FL393 representing full-length) (Santa Cruz Biotechnology, Inc), FoxO3, PARP, p53 (7F5 representing NH2terminus), Ac-p53 Lys 383, p21, PUMA, cleaved caspase-3, cleaved caspase-9, primary antibodies, and HRP conjugated appropriate secondary antibodies (Cell Signaling, Inc), followed by enhanced chemiluminescent detection (Thermo Fisher Scientific, Inc).

Techniques: Western Blot, Expressing, Binding Assay, Control, Activity Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis.

doi: 10.4161/cc.27523

Figure Lengend Snippet: Figure 5. Effects of combined class I/II and class III HDACIs in CTCL. (A) Combination of different classes of HDACIs results in more cell death in wtp53 MyLa than either single agent. Statistical significance (P < 0.05) when compared with no treatment control (*), 25 μM tenovin-1, (!) and 5 μm vorinostat (^). (B) Increased transcriptional activity of p53 in the wtp53 MyLa CTCL line was greatest with combination of both classes of HDACIs. The histogram represents statistical significance when com- pared with no treatment control (*), 25 μM tenovin-1, (!) and 5 μm vori- nostat (^) in 3 experiments with similar results (P < 0.05).

Article Snippet: Blots were exposed to SIRT1 and p53 (FL393 representing full-length) (Santa Cruz Biotechnology, Inc), FoxO3, PARP, p53 (7F5 representing NH2terminus), Ac-p53 Lys 383, p21, PUMA, cleaved caspase-3, cleaved caspase-9, primary antibodies, and HRP conjugated appropriate secondary antibodies (Cell Signaling, Inc), followed by enhanced chemiluminescent detection (Thermo Fisher Scientific, Inc).

Techniques: Control, Activity Assay

Journal: Cell cycle (Georgetown, Tex.)

Article Title: SIRT1 is upregulated in cutaneous T-cell lymphoma, and its inhibition induces growth arrest and apoptosis.

doi: 10.4161/cc.27523

Figure Lengend Snippet: Figure 6. The p53 knockdown in MyLa cells blunts the effects of HDACIs. (A) MyLa cells (NT), with p53 knocked down by lentiviral control (shNS) and shp53. Tenovin-1, vorinostat, and combination-treated shNS and shp53 MyLa cells showed treatment-dependent apoptosis by cleaved PARP (89 kDa band). GAPDH served as loading control. (B) HDACI medi- ated increase in dead cell luminescence (gray bars) and decrease in live cell luminescence (white bars) compared with no treatment (NT) control (*), T25 (tenovin-1; 25 μM) (!), V5 (vorinostat; 5 μm) (^), VT (combination of V5 and T25). (C) shp53 MyLa cells treated as in 6B, stained with Annexin V and PI and analyzed by flow cytometry for apoptosis. (D) Immunoblot analysis shows the effect of single and combination treatments on pro- tein levels of PUMA, p21, cleaved caspase-3 and cleaved caspase-9, and in shNS and shp53 MyLa cells. Densitometric analysis of protein bands was performed relative to GAPDH loading control.

Article Snippet: Blots were exposed to SIRT1 and p53 (FL393 representing full-length) (Santa Cruz Biotechnology, Inc), FoxO3, PARP, p53 (7F5 representing NH2terminus), Ac-p53 Lys 383, p21, PUMA, cleaved caspase-3, cleaved caspase-9, primary antibodies, and HRP conjugated appropriate secondary antibodies (Cell Signaling, Inc), followed by enhanced chemiluminescent detection (Thermo Fisher Scientific, Inc).

Techniques: Knockdown, Control, Staining, Flow Cytometry, Western Blot