Identical numbers of cells were plated into 6-well culture plate across the different treatment groups for each radiation dose. Forty-eight hours after treatment, the cells were irradiated with 6 megavoltage X-ray from a linear accelerator (Varian Medical System, Palo Alto, CA, USA) at a dose rate of 2.46 Gy/min and then incubated for colony formation for 14–23 days. Colonies were fixed with methanol and stained with 0.5% crystal violet. The colonies containing 50 or more cells at least were counted and the surviving fraction was calculated. The radiation-survival data was fitted to a linear-quadratic model using Kaleidagraph version 3.51 (Synergy Software, Reading, PA, USA). A sensitizer enhancement ratio of 0.05 (SER0.05) was calculated as the ratio of the dose resulting in a surviving fraction of 0.05 without drugs to that with drugs.
Kaleidagraph version 3
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KaleidaGraph is a data analysis and graphing software package. Version 3.51 provides tools for data import, visualization, and basic statistical analysis. The software supports a range of file formats and chart types to present data insights.
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Lab products found in correlation
Clinac 4 100, by Agilent Technologies (1 mentions)
Clinac 21ex, by Agilent Technologies (1 mentions)
Yeast trna, by Thermo Fisher Scientific (1 mentions)
Acetylated bsa, by Thermo Fisher Scientific (1 mentions)
P nitrophenyl phosphate substrate, by Merck Group (1 mentions)
Instat, by GraphPad (1 mentions)
10 protocols using kaleidagraph version 3
1
Radiation Sensitivity Assay Protocol
2
Clonogenic Assay for Radiosensitivity in miRNA Overexpression
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For the clonogenic assays, identical numbers of cells were plated across the different treatment groups for each radiation dose as previously reported [52 (link)]. Cells were seeded into each well of six-well culture plates and transfected with miRNA for 18 hours. After miRNA transfection, cells were irradiated with a 4-MV X-ray from a linear accelerator (Clinac 4/100, Varian Medical Systems, Palo Alto, CA, USA) at a dose rate of 2.46 Gy/minute and incubated for colony formation for 14 to 21 days. Colonies were fixed with methanol and stained with 0.5% crystal violet; the number of colonies containing at least 50 cells was determined and the surviving fraction was calculated. Radiation-survival data were fitted to a linear-quadratic model using Kaleidagraph version 3.51 (Synergy Software, Reading, PA, USA). The SER was defined as the ratio of the isoeffective dose at a surviving fraction (SF) of 0.5 and 0.05 in the overexpression of control miRNA to the overexpression of miR-200c.
3
Metastron-Induced Colony Formation
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A specified number of cells (n = 200 cells/each well) of each group were seeded into 24-well culture plates and cultured in DMEM medium with different concentrations (0.01, 0.1, 1, 10 µCi/ml, respectively) of Metastron (89SrCl2), which was obtained from Chengdu Gaotong Isotope Co., Ltd, China. Cells continued to be incubated for colony formation for 14–21 days. Colonies were then fixed with methanol and stained with 0.5% crystal violet. The number of colonies containing at least 50 cells was determined to calculate surviving fractions, and survival curves were then drawn using Kaleidagraph version3.51 (SynergySoftware, Reading, PA, USA).
4
Clonogenic Assay for Radiation Sensitivity
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Clonogenic assays were performed as previously described (26).
Identical numbers of cells were plated across the different treatment groups for each radiation dose. A specified number of cells were seeded into each wells of six-well culture plates and treated with temozolomide and/or Cpd188. After exposure of drugs, cells were irradiated with 6-MV X-ray from a linear accelerator (Clinac 21EX, Varian Medical Systems, Palo Alto, CA, USA) at a dose rate of 2.46 Gy/min and were incubated for colony formation for 14 to 21 days.
Colonies were fixed with methanol and stained with 0.5% crystal violet; the number of colonies containing at least 50 cells was determined and surviving fraction was calculated. Radiation survival data were fitted to a linear-quadratic model using Kaleidagraph version 3.51 (Synergy Software, Reading, PA, USA). Each point on the survival curves represents the mean surviving fraction from at least three dishes.
Sensitizer enhancement ratio (SER) was calculated as the ratio of the isoeffective dose at surviving fraction 0.5 and surviving fraction 0.05 in the absence of the drugs to that in the presence of the drugs.
Identical numbers of cells were plated across the different treatment groups for each radiation dose. A specified number of cells were seeded into each wells of six-well culture plates and treated with temozolomide and/or Cpd188. After exposure of drugs, cells were irradiated with 6-MV X-ray from a linear accelerator (Clinac 21EX, Varian Medical Systems, Palo Alto, CA, USA) at a dose rate of 2.46 Gy/min and were incubated for colony formation for 14 to 21 days.
Colonies were fixed with methanol and stained with 0.5% crystal violet; the number of colonies containing at least 50 cells was determined and surviving fraction was calculated. Radiation survival data were fitted to a linear-quadratic model using Kaleidagraph version 3.51 (Synergy Software, Reading, PA, USA). Each point on the survival curves represents the mean surviving fraction from at least three dishes.
Sensitizer enhancement ratio (SER) was calculated as the ratio of the isoeffective dose at surviving fraction 0.5 and surviving fraction 0.05 in the absence of the drugs to that in the presence of the drugs.
5
RNA-Protein Binding Kinetics Assay
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Oligoribonucleotides were labeled with [γ-32P]ATP and T4 polynucleotide kinase using standard conditions. A total of 10 nM 32P-labeled RNA was incubated with 0–10 µM protein in 20-μL reactions containing 250 mM KCl, 5 mM MgCl2, 25 mM Tris-HCl (pH 7.5), 10% glycerol, 1 mg/mL acetylated BSA (Ambion), 10 μM of yeast tRNA (Invitrogen). Reactions were incubated at 25°C for 30 min and separated on 1% agarose gel for 1 h at 150 V at room temperature using 1X TBE. For the His6-tagged N-terminal deletion construct and the His6-tagged full-length construct, we used binding conditions at pH 9.5 and 8.9, respectively, to accommodate their higher pI. The antibody concentrations used for supershift were 15 times higher than the protein concentration.
Image Gauge 4.1 (Fujifilm) was used to quantify the band intensity. The PhosphorImager-recorded fraction of bound RNA was calculated by dividing the intensities of mobility-shifted bands corresponding to the protein–RNA complexes by the sum of intensities by totaling both fractions of the labeled probe. The fraction of bound RNA was plotted against the protein concentration and fitted to the equation y = m1[1+(x/m2)m3], where y = fraction of bound RNA, x = protein concentration, m1 = maximum fraction bound RNA, m2 = Kd and m3 = cooperativity, using the software KaleidaGraph version 3.6 (Synergy Software).
Image Gauge 4.1 (Fujifilm) was used to quantify the band intensity. The PhosphorImager-recorded fraction of bound RNA was calculated by dividing the intensities of mobility-shifted bands corresponding to the protein–RNA complexes by the sum of intensities by totaling both fractions of the labeled probe. The fraction of bound RNA was plotted against the protein concentration and fitted to the equation y = m1[1+(x/m2)m3], where y = fraction of bound RNA, x = protein concentration, m1 = maximum fraction bound RNA, m2 = Kd and m3 = cooperativity, using the software KaleidaGraph version 3.6 (Synergy Software).
6
Fluorescence Spectroscopy of Proteins
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Data were recorded on a Cary Eclipse spectrofluorimeter
equipped with a Peltier cooling system, using an excitation wavelength
of 280 or 295 nm with slit widths set at 5 nm for both excitation
and emission. All measurements were taken at 20 °C in a 50 mM
Tris/25 mM MES/25 mM acetic acid buffer system, using 1 μM for
pH experiments and 0.8 μM for the urea denaturation experiments.
For the pH and urea experiments, only 295 nm excitation was used,
recording emission data for the WT at 330 or 333 nm for the 5-FTrp-labeled
PA or PA20 proteins, and the data are an average of five
scans from 300 to 600 nm. All samples were incubated overnight at
the respective pH or urea concentrations to allow for adequate equilibration.
For the pH experiments, the solid lines through the data points are
nonlinear least-squares fits of the data to the Henderson–Hasselbalch
equation to give an apparent pKa for the
pH transition. For the urea denaturation experiments, in the case
of the full-length PA proteins, the data were fit to a three-state
transition as described previously.19 (link) For
the PA20 urea denaturation experiments, the denaturation
curves were fit to a two-state model with sloping baselines according
to the model described by Clarke and Fersht.21 (link) The curves were fit using Kaleidagraph version 3.6 (Synergy Software,
Reading, PA).
equipped with a Peltier cooling system, using an excitation wavelength
of 280 or 295 nm with slit widths set at 5 nm for both excitation
and emission. All measurements were taken at 20 °C in a 50 mM
Tris/25 mM MES/25 mM acetic acid buffer system, using 1 μM for
pH experiments and 0.8 μM for the urea denaturation experiments.
For the pH and urea experiments, only 295 nm excitation was used,
recording emission data for the WT at 330 or 333 nm for the 5-FTrp-labeled
PA or PA20 proteins, and the data are an average of five
scans from 300 to 600 nm. All samples were incubated overnight at
the respective pH or urea concentrations to allow for adequate equilibration.
For the pH experiments, the solid lines through the data points are
nonlinear least-squares fits of the data to the Henderson–Hasselbalch
equation to give an apparent pKa for the
pH transition. For the urea denaturation experiments, in the case
of the full-length PA proteins, the data were fit to a three-state
transition as described previously.19 (link) For
the PA20 urea denaturation experiments, the denaturation
curves were fit to a two-state model with sloping baselines according
to the model described by Clarke and Fersht.21 (link) The curves were fit using Kaleidagraph version 3.6 (Synergy Software,
Reading, PA).
7
Statistical Analysis of Cell and Vesicle Characteristics
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Statistical analyses were performed using KaleidaGraph version 3.6 software (Synergy Software, Reading, PA, USA). Results were expressed as mean (m) ± SEM, when N > 50 and as mean (m) ± SD when N < 50. Each variable tested here was quantitative and continuous.
Normality of a group of values was checked against a theoretical normal distribution around the mean, using a single group Student t-test (KaleidaGraph, Student t-test, test value: mean value, single group). If p < 0.01, the distribution was considered normal.
For each quantitative variable (cell or vesicle area, cell elongation, PLT, fluorescence intensity, intrinsic rigidity (elastic modulus), and friction coefficient) 50 to 2000 values were analyzed. Six groups were considered: H, RA, OA (CTRL), and H, RA, OA (IL-17/TNF-α) and analyzed using an ANOVA test (KaleidaGraph, ANOVA). In case of significant differences between groups, a post hoc Tukey HSD test was used to compare groups by pairs. *** p < 0.0001, ** p < 0.001, * p < 0.05 and NS non-significant. In the case of cell culture kinetics, quantitative variables PLT and number of vesicles were analyzed. Five groups each of 50 values were analyzed with ANOVA and were found significantly different. The post hoc Tukey HSD test was used to compare groups by pairs. *** p < 0.0001 and NS non-significant.
Normality of a group of values was checked against a theoretical normal distribution around the mean, using a single group Student t-test (KaleidaGraph, Student t-test, test value: mean value, single group). If p < 0.01, the distribution was considered normal.
For each quantitative variable (cell or vesicle area, cell elongation, PLT, fluorescence intensity, intrinsic rigidity (elastic modulus), and friction coefficient) 50 to 2000 values were analyzed. Six groups were considered: H, RA, OA (CTRL), and H, RA, OA (IL-17/TNF-α) and analyzed using an ANOVA test (KaleidaGraph, ANOVA). In case of significant differences between groups, a post hoc Tukey HSD test was used to compare groups by pairs. *** p < 0.0001, ** p < 0.001, * p < 0.05 and NS non-significant. In the case of cell culture kinetics, quantitative variables PLT and number of vesicles were analyzed. Five groups each of 50 values were analyzed with ANOVA and were found significantly different. The post hoc Tukey HSD test was used to compare groups by pairs. *** p < 0.0001 and NS non-significant.
8
ELISA-based IMP-binding Assay Protocol
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An ELISA-based IMP-binding assay (Hubner et al., 1997 (link)) was performed using recombinant HIS-tagged PSPC1 (5 pmol/well) coated onto 96-well plates (cat. no. 260836; Nunc, distributed by ThermoFisher Scientific, Scoresby, Australia). Triplicate wells were incubated with different concentrations of GST, GST-tagged IMPs, or GST‑tag cleaved IMPs preincubated with GST‑IMPβ1. After washing, IMP binding was quantitated using an anti-GST antibody (cat. no. 27457701; GE Life Sciences) and a rabbit anti-goat secondary conjugated to alkaline phosphatase (cat. no. A-4187; Sigma-Aldrich). The absorbance change at 405 nm was recorded for 90 min after p-nitrophenyl phosphate substrate (Sigma-Aldrich, Castle Hill, Australia) addition. Kd and Bmax values were estimated by curve fitting using KaleidaGraph version 3.6 by Synergy.
9
Steroid Dose-Response Kinetics Analysis
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Unless otherwise noted, all experiments
were performed in triplicate multiple times. KaleidaGraph version
3.5 (Synergy Software, Reading, PA) was used to determine a least-squares
best fit of the experimental data to the theoretical dose–response
curve, which is given by the equation derived from Michaelis–Menten
kinetics of y = [free steroid]/[free steroid + dissociation
constant (Kd)] (where the concentration
of total steroid is approximately equal to the concentration of free
steroid because only a small portion is bound), to yield a single
EC50 value. The values of n independent
experiments were then analyzed for statistical significance by the
two-tailed Student’s t test using InStat version
2.03 for Macintosh (GraphPad Software, San Diego, CA). The Mann–Whitney
test or the Alternate Welch t test is used when the
difference between the standard deviations of two populations is statistically
significant. The Bayesian Information Criterion was used to determine
the better of two types of fits for a particular graph (e.g., linear
vs quadratic).
were performed in triplicate multiple times. KaleidaGraph version
3.5 (Synergy Software, Reading, PA) was used to determine a least-squares
best fit of the experimental data to the theoretical dose–response
curve, which is given by the equation derived from Michaelis–Menten
kinetics of y = [free steroid]/[free steroid + dissociation
constant (Kd)] (where the concentration
of total steroid is approximately equal to the concentration of free
steroid because only a small portion is bound), to yield a single
EC50 value. The values of n independent
experiments were then analyzed for statistical significance by the
two-tailed Student’s t test using InStat version
2.03 for Macintosh (GraphPad Software, San Diego, CA). The Mann–Whitney
test or the Alternate Welch t test is used when the
difference between the standard deviations of two populations is statistically
significant. The Bayesian Information Criterion was used to determine
the better of two types of fits for a particular graph (e.g., linear
vs quadratic).
10
Peptide DOSY Experimental Protocol
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The peptide concentration in DOSY experiment was 120 μM, and 128 scans were acquired, where the gradient strength was varied linearly. Translational self-diffusion measurements were performed with the pulsed-gradient spin-echo sequence in the presence of 100% D2O. Experimental details have been described elsewhere [46 (link)]. The gradient strength was varied in sixteen linear steps between 2 and 95% of the total power of the gradient coil. The gradient strength was calibrated using the value of the translational diffusion coefficient, D, for the residual proton water signal in a sample containing 100% D2O in a 5 mm tube [53 (link)]. The length of the gradient was 2.25 ms, the time between the two pulse gradients in the pulse sequence was 200 ms, and the recovery delay between the bipolar gradients was 100 μs. The methyl groups with signals between 1.0 and 0.80 ppm were used for peak integration (Section 2.2.1 ). Fitting of the exponential curves, obtained from experimental data as previously described [46 (link)], was carried out with KaleidaGraph version 3.5 (Synergy Software, Reading, PA, USA). A final concentration of 1% of dioxane, which was assumed to have a hydrodynamic radius Rh = 2.12 Å [53 (link)], was added to the peptide solution.
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