Kaleidagraph v4

The spectral 2D data were analyzed using the KaleidaGraph v.4.0 (Synergy Software, Reading, PA, USA) and 3D-surface emission plot was created in Excel version 2207 (Microsoft 365 package). Mass spectrometry data were analyzed using a Bruker’s Flex Analysis v.3.4 software (Bruker Daltonics, Bremen, Germany). The AFM data acquisition and analysis were performed using Nova 1.1.1.19891 and Image Analysis 3.5.0.20102 software developed by NT-DMT Spectrum Instruments.

CD spectroscopy experiments were conducted based on a previously published protocol [38 (link)] using a Jasco CD J810 Spectropolarimeter and analyzed with KaleidaGraph v.4.5.2 (Synergy Software). RNA oligonucleotides were prepared to a concentration of 2.5 μM in 10 mM LiCacodylate (pH 7.0) by the dialysis procedure described above. Prior to CD, RNAs were denatured at 95°C for 2 min and renatured at room temperature for 15 min for equilibration. Spectra were acquired every nanometer from 220–310 nm at 25°C. Each reported spectrum is an average of 2 scans with a response time of 2 s/nm. Data were normalized to concentration, oligonucleotide length and cuvette pathway to provide molar residue ellipticity values and smoothed over 5 nm [39 (link)].

CD-detected titrations were performed with KCl to determine the concentration of potassium ion (K⁺) required to drive G-quadruplex formation. To determine K⁺_1/2 values, ellipticity data as a function of K⁺ concentration were fit with KaleidaGraph v. 4.5.2 (Synergy software) according to the apparent three-state Hill equation as previously described [38 (link)].
$\epsilon =\frac{{\epsilon }_{\text{U}}+{\epsilon }_{\text{I}}{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_1}\right)}^{n_1}+{\epsilon }_{\text{F}}{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_1}\right)}^{n_1}{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_2}\right)}^{n2}}{1+{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_1}\right)}^{n_1}+{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_1}\right)}^{n_1}{\left(\frac{{[\text{K}}^{+}]}{{{[\text{K}}^{+}{}_{1/2}]}_2}\right)}^{n2}}$
where ε is the normalized molar ellipticity, ε_U is the normalized CD signal for fully unfolded RNA, ε_I is the normalized CD signal for intermediate state RNA, and ε_F is the normalized CD signal for fully folded RNA. [K⁺_1/2]₁ and n₁ are the K⁺_1/2 and Hill coefficient values for the U-to-I transition, while [K⁺_1/2]₂ and n₂ are the values for the I-to-F transition. Data were collected at the maximum wavelength (λ_max) in the spectrum.

Third instar larval body wall muscle 6 was chosen for all nuclear clustering analysis. Images were opened in ImageJ and nuclei clusters in muscle 6 were counted. We defined a “cluster” as two or more nuclei in which the distance between two nuclear borders was less than five microns. We analyzed six larvae from hemisegments A2-A4 (18 hemisegments total) for each genotype.
Microsoft excel was used to import all nuclear clustering data. “Normal” muscles contained no clusters. For muscles that contained cluster(s), we determined cluster number and nuclei number per cluster. Cluster sizes for each genotype were plotted on a frequency histogram. The percentage of normal hemisegments for each genotype was plotted on a column graph. For each hemisegment we calculated the percentage of nuclei in clusters using the following calculation: number of nuclei within clusters divided by total number of nuclei in muscle 6 multiplied by 100. Then, the average of three hemisegments per larva was calculated and plotted on a column graph. Data was visualized and analyzed using GraphPad Prism v9.3.1 or Synergy Software KaleidaGraph v4.5.2.

Statistical analysis of microarray data except for Z-scores was performed using Gene-Spring GX14.9.1 software (Agilent). Z-score was analysed by calculating the standard deviations of log fold change results from microarray data using Excel software (Microsoft, Redmond, WA, USA). Unpaired two-tailed t-tests of qPCR data were performed using KaleidaGraph v4.5.2 software (Synergy Software, MN, USA).