TFF1 and TFF3 expressions show opposite trends during the E2 signaling time-course
A. 60X Representative images from single molecule RNA FISH experiment showing transcripts for TFF1 and TFF3. The probe was designed against the unspliced RNA containing the intronic region. The scale bar is 5 microns.
B. The mean RNA numbers are depicted. These are counted using an in-house MATLAB code which uses the DAPI-stained nuclei as the mask to count the RNA present in the nucleus. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n= 665, N=3). p-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p < 0.001,** denotes p < 0.01,* denotes p < 0.05, ns denotes p > 0.05.
C. Scatter plots showing the distribution of InTFF1 and InTFF3 on a cell-by-cell basis (n= 665, N=3). The absolute RNA numbers are combined from three different repeats. Density plots have been used to clearly visualize overlapping data points.
D. 60X Representative images from single molecule RNA FISH experiment showing transcripts for InTFF1 and ExTFF1. Scale bar is 5 microns.
E. The mean RNA numbers for InTFF1 and ExTFF1 are depicted. Separate probes were used to target unspliced (InTFF1) and mature (ExTFF1) RNA. These are counted using an in-house MATLAB code which uses the DAPI-stained nuclei as the mask to count the intronic RNA present in the nucleus and a free-drawn region to designate the cell to count the exonic RNA present in the nucleus as well as the cytoplasm. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n>360, N=3). p-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p < 0.001,** denotes p < 0.01,* denotes p < 0.05, ns denotes p > 0.05.
F. The mean RNA numbers for InTFF3 and ExTFF3 are depicted. Separate probes were used to target unspliced (InTFF3) and mature (ExTFF3) RNA. The graph shows the mean of means from three different repeats of the experiment, and error bars denote SEM (n>210, N=3). p-values were calculated by Student’s two-tailed unpaired t-test, and the significance is represented as: *** denotes p < 0.001,** denotes p < 0.01,* denotes p < 0.05, ns denotes p > 0.05.
G. Violin plots showing the ratio of intronic to exonic TFF1 counts are depicted. The graph shows the distribution of ratios combined from three different repeats (n>360, N=3). p-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p < 0.001,** denotes p < 0.01,* denotes p < 0.05, ns denotes p > 0.05.
H. Violin plots showing the ratio of intronic to exonic TFF3 counts are depicted. The graph shows the distribution of ratios combined from three different repeats (n>210, N=3). p-values were calculated by the Mann-Whitney test, and the significance is represented as: *** denotes p < 0.001,** denotes p < 0.01,* denotes p < 0.05, ns denotes p > 0.05.