Figure 1.
STAT5AB protein expression in mouse Stat5ab+/+, Stat5ab+/Δ and Stat5abΔ/Δ livers.
A STAT5AB protein expression in Stat5ab+/+ (n = 2), Stat5ab+/Δ (n = 2) and Stat5abΔ/Δ (n = 2) livers determined by Western blot (see Figure S1 in File S1 for non-cropped Western blot) and HSC70 as loading control. B IHC staining of STAT5AB in histological sections of the livers (n = 3) used in A. Red, AEC; blue, hematoxylin. Insets are higher magnifications. White stars indicate STAT5AB expression in Kupffer cells and endothelial cells, whereas the hepatocytes are negative for STAT5AB expression. Size bar: 100 µm. C Densitometric scanning of the Western blot and quantification of STAT5AB levels (n = 2). Error bars indicate the data range. D Dot-blot of STAT5AB IHC quantification from Stat5ab+/+, Stat5ab+/Δ and Stat5abΔ/Δ livers. E Whisker-box blots depicting the quantification of STAT5AB protein levels from D using the HistoQuest software. The box indicates the interquartile range; the horizontal line in the box depicts the median; whiskers indicate the data range. 3788 Stat5ab+/+, 3041 Stat5abΔ/+ and 753 Stat5abΔ/Δ cells (hepatocytes) were analyzed in total. One-way ANOVA testing proved that the measured differences were of significance (p<0.001).
Figure 2.
p-STAT5Tyr694 and p-STAT3Tyr705 expression upon GH treatment compared to control mice.
A Western Blot of p-STAT5Tyr694 expression in Stat5ab+/+ (n = 2), Stat5ab+/Δ (n = 2) and Stat5abΔ/Δ (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. B IHC of p-STAT5Tyr694 in sections of the livers (n = 3) used in A. Red, AEC; blue, hematoxylin. Size bar: 100 µm. High magnifications (60x) are shown in insets. C Quantification of p-STAT5Tyr694 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software (Stat5ab+/+, control; number of cells n = 3276, GH; number of cells n = 2286; Stat5ab+/Δ control; number of cells n = 2577, GH; number of cells n = 3549; Stat5abΔ/Δ control; number of cells n = 3299, GH; number of cells n = 2200). The highest arbitrary expression values from A and B were set to 100 for direct comparison. D Western blot of p-STAT3Tyr705 expression in Stat5ab+/+ (n = 2), Stat5ab+/Δ (n = 2) and Stat5abΔ/Δ (n = 2) livers of GH treated and control mice. HSC70 is shown as loading control. E IHC of p-STAT3 Tyr705 in sections of the livers (n = 3) used in D. Red, AEC; blue, hematoxylin. Size bar: 100 µm. High magnifications are shown in insets (60x). F Quantification of p-STAT3Tyr705 levels from Western blots (grey) by densitometric scanning and IHC (magenta) using the HistoQuest software (Stat5ab+/+, control; number of cells n = 2634, GH; number of cells n = 2286; Stat5ab+/Δ control; number of cells n = 22230, GH n = 2562; Stat5abΔ/Δ control; number of cells n = 2414, GH n = 2716). The highest arbitrary expression values from A and B were set to 100 for direct comparison.
Figure 3.
Detection and quantification of transcription factor translocation into the nucleus in mouse livers.
A IHC of STAT5AB expression in Stat5ab+/+ (n = 3), Stat5ab+/Δ (n = 3) and Stat5abΔ/Δ (n = 3) livers of GH treated and control mice. Size bar: 50 µm. B Scattergram of the analyzed IHC samples used in A. Discrimination of hepatocytes due to their hematoxylin area and mean intensity depicted in gate 4. Image of gated (hepatocytes, red circled) and of non-hepatocytic cells, mainly Kupffer cells (green circled) in an IHC stained liver sample. C Nuclear and cytoplasmic mask of cells as they are recognized by HistoQuest. The bar graph shows the ratios of nuclear versus cytoplasmic STAT5AB expression levels. Error bars are S.D. Student's t-test was performed to demonstrate statistical significance. D Histograms of nuclear/cytoplasmic STAT5AB intensities are shown; Stat5ab+/+ (blue), Stat5ab+/Δ (red) and Stat5abΔ/Δ (green). The corresponding values in the histograms (black values) showed the gradual decrease of the mean intensities. Based on the fact that Stat5abΔ/Δ mice have no hepatic STAT5AB their values in the histograms can be set as background intensity. After background subtraction the numbers displayed the expected 2∶1∶0 ratios (red values). E Whiskers-box plots depicting the quantification of STAT5AB positive hepatocytes (cytoplasmic C; nuclear N) from Stat5ab+/+ with and without GH stimulation using the HistoQuest software. The box indicates the interquartile range; the horizontal line in the box depicts the median. Whiskers indicate the data range. Student's t-test was used to demonstrate statistical significance.
Figure 4.
Analysis and evaluation of STAT5AB in human HCC samples and the detection of interobserver variabilties.
A Representative images of human HCC with different nuclear STAT5AB expression levels (0, 1, 2, 3) which were scored corresponding to pathological evaluation. Size bar: 100 µm. B Histograms from image analysis based quantification of nuclear STAT5AB in the representative images from A. Mean intensity (MI) values and scoring zones are depicted. C Venn diagram depicting the percent overlaps from two pathologists and image analysis on the assessment of nuclear STAT5AB. D Linear weighed kappa analysis depicting the grade of agreement between the evaluating pathologists and the HistoQuest software before (black values) and after software-assisted re-evaluation (red values).