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Page 6 of 19 Mondal et al. Neuroimmunol Neuroinflammation 2018;5:34 I http://dx.doi.org/10.20517/2347-8659.2018.13

Figure 1. Comparative study of expression of CD34 in HSCs of five experimental animal groups, viz., normal, healthy control rats (N),
glioma-bearing rats (ENU), and rats having received the first (ET1), second (ET2) and third (ET3) doses of T11TS. Flowcytometric studies
of the expression of CD34 in HSCs cells isolated from bone marrow of normal control and of glioma-bearing rats before and after T11TS
treatment have been shown. A: Flowcytometric analysis of CD34 percent positive cells in the LDC using BD Cell Quest Pro Software
by using scatter from a single representative experiment. The percentage of the expression refers to the percent positive cells out of
10,000 cells analyzed represented in graphical form in bar diagram. Column values represent mean ± SD (n = 6 animals per group);
B: Flowcytometric analysis of CD34 percent positive cells in the HDC using BD Cell Quest Pro Software by using scatter from a single
representative experiment. The percentage of expression refers to the percent positive cells out of 10,000 cells analyzed represented
in graphical form in bar diagram. Column values represent mean ± SD (n = 6 animals per group). Comparison of Distribution of CD34 +
Cells between LDC and HDC: The line diagram shows both the LDC and HDC HSCs there is a steep high rise in their expression level of
+
CD34 cells following T11TS administration, but compared to HDC the LDC cells show the high level of proliferation as compared to ENU
and normal groups probably hinting arousal of HSCs from niche as CD34 expressed mainly in primitive HSCs; C: representative images
(100× magnification) showing immunofluorescent staining of cell surface CD34 expression on bone marrow derived hematopoietic
stem cells of normal (N) and glioma-bearing rats before (ENU) and after T11TS treatment (ET1, ET2 and ET3) in both the LDC and HDC
compartments. Each condition was observed in triplicate and six images were taken for each sample. Figures are representative of the
group. Results were expressed as the percent CD34-positive cells out of the total number of cells counted for each experimental group.
Column CD34: FITC-stained CD34 expression in bone marrow derived hematopoietic stem cells green in color due to the presence of
CD34. Column DAPI: nuclei appear blue due to staining with DAPI. Column merged: FITC-stained fluorescence intensity of each group
was analyzed with Nikon’s Nis - Elements D3.00 software and the mean intensity was expressed in bar diagrams. Individual bar values
represent mean fluorescence intensity ± SD of the respective group. At the bottom the comparison of the mean fluorescence intensity
of CD34 positive cells between LDC and HDC has been also depicted. *Significant (P < 0.001) decrease in ENU compared with that of
#
normal control group. Significant (P < 0.001) increase, when individually comparing T11TS treated groups with glioma-bearing ENU
group in both the compartments. HSCs: hematopoietic stem cells; LDC: low density compartment; HDC: high density compartments;
ENU: N-ethyl-N-nitrosourea

and HDC. T11TS administration in glioma-bearing animal significantly (P < 0.001) up-regulated c-kit
expression in glioma associated BMHSCs of both the groups in all three dose levels both in the mature
and immature compartments as compared to the significantly low expression in the ENU induced glioma-
bearing group.

Immunofluorescence imaging studies also confirmed our findings. Immunofluorescence staining and
imaging [Figure 3C] of cytoplasmic c-kit expression of bone marrow-derived hematopoietic stem cells
were evident from robust (P < 0.001) decrease in cytoplasmic mean fluorescence intensity observed in
bone marrow-derived hematopoietic stem cells of ENU group both in the LDC and HDC. Normal bone
marrow-derived hematopoietic stem cells showed moderate fluorescence staining and hence medium