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

Statistical analysis
Data shown are representative of six independent experiments (n = 6) and values are expressed as mean ± SD
unless otherwise stated. For statistical comparison, the one-way analysis of variance was performed followed
by application of the Tukey’s post-hoc test. A P value < 0.05 was considered to be statistically significant.

RESULTS
Bone marrow-derived CD34 cell, Sca-1 cell and c-kit cell, Ang-1 cell and Tie-2 cell: density specific
+
+
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compartmentalization
During our phenotyping characterization of HSCs from the bone marrow, densitometric centrifugation
technique resulted in two well distinct categories of cells. Cells at LDC (1.050) indicate immature HSCs and the
other at HDC (1.077) denote mature HSCs , which will lead to the generation of the progenitors. The harvested
[48]
cells were subjected to CD34, Sca-1, c-kit, Ang-1, and Tie-2 positivity in a cell sorter.

Effect of T11TS on the phenotypic markers CD34 of BMHSCs
Bone marrow-derived CD34 -enriched cell population claimed to be one of the most critical markers for
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HSCs and their expression is down-regulated as they differentiate into mature cells . Single staining was
[50]
[49]
done for CD34. Flowcytometric analysis [Figure 1A and B] showed a higher enrichment of CD34 cells in the
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LDC level than in the HDC. In normal rats, there was a higher level of expression of CD34 both in the LDC
and HDC in comparison to the ENU treated glioma-bearing rat where BMHSCs showed a significantly (P
< 0.001) decreased level of expression both in the LDC and HDC. However, there was a very significant (P <
0.001) increase in expression levels of both in the LDC and HDC in all three dose levels, i.e., ET1, ET2 and
ET3 in T11TS treated glioma-bearing rats.

In situ immunofluorescence imaging studies [Figure 1C] further confirmed the CD34 FACS findings.
In the ENU group, the majority of CD34 cells showed a significant (P < 0.001) decrease in their expression of
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fluorescence intensity both in the LDC and (1.692 ± 0.439) in the HDC as compared to the normal group in both
compartments. The intensity gradually increased following T11TS therapy in dose dependent manner ET1. ET2
and a significant up-regulation were noted at the third dose, i.e., ET3 in both the LDC and HDC compartments.

Effect of T11TS on the phenotypic markers Sca-1 of BMHSCs
Sca-1, an important marker of emerging HSCs, regulates the overall developmental program of HSCs
towards self-renewal, lineage fate and c-kit expression [25,51] .

Flowcytometric studies [Figure 2A and B] showed that there was significant down regulation (P < 0.001) of
Sca-1 expression both in the LDC and HDC (in glioma-bearing rats as compared to the normal groups in
both the LDC and HDC. Following T11TS therapy, there was significant up-regulation (P < 0.001) of the
expression of Sca-1 at all dose levels (ET1, ET2 and ET3) both in the immature and mature compartments
compared to glioma-bearing groups. Immunoblot data [Figure 2C and D] corroborated the flowcytometric
finding and confirmed that the expression of Sca-1 increased significantly (P < 0.0001) in dose-dependent
manner in glioma associated BMHSCs of T11TS treated groups both in the LDC and HDC compared to that
in HSCs of glioma-bearing ENU group both in LDC and HDC.

Changes in the expression pattern of c-kit following T11TS therapy
C-Kit, a tyrosine kinase receptor for stem cell factor, acts as a unique marker for HSCs and regulates the
[52]
fate of HSCs . Even small changes in the expression pattern of c-kit resulted in dramatic phenotypes and
[53]
profoundly altered the developmental rhythm of hematopoiesis .
[54]
Flowcytometric studies showed [Figure 3A and B] that following glioma induction, expression of c-kit in
BMHSCs of ENU group, there was a remarkable (P < 0.001) decrease in the expression level, both in LDC