Fig 1: Stemness and Bcl2 are independent targets of Shh signaling.A Flowchart representing method for obtaining CD34(+) and CD34(–) cells from K562 and Shh-K562 cell lines. B Graph depicts RT-PCR-based fold change in expression levels of CD34, Shh, Gli-1, and BCl2 in fractionated CD34(+) (green bars) and negative cells (red bars). P values derived from two biological repeats. C Western blot representing Bcl2 protein levels in CD34(+) and CD34(–) Shh-K562 and K562 cells. Actin used as loading control. Graph represents densitometric analysis of relative Bcl2 levels in Shh-K562-CD34(+) (red bar) and Shh-K562-CD34(–) (green bar) cells using K562-CD34(–) cells as normalizing control. Bar represents mean ± SD from two biological repeats. D Viability of Shh-K562 (in green) and K562 cells (in black; control) after treatment with different concentration of YC137 (Bcl2 inhibitor II) to identify IC50 at 72 h. Untreated cells at respective time-point used as reference. Line graph represents mean ± SD from three biological repeats. E Percentage viability of Shh-K562 (in green) and K562 cells (in black; control) after treatment with Imatinib (0.5 μM) + different concentration of YC137 (Bcl2 inhibitor II) to identify lowest effective concentration. Untreated cells at respective time-point used as reference. Line graph represents mean ± SD from three different experiments. P values, *<0.05, **<0.01, and ***<0.001. Statistical analysis done using t-test.
Fig 2: Shh expression and signaling are significantly upregulated in CML-variants and non-compliant patients.A Graph representing fold change in Shh and Gli1-mRNA (in log2 scale) from chronic phase CML (CML-CP) patients (n = 3; blue data points); chronic phase CML with variant chromosomal translocation (n = 5; orange) and non-compliant patients (n = 1; red) using RT-PCR. Reference line (black dotted line) represents the expression in normalizing control (K562 cells). P values, *<0.05. Statistical analysis done using t-test. B Immunohistochemistry for Shh and Patched1 using bone biopsy samples of CML patients in CML-CP (9:22; CML-3&8) and chronic phase CML with variant chromosomal translocation (CML-7, CML12). Blue, hematoxylin. Scale bar 20 μm. C Heatmap showing expression of Shh signaling pathway genes (as per KEGG pathway) using RNA-seq data from high-risk CML patients1. Patients were in Chronic Phase at diagnosis (CP Dx.) with progression to lymphoid blast crisis (LBC) or myeloid blast crisis (MBC). Three patients were in MBC at diagnosis (MBC Dx.). Two patients were in accelerated phase at diagnosis (AP Dx.) and progressed to MBC. The gene expression profiles depict the clear distinction between CML-CP from other stages.
Fig 3: Shh signaling promotes stemness and imparts Imatinib resistance.A Cartoon representing cell line model used, K562 (in black) and Shh-K562 (in green). B Shh and Gli-1-mRNA expression in Shh-K562 determined using RT-PCR, normalized to K562. Bar represents mean ± SD from three biological repeats. Western blot demonstrating expression of Gli-1 and Shh proteins in K562 and Shh-K562. Actin is used as loading control. C Levels of stemness markers, CD73, CD34, CD117, and CD90, and myeloid differentiation marker (CD38) in the Shh-K562 cell line determined using RT-PCR, normalized with K562. Bar represents mean ± SD from two biological repeats. D Representative images of FACS-based analysis of CD90, CD34, and CD73 expression in K562 and Shh-K562. Experiment was done with two biological and technical repeats with similar results. E Concentration curve for Imatinib to identify IC50 at 72 h, Shh-K562 (green line) compared to K562 (control; black line graph). Line graph represents mean ± SD from three biological repeats. F Images demonstrating colony forming units (CFU) using K562, Imatinib-treated K562 (TK562), Shh-K562, and Imatinib-treated Shh-K562 (TShh-K562). Graph represents average area of the colonies (30–100 colonies per condition) from duplicate experiments, in Log10 scale. For all images, P values, *<0.05, **<0.01, and ***<0.001. Statistical analysis done using t-test.
Fig 4: Rack1 deficiency in SHH‐MB inhibits SHH signaling pathway. (A) Western blots detected the expression of Gli1 and HDAC2 in cerebellar lysates from different genotypes. (B) Quantitative analysis of Western blot results indicates the significantly reduced expression of Gli1 and HDAC2 in rescue mice compared to SHH‐MB tumor mice. mean ± SEM; *p < 0.05, **p < 0.01, n ≥ 3
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