Fig 2: GRAMD1B and JAK/STAT signaling act synergistically to regulate anti-apoptotic gene expression(A and B) Knockdown of Gramd1b causes a decrease in both total STAT3 and pSTAT3 (Tyr705) levels in AGS and NUGC3 cells. (C-F) Cellular assays were conducted using AGS cells. (C) MTS assay reveals that Gramd1b knockdown decreases cell growth by approximately 20% compared to control. (D) PI staining suggests the synergistic effects of GRAMD1B and JAK/STAT signaling on cell survival. (E) AO/EB staining suggests that inhibition of both GRAMD1B and JAK/STAT signaling synergistically increases the number of apoptotic cells (white arrows). (F) Decreased Bcl-xL expression on Gramd1b knockdown is further enhanced by co-treatment with AG490. The increase in cleaved PARP levels on Gramd1b knockdown is also further enhanced by co-treatment with AG490. **, P<0.01; ****, P<0.0001.

Fig 3: TC-PTP deficiency confers a resistance against DMBA-induced apoptosis and an enhanced TPA-induced cell proliferation through the regulation of STAT3 and AKT signaling.(A) Western blot analysis of p-EGFR, p-STAT3, p-AKT, p-ERK1/2, cyclin D1, Bcl-xL, Bax in the epidermis from K14Cre.Ptpn2w/w and K14Cre.Ptpn2fl/fl mice after TPA treatment. Mice were sacrificed at the indicated time after TPA treatment and epidermal cell lysates were prepared. (B) Western blot analysis of p-EGFR, p-STAT3, p-AKT, p-ERK1/2, cyclin D1, Bcl-xL, Mcl-1 in primary keratinocytes from both genotypes after TPA treatment. Cells were collected at the indicated time after TPA treatment and total cell lysates were prepared.

Fig 4: Shh signaling imparts Imatinib resistance via Bcl2 upregulation.A RT-PCR-based fold change determination in expression of various genes known for their role in drug resistance. Imatinib-treated Shh-K562 cells (TShh-K562) and treated K562 (TK562) normalized to their respective untreated controls, at 72 h. Bars represents mean ± SD from three biological repeats. B mRNA-fold change of three anti-apoptotic genes, Bcl2, Mcl-1, Bcl-xL, measured using RT-PCR in Imatinib-treated Shh-K562 (TShh-K562) normalized to Imatinib-treated K562 (TK562). Bars represents mean ± SD from two biological repeats. C Western blot representing expression of Bcl2 and Mcl-1 from untreated and Imatinib-treated cells (K562, Shh-K562, TK562, and TShh-K562). GAPDH is used as loading control. The graph represents densitometric analysis of relative protein levels normalized to untreated K562. Bar represents mean ± SD from three biological repeats. D Time-curve representing viability (MTT assay) of K562 (black dotted line) and Shh-K562 (green dotted line) cell lines after treatment with Imatinib (0.5 µM); K562 (black solid line) and Shh-K562 (green solid line) for Imatinib (0.5 µM) + Cyclopamine (25 nM) treated conditions; gray solid line represents viability of K562 after Cyclopamine treatment (25 nM). Graph represents mean ± SD from three biological repeats. E RT-PCR data determining the level of Bcl2-mRNA in K562 and Shh-K562 after treatment for 72 h with either vehicle control (untreated), Imatinib-alone, or Imatinib + Cyclopamine normalized to their respective K562 controls (black dotted line at 1). Bar represents mean ± SD from two biological repeats. For all assays, P values, *<0.05, **<0.01, and ***<0.001. Statistical analysis done using t-test.

Fig 5: Study of the sensitivity of four human HNSCC cell lines to ABT-737(A) Correlation between the sensitivity to ABT-737 (IC50) and the radiosensitivity of cell lines (SF2). (B) Expression of Bcl-2 family proteins of the four cell lines and the CSC sub-population (SP+/CD44high/ALDHhigh) was performed by Western blotting experiments. The correlation between sensitivity to ABT-737 (IC50) and (C) Bak expression, or (D) Bcl-XL expression were normalized to the expression of a-tubulin.