Fig 2: Gli2 knockdown reversed cisplatin (DDP) resistance and inhibited proliferation of DDP-resistant ovarian cancer cells. (A, B) Western blot analysis of the protein expression levels of Gli2 in different groups after Gli2 knockdown in SKOV3/DDP cells. (C) Survival rate and IC50 of SKOV3/DDP cells transfected with sh-control or sh-Gli2 #3 and #5 after 48 h DDP treatment determined using CCK-8 assays. (D–G) Western blot analysis of the expression levels of MDR1 and ?H2AX in SKOV3/DDP cells transfected with empty plasmid or Gli2 specific short hairpin RNA following DDP treatment (0.5 µg/mL). n.s, not significant, * P < 0.05, **P < 0.01.

Fig 3: ZIP1+ fibroblasts interconnect lung cancer cells via upregulating CX43 and promote chemoresistance by Zn2+ transfer.Chemotherapy treatment induces Zn2+ released from dying cancer cells and the enrichment of ZIP1+ fibroblasts. ZIP1+ fibroblasts interconnect cancer cells with gap junctions by upregulating CX43. Upon treatment, the ability of Zn2+ uptake by cancer cells is suppressed. ZIP1+ fibroblasts act as a reservoir to absorb and transfer Zn2+ to lung cancer cells via gap junctions. In ZIP1+ fibroblasts, imported Zn2+ stimulates degradation of PTEN and activation of AKT to upregulate the expression of gap junction protein CX43. In lung cancer cells, the elevation of Zn2+ enhances the expression of ABCB1 and reduces the accumulation of drugs, resulting in chemoresistance.

Fig 4: SY-1320 promotes ADR accumulation in MCF-7ADR cells by targeting P-gp.A, B Levels of P-gp protein and mRNA in MCF-7 or MCF-7ADR cells were detected by western blot and RT-qPCR, respectively. C MCF-7ADR cells were treated with ADR (2 µM) alone or together with TQ (0.5 µM) for 48 h. Cell viability was detected using the CCK8 assays. D, E Intracellular ADR accumulation was detected by flow cytometry (D) and fluorescence (E). Scale bars, 50 µm. F Both SY-1320 and TQ increased the sensitivity of MCF-7ADR cells to etoposide (VP16). Data are presented as mean ± SD of three independent experiments. *P < 0.05, ***P < 0.001, two-tailed Student’s t test.

Fig 5: MDR1 is the direct downstream target of Gli2. (A) Consensus sequence of Gli2-binding sites (GBS). The size of each base in the figure indicates its conservation. The larger the base, the stronger the conservation and more important its function. (B) Within the range of -2,002 to 0 in the promoter region of MDR1 and adjacent regions (the first base at the 5' end of the MDR1 mRNA NM_000927.5 was recorded as +1), seven candidate GBS were predicted using software analysis. They are numbered separately, and the position and sequence are indicated. The consensus sequence with GBS is underlined in red with the corresponding sequence, and the base list differing from the GBS consensus sequence is in red font. (C) Schematic illustration of the distribution of candidate GBS within the MDR1 promoter and luciferase reporter constructs Frag-I, -II, –II-1, –II-2, –II-1-a, and –II-1-b containing the indicated GBS. (D–F) Gli2 activating effects on Frag-I, -II, –II-1, –II-2, –II-1-a, and –II-1-b reporter constructs detected using a dual-luciferase assay in Gli2-transfected and control HEK293 cells. (G) Wild-type and mutant sequences of the GBS-4 locus in Frag–II-1-a. (H) Compared with the wild-type, the activation effect of Gli2 on the mutant Frag–II-1-a was significantly reduced after GBS-4 point mutation. “Mut” indicates a mutation, and the letters after it denote different GBS sites. Data are represented as the mean ± SD of three independent experiments. n = 3, *P < 0.05, **P < 0.01, ***P < 0.001.