Fig 2: The effects of P5 on HC permeability (a, b) and Cx expression (c, d) of alveolar cells in vivo. (a, b) At 20 hr after LPS challenge, saline or P5 was administered (ip) twice at 2 and 16 hr post-ALI induction. After 10 min, mouse lungs were perfused with PBS, fixed with PF, and then cut into sections that were subsequently stained with DAPI. LY-containing cells appeared in green, showing LY uptake through HCs. Nuclei of all alveolar cells were stained with DAPI and appeared in blue. The ratio of LY/DAPI fluorescence signals was used to determine HC activity. a.u.: arbitrary unit. (c, d) Saline or P5 was administered (ip) twice at 2 and 16 hr post-ALI induction. After BALF extraction, lungs were harvested and homogenated to determine the expression of Cx37, Cx40, and Cx43 by Western blotting.

Fig 3: High glucose reduces Cx43 expression and GJIC activity through RhoA/ROCK1/pMLC signaling. (A) Immunofluorescence staining of Cx43 (green) and DAPI staining (blue) in mRMVECs. Cx43 was distributed mainly in the membrane of mRMVECs (arrows) in the normal glucose group or ROCK inhibitor group. Cx43 distribution decreased in the cell membrane and it was translocated to the cytoplasm (asterisks) in the high glucose group or RhoA over expression plasmid group. (B, C) GJIC function was evaluated by counting cell layers stained with Lucifer Yellow CH dye. Scale bar: 100 µm. EV, empty vector. ns, no significance. ****P < 0.0001 compared with the control group; ###P < 0.001, ####P < 0.001 compared with the high glucose (30 mM) group. N = 3. The bars are the mean ± SD.

Fig 4: Effect of TAK-242, PDTC or SR11302 on LPS-induced Cx43 expression in cultured astrocytes. (A) Double immunofluorescence indicates the expression of TLR4 on Cx43-labeled dorsal spinal cord astrocytes. Scale bar: 20 μm. (B) RT-qPCR quantitative analysis showed that TAK-242 significantly inhibited LPS-induced Cx43 mRNA expression. *p < 0.05 vs. Veh-treated group, +p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (C) RT-qPCR quantitative analysis showed that PDTC or SR11302 significantly inhibited LPS-induced Cx43 mRNA expression. *p < 0.05 vs. Veh-treated group, +p < 0.05 vs. Veh + LPS-treated group, n = 5 per group. (D) WB results showed that TAK-242 significantly inhibited LPS-induced Cx43 and p-Cx43 expression. The top panel was the target band, Cx43 and p-Cx43 (Ser368), and the bottom one was for the loading control GAPDH. (E) WB results showed that PDTC or SR11302 significantly inhibited LPS-induced Cx43 and p-Cx43 expression. The top panel was the target band, Cx43 and p-Cx43 (Ser368), and the bottom one was for the loading control GAPDH. (F) Statistical analysis shows the effect of TAK-242 on Cx43 and p-Cx43 protein expression [intensity ratios (normalized to GAPDH expression) relative to the Veh-treated group]. *p < 0.05 vs. Veh-treated group, +p < 0.05 vs. Veh + LPS-treated group, n = 4 per group. (G) Statistical analysis shows the effect of PDTC or SR11302 on Cx43 and p-Cx43 protein expression [intensity ratios (normalized to GAPDH expression) relative to the Veh-treated group]. *p < 0.05 vs. Veh-treated group, +p < 0.05 vs. Veh + LPS-treated group, all data were expressed as mean ± standard deviation of at least four independent experiments (n = 4 per group). (H) EMSA analysis was performed on binding reactions (the binding of AP-1 to Cx43 promoter) conducted in the presence of the indicated μg protein amount of Veh + LPS-treated cells (left). NP, no protein; UP, unlabeled probe. AP-1 DNA binding activity was analyzed (right). *p < 0.05 vs. 4 μg group, +p < 0.05 vs. 8 μg group, n = 4 per group. (I) EMSA analysis was performed on binding reactions (the binding of AP-1 to Cx43 promoter) conducted in LPS-treated cells pretreated with or without PDTC or SR11302. This is a representative of three separate experiments (left). For competition, a 100-fold molar excess of unlabelled probe (comp) was added to nuclear extracts from Veh + LPS group. AP-1 DNA binding activity in different groups was analyzed (right). *p < 0.05 vs. Veh-treated group, +p < 0.05 vs. Veh + LPS-treated group, n = 4 per group.

Fig 5: 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.