Fig 1: YAP ISGylation stimulates 6PGL transcription.A Co-IP experiments analyzing YAP ISGylation in WT or ISG-/- A549 and H1299 cells with or without overexpressing YAP, and YAP and 6PGL expression in Input sample were analyzed by IB. The FLAG level in YAPFLAG overexpressed co-IP samples was adjusted to the same protein content. B 6PGL mRNA level was measured in WT or ISG-/- A549 and H1299 cells with or without overexpressing YAP. C, D Luciferase activity was analyzed in WT or ISG15-/- A549 (C) and H1299 (D) cells co-expressing indicated 6PGL promoter–reporter with or without overexpressing YAP. E, F The enrichments of YAP at indicated regions of 6PGL promoter were calculated as the percentage of Input chromosomal DNA via ChIP in WT or ISG-/- A549 and H1299 cells with or without overexpressing YAP (E). Anti-IgG was used as parallel control (F). G Co-IP experiments analyzing YAP ISGylation in tumor xenografts overexpressing YAP with or without ISG15 knockout, and YAP and 6PGL expression in Input sample were analyzed by IB. The YAP level in each co-IP sample was adjusted to the same protein content. H The enrichments of YAP at indicated regions of 6PGL promoter, NADPH, and Rib-5-P level were analyzed in the same tumor xenografts as those in Panel G. I Co-IP experiments analyzing YAP ISGylation in PDX#1 and PDX#2, and YAP and 6PGL expression in Input sample were analyzed by IB. The YAP level in each co-IP sample was adjusted to the same protein content. J The enrichments of YAP at indicated regions of 6PGL promoter, NADPH, and Rib-5-P level were analyzed in PDX#1 and PDX#2. The data are shown as the mean ± SD from three biological replicates (including IB). Data in B–F were analyzed using a one-way ANOVA test. Data in H and J were analyzed using a student’s t test. **P < 0.01, NS nonsignificant.
Fig 2: SMAD and TEAD stimulate 6PGL transcription in a YAP-dependent manner.A Predicting 6PGL promoter binding YAP-associated transcription factors by JASPAR database. B 6PGL mRNA was measured in A549 and H1299 cells with indicated plasmids overexpressed. C The enrichments of YAP at indicated regions of the 6PGL promoter were calculated as the percentage of Input chromosomal DNA via ChIP in A549 cells. Anti-IgG and anti-TFCP2 were used as parallel control. D, E Luciferase activity was analyzed in A549 cells co-expressing indicated 6PGL promoter–reporter with or without overexpressing (D) or knocking out YAP (E). F, G Co-occupancies of YAP and SMAD2 (F) and YAP and TEAD4 (G) were measured by ChIP and Re-ChIP experiments in A549 cells using indicated antibodies. H Nuclear extracts from A549 cells were incubated with indicated 6PGL promoter probes with or without the presence of indicated antibodies. The DNA–protein interactions were measured using EMSA. The data are shown as the mean ± SD from three biological replicates (including IB). Data in B–E were analyzed using a one-way ANOVA test. **P < 0.01, *P < 0.05, NS nonsignificant.
Fig 3: Clinical association among YAP ISGylation, YAP, and 6PGL.A YAP protein level in LUAD and adjacent normal tissues as measured by ELISA. B 6PGL mRNA level in LUAD and adjacent normal tissues as measured by qPCR. C Association between YAP protein and 6PGL mRNA level. D Co-IP experiments analyzing YAP ISGylation in LUAD and adjacent normal tissues, and YAP and 6PGL expression in Input sample were analyzed by IB. The YAP level in each co-IP sample was adjusted to the same protein content. E, F Association between YAP bound ISG15 intensity and YAP intensity (E), as well as YAP, bound ISG15 intensity and 6PGL intensity (F) in LUAD tissues. G, H YAP protein and 6PGL mRNA level in stage I–III LUAD samples (G), <3 cm and >3 cm diameter LUAD samples (H). I Survival analysis for LUAD patients with high or low YAP protein and 6PGL mRNA level. J YAP protein and 6PGL mRNA level in high and low glucose LUAD samples. K Co-IP experiments analyzing YAP ISGylation in high and low glucose LUAD tissues, and YAP and 6PGL expression in Input sample were analyzed by IB. The YAP level in each co-IP samples was adjusted to the same protein content. L, M Association between YAP bound ISG15 intensity and YAP intensity, as well as YAP, bound ISG15 intensity and 6PGL intensity in high (L) or low (M) glucose LUAD tissues. The data are shown as the mean ± SD from three biological replicates (including IB). Data in A, B, H, I were analyzed using a student’s t test. Data in C, E, F, L, M were analyzed using the Spearman rank correlation analysis. Data in G were analyzed using a one-way ANOVA test. Data in I were analyzed using a log-rank test. **P < 0.01.
Fig 4: YAP stimulates PPP by activating 6PGL.A Schematic presentation of PPP from glucose to Rib-5-P. B, C NADPH (B) and Rib-5-P (C) concentration in control, YAP overexpression or knockout A549 and H1299 cells. D, E Indicated mRNA levels in control, YAP overexpression or knockout A549 (D) and H1299 (E) cells. F 6PGL and YAP protein levels in control, YAP overexpression or knockout A549 and H1299 cells. G–J NADPH (G), Rib-5-P concentration (H), and colony formation (I, J) were measured in WT or YAP-/- A549 and H1299 cells with or without 6PGL overexpression. Scale bar, 200 µm. K, L NADPH (K) and Rib-5-P concentration (L) were measured in WT or 6PGL-/- A549 and H1299 cells with or without YAP overexpression. The data are shown as the mean ± SD from three biological replicates (including IB). Data in B–E, G–I, K, L were analyzed using a one-way ANOVA test. **P < 0.01, NS nonsignificant.
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