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. 2023 Oct 5;26(11):108142. doi: 10.1016/j.isci.2023.108142

Figure 6.

Figure 6

2-Hydroxyisobutyrylation at lys-260 of ALDH1A1 affects tumor growth in vivo and was associated with prognosis in bladder cancer

(A and B) Flag-tagged ALDH1A1 was transfected into T24 cells, and the cells were treated with serum starvation and EBSS stresses for the indicated time to observe the level of 2-hydroxyisobutyrylation.

(C) T24 cells were treated with GC for the indicated time to observe the level of 2-hydroxyisobutyrylation. The final concentration of gemcitabine and cisplatin were 0, 0.1 μg/mL and 0.3 μg/mL respectively.

(D) Xenograft was performed in BALB/c nude mice injected with T24 ALDH1A1WT or ALDH1A1K260T rescued cells (n = 6 per group). The mice were sacrificed 2 weeks after injection, and the tumors were obtained and photographed. The volume of tumors were measured every 3 days after T24 ALDH1A1WT or ALDH1A1K260T rescued cells injection (p < 0.05).

(E) Representative pulmonary CT images acquired from mice with intravenous injection of ALDH1A1WT/ALDH1A1K260T at 5 weeks. Black arrows revealed the detection of the metastatic tumors in the lungs (n = 5 per group). Mice were sacrificed, the lungs were obtained and photographed. Red arrows revealed the detection of the metastatic nodesin the lungs (n = 5 per group).

(F) Then, the lungs were also fixed in 4% paraformaldehyde for HE staining. Up: Scale bar = 500 μm. Down: Scale bar = 50 μm.

(G) IHC analysis of the levels of ALDH1A1 and ALDH1A1-K260hib in bladder cancer tissues. Scale bar = 200 μm.

(H) Chi-square test was used to analyze the correlation between the expression levels of ALDH1A1 and ALDH1A1-K260hib in bladder cancer (p < 0.05).

(I) The lower expression of ALDH1A1K260hib was associated with an advanced tumor stage in 105 patients with bladder cancer from our hospital.

(J) Kaplan–Meier analysis of OS with 105 patients with bladder cancer was assessed between strong and weak staining of ALDH1A1-K260hib (log rank of p < 0.05).

(K) Kaplan–Meier analysis of RFS was evaluated according to the level of ALDH1A1 K260hib in 105 patients with bladder cancer. For cell experiments, each experiment was performed at least three times. For animal experiments, five to six per group were used for in vivo studies. Mean ± SEM, ∗p < 0.05, ∗∗∗p < 0.001 by two-sided Student’s t test.