After treating the U2OS cell line with three different concentration gradients of the TLK2 inhibitor UNC-CA2-103 (HY-162426, MCE) for 48 hours, I extracted proteins and performed Western blotting (WB). However, I observed no effect on protein expression levels.
We only have antibodies related to DNA damage response, and previous literature has reported that TLK2 knockdown leads to increased phosphorylation of H2AX (γH2AX)—a marker of DNA damage. Yet, I did not detect any increase in phosphorylation either.
Yan Yan Zhao , the TLK2 inhibitor you used did not produce a significant inhibitory effect. From the literature I found:
1. Doxorubicin damages U2OS cells, which leads to a decrease in TLK2 expression; knocking down TLK2 inhibits damage repair.
https://pmc.ncbi.nlm.nih.gov/articles/PMC5341517/
2. TLK2 is a DNA repair protein. Theoretically, under the condition of DNA damage + inhibition of TLK2, it will have a significant effect on cell proliferation.
https://pmc.ncbi.nlm.nih.gov/articles/PMC11071368/
3. I have not found any literature that directly affects the proliferation of U2OS cells by inhibiting TLK2 activity. However, I have found literature reports that knocking down/inhibiting TLK2 will slow the growth of some cancer cells and inhibit invasion.
https://pmc.ncbi.nlm.nih.gov/articles/PMC11421834/
So it is recommended that you detect the expression and phosphorylation level of the downstream protein of the target. Confirm whether the target is inhibited. In addition, the compound you use is relatively new and there are no more references. Regarding the setting of the treatment concentration,
you can try it at the common concentration of conventional compound cell experiments, around 10 μM.
The answer to this question comes from MedChemExpress (MCE) Technical Support.
Thanks for sharing your experience. I’m also considering using UNC‑CA2‑103 for TLK2-related studies and have been looking into its intracellular activity.
So far, I haven’t found any published data confirming whether UNC‑CA2‑103 can inhibit TLK2 autophosphorylation or the phosphorylation of its well-known substrate ASF1 (ASF1A/B) in cells. I was wondering—have you tested either of these readouts in your system? Or do you happen to have any related information about its functional validation in a cellular context?
Would really appreciate any insight you could share!