Fig. 6: KIRA6 modulates CXCL8 production in an IRE1α-independent pathway.

A KIRA6 (1 μM) mediated blocking of CXCL8 production and intracellular accumulation 4 h after treatment with MTX or Hyp-PDT in both IRE1α^−/− or IRE1α^+/+ A375 cells. B CXCL8 secretion was measured by ELISA 24 h after treatment with MTX in conditioned medium of IRE1α^−/− or IRE1α^+/+ A375 cells with or without incubation with KIRA6 (1 μM). C Molecular structure of the modified KIRA6 (KIRA6 Affinity-based Probe, KIRA6 AfBP) used for off-target protein identification. D Comparison of the ability of KIRA6, KIRA6 AfBP and the intermediate tag-free KIRA6 AfBP (without photoaffinity group and clickable handle) in inhibiting CXCL8 production and IRE1α phosphorylation 4 h after treatment with MTX in A375 cells at the indicated concentrations. E CXCL8 secretion was measured by ELISA in conditioned medium of A375 cells 24 h after treatment with MTX with or without co-incubation with KIRA6 AfBP (10 μM). F Scheme of the experimental workflow used to identify the KIRA6 off-targets by using the modified KIRA6 AfBP. G Representative gel assessing the efficacy of protein off-target identification workflow in protein lysates of A375 cells. Lane 1 shows multiple fluorescent bands indicating KIRA6 AfBP (10 μM) labelled proteins. Lane 2 shows selective depletion of KIRA6 AfBP labeled proteins following azide beads pull-down indicated by decrease of fluorescent signal but a comparable amount of proteins (Coomassie). Lane 3 shows efficient removal of the most abundant aspecific proteins (Coomassie) but absence of removal of labeled proteins (fluorescence). Lane 4 shows that KIRA6 AfBP protein binding is outcompeted by co-incubation with KIRA6 (10 μM) suggesting overlapping targets. (Table 1) Top 15 off-target candidates obtained by LC-MS/MS scored by total spectral counts. In the graphs values are presented as mean ± SD of at least n = 3 independent biological replicates and analyzed by two-tailed Student’s t-test,****p < 0.0001, ns not significant.