Fig. 7: Target identification and validation disclose reticulon 4 as responsible molecular target. | Cell Death Discovery

Fig. 7: Target identification and validation disclose reticulon 4 as responsible molecular target.

From: Neocarzilin A induces apoptosis and mitochondrial disturbance by targeting reticulon 4-mediated endoplasmic reticulum stress

Fig. 7

A Chemical structure of target probe NC-4, drawn with ChemDraw 20.0 B Target identification by ABPP with NC-4 target probe in HeLa cells, C respective competition experiment. D, E Representative images of immunofluorescence staining of HeLa cells D incubated with NC-4 and clicked to Alexa FluorTM 647 azide or E treated with NCA or DMSO as indicated and D, E immunostained for Rtn4. D, E Nuclei shown in blue, Rtn 4 in green, E Alexa FluorTM 647 coupled NC-4 in red (D, E scale bar 25 µm), n = 3. F Molecular docking of nogo-66 and NCA by UCSF Chimera and AutoDock Vina plugin. Contact sites are marked yellow, hydrogen bond is marked red, with interacting residues labeled. Flow cytometry experiments in Rtn4 knockdown HeLa cells to determine ROS generation (G), mitochondrial membrane potential (H), and apoptosis induction (I) upon NCA or DMSO treatment as indicated. Data are presented A,B in volcano plots, C box and whiskers (min to max), F–H bar graphs as mean ± SD, B, C, G n = 4, D, H, I n = 3. Statistical significance was analyzed by two-way ANOVA with Tukey’s posttest (ns not significant, **P < 0.01, ***P < 0.001).

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