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. 2021 Sep 10;41(1):15–25. doi: 10.1038/s41388-021-02006-x

Fig. 3. SAMMSON inhibition affects translation and mitochondrial function.

Fig. 3

A P32, XRN2 and MRPL13 were identified as SAMMSON interacting proteins by means of RIP-qPCR in OMM1. Data presented as the mean ± standard error (SE) of qPCR replicates. B Volcano plot depicting SAMMSON interacting proteins identified by ChIRP-MS (left). Probes targeting LacZ were included as a control (right). Mitochondrial ribosomal proteins (MRPs) are indicated on the graph. Significance was calculated using two-sided t-test. C Pathway enrichment analysis for ChIRP results showed participation of SAMMSON in mitochondrial translation pathways. D Representative images of WB-SUnSET analysis of UM cells treated with cycloheximide (translation inhibitor, positive control), scrambled ASO (NTC) (without puromycin, negative control), NTC ASO or ASO 3 (50 nM). Quantification of protein synthesis measured by calculating the intensity of the puromycin signal on WB. The individual data points (n = 3 biological replicates) and mean are presented. P values were calculated using two-way ANOVA with Tukey’s multiple comparisons test. E Representative images of WB-SUnSET analysis of UM cells treated with scrambled ASO (NTC) or ASO 3 (100 nM) followed by mitochondrial (mito) and cytosolic (cyto) fractionation. Quantification as described in D. The individual data points (n = 3 biological replicates) and mean are presented. P values were calculated using one-way ANOVA with Tukey’s multiple comparisons test. F Oxygen Consumption Rate (OCR) measurements over time after sequential injections of oligomycin, fluoro-carbonyl cyanide phenylhydrazone (FCCP) and rotenone/antimycin A in UM cell lines treated for 24 h with NTC ASO or ASO 3 (100 nM). Data are represented as the mean of three replicates ± s.d. Spare respiratory capacity (SRC) was obtained by subtracting the basal respiration from the maximal respiration. The individual data points and mean are presented. P values were calculated using unpaired two-tailed t-test. G 5,5’,6,6’-Tetraethylbenzimidazolyl-carbocyanine iodide (JC-1) staining in UM cells treated with NTC ASO or ASO 3 (100 nM) (magnification of the images x400). Quantification of the electric membrane potential (ΔΨ) as the red over green fluorescence of 10 random selected fields. The individual data points and mean are presented. P values were calculated using unpaired two-tailed t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.