Fig. 2: Interaction between Serpinb9 and SMS prevents SMS migration into the lysosome.
From: Targeting spermine metabolism to overcome immunotherapy resistance in pancreatic cancer
a KEGG enrichment analysis of differentially expressed proteins in Serpinb9 KD vs. Serpinb9 NC KPC cells (n = 3 biologically independent samples). b Western blotting analysis of proteins within three metabolic pathways in Serpinb9 NC and KD KPC cells. c Correlation analysis of Serpinb9 and SMS expression in PDAC tissue microarrays (n = 148 patients). d Co-IP and western blotting analysis showing the interaction between HA-tagged SMS and Flag-tagged Serpinb9. e GST-pull-down assay of SMS-His and Serpinb9-GST proteins. f Duolink immunofluorescence images of Serpinb9-SMS interaction in KPC and BxPC-3 cells; red dots indicate interaction. Scale bars: 10 μm. g The predicted complex of the Serpinb9 (blue) and SMS (pink), showing the relative binding free energy difference (ΔΔG) from alanine scanning of crucial residues in Serpinb9 (n = 10 biologically independent samples). h Co-IP and quantification of SMS binding to wild-type or mutant Serpinb9 (F336A; E306A/R307A/D308A). i SMS stability in Serpinb9 NC and KD KPC cells after CHX treatment (200 μg/mL). j SMS levels in KPC cells treated with MG132 (5 μM, 12 h) or CQ (50 μM, 12 h). k Representative images and quantification of lysosomes in KPC cells (n = 5 biologically independent samples). Scale bars: 0.5 μm. l SMS distribution in Serpinb9 NC vs. KD cells via confocal microscopy. Scale bar: 25 μm. m SMS expression in CQ-treated NC and KD cells. n Co-IP of HA-tagged SMS in KPC cells. o SMS interaction with Lamp2A in Serpinb9 NC vs. KD cells. p Schematic and binding free energy of Serpinb9 and Lamp2A competitively binding to SMS (n = 50 biologically independent samples). For h, i, j, m, o data were presented as mean ± S.D., n = 3 biologically independent samples. For k data were presented as mean ± S.D., n = 5 biologically independent samples. For g data were presented as mean ± S.D., n = 10 biologically independent samples. For p, data were presented as mean ± SEM, n = 50 independent samples. For c Spearman correlations and P values were calculated using a two-sided Spearman’s test. Statistical significance was determined using two-sided unpaired Student’s t-test or one-way ANOVA followed by Tukey’s post hoc test. NS, no significance. Source data are provided as a Source Data file.
