Extended Data Fig. 6: NPLOC4 and UFD1 are the primary cofactors involved in p97’s targeting of bacteria.
A. Percentage of p97 positive intracellular SPN associating with various cofactors, such as, NPLOC4, UFD1, FAF1 or NPLOC4 positive SPN associating with UFD1. n > 50 events. Data are means ± SD of N = 3, independent biological replicates. B. Structural illumination microscopy (SIM) showing the co-localization of SPN (Cyan) with UFD1 (White), NPLOC4 (Magenta) and p97 (Yellow). Scale bar, 1 μm. 3D reconstruction of the merged image using IMARIS is also represented. Bacteria were stained with DAPI, p97 was conjugated to GFP, NPLOC4 and UFD1 were stained with respective primary Ab, followed by Alexa Fluor 633 or 555 conjugated to secondary Ab. C. Immunoblot showcasing the accumulation of K48-Ub substrates in host cells expressing p97∆54-241. Host cells were stably transfected with truncated p97 variant and inability to clear ubiquitinated proteins was detected by immunoblotting with anti-K48-Ub Ab. Densitometric analysis of K48-Ub band normalized to β-actin levels revealed ~ 1.4-fold enrichment of K48-Ub in p97∆54-241 cells compared to p97WT. D. Quantitative analysis showcasing association of p97 with intracellular SPN in host cells expressing truncated p97∆54-241 compared to wild-type. n > 100 bacteria/coverslip. Data are means ± SD of N = 3, independent biological replicates. E. Fold change in intracellular burden of SPN in host cells expressing truncated p97∆54-241 compared to wild-type cells. Host cells are infected with SPN (MOI ~ 25) for 9 h and fold change in intracellular bacterial burden is depicted as ratio of intracellular bacterial CFU of the test to that of control at specified time point. Data are means ± SD of N = 6, independent biological replicates. Statistical significance was assessed by Two-tailed unpaired student’s t-test (nonparametric) for D, E, with correction for multiple comparisons. **P < 0.01; ***P < 0.001.
