Figure 2A . KRI1 localized to the nucleoli in MCF - 7 cells ( HPA043574 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . B . GPATCH4 localized to the nucleoli in U - 2 OS cells ( HPA054319 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . C . FOXL2NB localized to the fibrillar center in SiHa cells ( HPA061017 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . D . FAM71E1 localized to the fibrillar center in U - 2 OS cells ( HPA048111 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . E . LRRC23 localized to the nucleoli in U - 2 OS cells ( HPA057533 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . F . ZNF853 localized to the nucleoli in U - 2 OS cells ( HPA067690 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm . G . METTL5 localized to the nucleoli in U - 2 OS cells ( HPA038223 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI in blue and the reference marker of microtubules in red . Scale bar 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. KRI1 localized to the nucleoli in MCF-7 cells (HPA043574). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.B. GPATCH4 localized to the nucleoli in U-2 OS cells (HPA054319). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.C. FOXL2NB localized to the fibrillar center in SiHa cells (HPA061017). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.D. FAM71E1 localized to the fibrillar center in U-2 OS cells (HPA048111). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.E. LRRC23 localized to the nucleoli in U-2 OS cells (HPA057533). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.F. ZNF853 localized to the nucleoli in U-2 OS cells (HPA067690). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.G. METTL5 localized to the nucleoli in U-2 OS cells (HPA038223). Data information: Protein of interest is shown in green, nuclear marker DAPI in blue and the reference marker of microtubules in red. Scale bar 10 µm.


Figure 3B . IF staining of MKI67 shows localization to nucleoli rim in U - 251 cells ( CAB000058 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm . C . IF staining of GNL3 shows localization to nucleoli rim in U - 2 OS cells ( HPA036743 ) . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm . D . HEK 293T cells expressing endogenous levels of N - terminus mNG - MKI67 show a faint nucleoli rim localization , although still visible . White arrows indicate cells where the rim could be seen . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm . E . Fixed HEK 293T cells expressing mNG - tagged MKI67 show a clear nucleoli rim localization . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm . F . HEK 293T cells expressing endogenous levels of C - terminus tagged GNL3 show nucleoli rim localization . White arrows indicate cells where the rim could be seen . Data information : Protein of interest is shown in green , nuclear marker DAPI / Hoechst in blue and microtubules reference marker in red . Scale bar 10 µm .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3B. IF staining of MKI67 shows localization to nucleoli rim in U-251 cells (CAB000058). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.C. IF staining of GNL3 shows localization to nucleoli rim in U-2 OS cells (HPA036743). Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.D. HEK 293T cells expressing endogenous levels of N-terminus mNG-MKI67 show a faint nucleoli rim localization, although still visible. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.E. Fixed HEK 293T cells expressing mNG-tagged MKI67 show a clear nucleoli rim localization. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.F. HEK 293T cells expressing endogenous levels of C-terminus tagged GNL3 show nucleoli rim localization. White arrows indicate cells where the rim could be seen. Data information: Protein of interest is shown in green, nuclear marker DAPI/Hoechst in blue and microtubules reference marker in red. Scale bar 10 µm.


Figure 4A . MKI67 ( HPA000451 ) . B . NOC2L ( HPA044258 ) . C . EMG1 ( HPA039304 ) . D . BMS1 ( HPA043081 ) . E . BRIX1 ( HPA039614 ) . F . RSL1D1 ( HPA043483 ) . Data information : Protein of interest is shown in green , microtubules in red and DAPI in blue . Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells . Scale bar 10 μm .

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Figure 4A. MKI67 (HPA000451). B. NOC2L (HPA044258). C. EMG1 (HPA039304). D. BMS1 (HPA043081). E. BRIX1 (HPA039614). F. RSL1D1 (HPA043483). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


Figure 5C - E . IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered . Protein of interest shown in green and DAPI in blue . Scale bar 10 µm . C . CCDC86 ( HPA041117 ) , 100 % predicted disorder level . D . RRP15 ( HPA024639 ) , 87 % predicted disorder level . E . PRR19 ( HPA070350 ) , 67 % predicted disorder level .

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Figure 5C-E. IF stainings of previously uncharacterized mitotic chromosome proteins predicted to be highly disordered. Protein of interest shown in green and DAPI in blue. Scale bar 10 µm. C. CCDC86 (HPA041117), 100% predicted disorder level. D. RRP15 (HPA024639), 87% predicted disorder level. E. PRR19 (HPA070350), 67% predicted disorder level.


Figure 6A . MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category . B - F . IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase , showing a typical late recruitment expression pattern . B . NOL12 ( HPA003547 ) . C . TIGD1 ( HPA041717 ) . D . BYSL ( HPA031219 ) . E . ACTL6B ( HPA045126 ) . F . ZNF202 ( HPA059229 ) . Data information : Protein of interest is shown in green , microtubules in red and DAPI in blue . Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells . Scale bar 10 μm .

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Figure 6A. MKI67 is detected on the chromosomes throughout all mitotic phases and exemplifies proteins in the early recruitment category. B-F. IF stainings of proteins where localization to mitotic chromosomes is seen after metaphase, showing a typical late recruitment expression pattern. B. NOL12 (HPA003547). C. TIGD1 (HPA041717). D. BYSL (HPA031219). E. ACTL6B (HPA045126). F. ZNF202 (HPA059229). Data information: Protein of interest is shown in green, microtubules in red and DAPI in blue. Images of interphase cells were acquired from a different experiment and staining intensities cannot be compared between interphase and mitotic cells. Scale bar 10 μm.


Figure 7A - B . GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells . A . GNL3 ( HPA036743 ) . Median overlapKO = 0 . 539 , median overlapWT = 0 . 795 ( P = 9 . 34x10 - 9 ) and median Pearson correlationKO = - 0 . 335 , median Pearson correlationWT = 0 . 134 ( P = 3 . 58x10 - 6 ) . B . PES1 ( HPA040210 ) . Median overlapKO = 0 . 516 , median overlapWT = 0 . 755 ( P = 1 . 77x10 - 13 ) and median Pearson correlationKO = - 0 . 293 , median Pearson correlationWT = 0 . 129 ( P = 4 . 06x10 - 13 ) . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used . C . No protein aggregates were observed in MKI67 KO cells for PWP1 ( HPA038708 ) , despite being localized to mitotic chromosomes in WT cells . Median overlapKO = 0 . 523 , median overlapWT = 0 . 861 ( P = 1 . 05x10 - 13 ) and median Pearson correlationKO = - 0 . 190 , median Pearson correlationWT = 0 . 515 ( P = 8 . 71x10 - 15 ) . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used . Box limits mark 1st and 3rd quantile , whiskers the 1 . 5x interquartile range and center line the median . D - E . Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells . White arrows indicate where protein aggregates can be seen . D . DENND4A ( HPA065343 ) . Median overlapKO = 0 . 671 , median overlapWT = 0 . 803 ( P = 1 . 02x10 - 4 ) and median Pearson correlationKO = - 0 . 224 , median Pearson correlationWT = 0 . 236 ( P = 1 . 91x10 - 5 ) . E . NOL12 ( HPA003547 ) . Median overlapKO = 0 . 468 , median overlapWT = 0 . 820 ( P = 1 . 01x10 - 17 ) and median Pearson correlationKO = - 0 . 318 , median Pearson correlationWT = 0 . 381 ( P = 1 . 81x10 - 10 ) . White arrow indicates metaphase cell . Data information : Protein of interest is shown in green and DAPI in blue . Scale bar 10 μm . For the colocalization analysis data ; biological replicate 1 highlighted in grey and biological replicate 2 in green . Both the overlap and Pearson correlation between DAPI / Protein staining was measured and a two - tailed unpaired Wilcoxon test was used . Box limits mark 1st and 3rd quantile , whiskers the 1 . 5x interquartile range and center line the median .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7A-B. GNL3 and PES1 are localizing to protein aggregates around the chromosomes in the MKI67 KO cells. A. GNL3 (HPA036743). Median overlapKO = 0.539, median overlapWT = 0.795 (P = 9.34x10-9) and median Pearson correlationKO = -0.335, median Pearson correlationWT = 0.134 (P = 3.58x10-6). B. PES1 (HPA040210). Median overlapKO = 0.516, median overlapWT = 0.755 (P = 1.77x10-13) and median Pearson correlationKO = -0.293, median Pearson correlationWT = 0.129 (P = 4.06x10-13). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used.C. No protein aggregates were observed in MKI67 KO cells for PWP1 (HPA038708), despite being localized to mitotic chromosomes in WT cells. Median overlapKO = 0.523, median overlapWT = 0.861 (P = 1.05x10-13) and median Pearson correlationKO = -0.190, median Pearson correlationWT = 0.515 (P = 8.71x10-15). Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.D-E. Late recruited proteins do not follow the same recruitment pattern in the MKI67 KO cells compared to WT HeLa cells. White arrows indicate where protein aggregates can be seen. D. DENND4A (HPA065343). Median overlapKO = 0.671, median overlapWT = 0.803 (P = 1.02x10-4) and median Pearson correlationKO = -0.224, median Pearson correlationWT = 0.236 (P = 1.91x10-5). E. NOL12 (HPA003547). Median overlapKO = 0.468, median overlapWT = 0.820 (P = 1.01x10-17) and median Pearson correlationKO = -0.318, median Pearson correlationWT = 0.381 (P = 1.81x10-10). White arrow indicates metaphase cell. Data information: Protein of interest is shown in green and DAPI in blue. Scale bar 10 μm. For the colocalization analysis data; biological replicate 1 highlighted in grey and biological replicate 2 in green. Both the overlap and Pearson correlation between DAPI/Protein staining was measured and a two-tailed unpaired Wilcoxon test was used. Box limits mark 1st and 3rd quantile, whiskers the 1.5x interquartile range and center line the median.


Figure 1A - B . Wild - type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT , combined and quantified by LC - MS , using the published 2D - TPP protocol ( Mateus et al . , 2018 ) . Shown are volcano plots of two replicates depicting changes in : protein abundance ( A ) and thermostability ( B ) . A local FDR ( false discovery rate ) < 0 . 01 was set as a threshold for significance . Highlighted proteins : outer membrane proteins ( OMPs , light green ) , β - barrel assembly machinery ( BAMs , red ) , PG synthases / regulators ( green ) , PG hydrolases and regulators ( blue ) and the Tol - Pal complex ( violet ) . All other proteins were colored grey and not labelled to increase the plot clarity . C - D . Affinity chromatography with immobilised NlpI . Membrane extracts from E . coli were incubated in low and high salt binding conditions ( 50 and 400 mM NaCl , respectively ) , and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label - free LC - MS analysis . The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control , versus the log10 p - value , in low ( 4 replicates ) ( C ) and high ( 2 replicates ) ( D ) salt . Highlighted points are all interactions with PG enzymes and their regulators , as well as members of the divisome . All other proteins were colored grey and not labelled to increase the plot clarity ; many were non - physiological interactions with abundant cytoplasmic proteins .

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Figure 1A-B. Wild-type and ΔnlpI cells were heated at a range of temperatures and the soluble components were labelled by TMT, combined and quantified by LC-MS, using the published 2D-TPP protocol (Mateus et al., 2018). Shown are volcano plots of two replicates depicting changes in: protein abundance (A) and thermostability (B). A local FDR (false discovery rate) <0.01 was set as a threshold for significance. Highlighted proteins: outer membrane proteins (OMPs, light green), β-barrel assembly machinery (BAMs, red), PG synthases/regulators (green), PG hydrolases and regulators (blue) and the Tol-Pal complex (violet). All other proteins were colored grey and not labelled to increase the plot clarity.C-D. Affinity chromatography with immobilised NlpI. Membrane extracts from E. coli were incubated in low and high salt binding conditions (50 and 400 mM NaCl, respectively), and then eluted with 1 M NaCl or 2 M NaCl to identify possible interaction partners by label-free LC-MS analysis. The plot shows the log2 fold enriched proteins when compared to those eluted from a parallel empty column control, versus the log10 p-value, in low (4 replicates) (C) and high (2 replicates) (D) salt. Highlighted points are all interactions with PG enzymes and their regulators, as well as members of the divisome. All other proteins were colored grey and not labelled to increase the plot clarity; many were non-physiological interactions with abundant cytoplasmic proteins.


Figure 2A . Dissociation constants for interactions between NlpI with MepM , MepS , PBP4 , PBP7 as determined by Microscale thermophoresis ( MST ) . The values are mean ± standard deviation of three independent experiments . B . MepS - NlpI interaction by MST as an example plot for Fig 2A . MST curve plotted is the mean data + / - SD of three independent experiments . Fl , fluorescently labelled ; FNorm , normalized fluorescence . NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI - PBP4 ( C ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 ) NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI - PBP7 ( D ) complexes by a fixed concentration MST assay . Values are mean ± standard deviation of 3 - 6 independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 )

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2A. Dissociation constants for interactions between NlpI with MepM, MepS, PBP4, PBP7 as determined by Microscale thermophoresis (MST). The values are mean ± standard deviation of three independent experiments. B. MepS-NlpI interaction by MST as an example plot for Fig 2A. MST curve plotted is the mean data +/- SD of three independent experiments. Fl, fluorescently labelled; FNorm, normalized fluorescence.NlpI has different binding sites for MepS and PBP4 as shown by the ability of labelled MepS to bind preformed NlpI-PBP4 (C) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)NlpI has different binding sites for MepS and PBP7 as shown by the ability of labelled MepS to bind preformed NlpI-PBP7 (D) complexes by a fixed concentration MST assay. Values are mean ± standard deviation of 3-6 independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)


Figure 3A . HPLC - based PG digestion assay representing EPase activity . The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods . MepM and PBP4 were incubated with sacculi , whilst MepS and PBP7 were incubated with soluble muropeptides , both from E . coli MC1061 , respectively . Values are mean ± standard deviation of three independent experiments . To calculate significance , the data were fit using a linear model . Calculated means were compared using Tukey ' s HSD test , resulting in p - values corrected for multiple testing . Relevant p - values are highlighted directly in the figure ( * < 0 . 05 ; * * < 0 . 01 , * * * < 0 . 001 ) B . Genetic interactions of nlpI with EPase genes . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants . An example of a 384 plate is shown . Double mutants were made twice , swapping the resistance markers to the two single mutants . Colony integral opacity was quantified as a fitness readout , using the image analysis software Iris ( Kritikos et al . , 2017 ) . Bar plots shows the averaged values of 2 biological experiments , each having 96 technical replicates ( i = 2 , n = 192 ) . The error bars represent the 95 % confidence interval . C . nlpI deletion changes the morphology of EPase - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , dacB encodes PBP4 , pbpG encode PBP7 , mepM encodes MepM , meps encodes MepS . D . Inducible mepS expression system ( pBAD30 ) strongly overproduces MepS . Strains were grown in LB at 30 ° C and cells were collected at OD600 ≈ 0 . 4 . The level of MepS contained in the membrane fraction was detected using purified anti - MepS antibody . E . Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy . Cultures were grown in LB at 30 ° C and aliquots of culture were taken at OD600 ≈ 0 . 1 . The scale bar equals 5 μm . F . MepS level modulates cell width . The graph shows the distribution of mean cell width for each cell , with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w . G . Relative fitness of ΔnlpI , ΔmepS and ΔnlpIΔmepS mutants . Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10 % sucrose , or LB agar plates containing 0 mM or 500 mM NaCl . Plates were incubated for 12 h at 37 ° C . Each plate contained 384 colonies , 96 from the wildtype , single mutants and double mutants . H . Cells of wildtype ( WT ) , ΔnlpI , ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity . CPRG ( yellow ) cannot penetrate intact Gram - negative envelopes . Its conversion by intracellular β - galactosidase to CPR ( red ) indicates loss of envelope integrity .

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Figure 3A. HPLC-based PG digestion assay representing EPase activity. The graph shows the relative percentage of the muropeptide TetraTetra present at the end of the incubation period for each protein as described in materials and methods. MepM and PBP4 were incubated with sacculi, whilst MepS and PBP7 were incubated with soluble muropeptides, both from E. coli MC1061, respectively. Values are mean ± standard deviation of three independent experiments. To calculate significance, the data were fit using a linear model. Calculated means were compared using Tukey's HSD test, resulting in p-values corrected for multiple testing. Relevant p-values are highlighted directly in the figure (*<0.05; **<0.01, ***<0.001)B. Genetic interactions of nlpI with EPase genes. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates and incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants. An example of a 384 plate is shown. Double mutants were made twice, swapping the resistance markers to the two single mutants. Colony integral opacity was quantified as a fitness readout, using the image analysis software Iris (Kritikos et al., 2017). Bar plots shows the averaged values of 2 biological experiments, each having 96 technical replicates (i=2, n=192). The error bars represent the 95% confidence interval.C. nlpI deletion changes the morphology of EPase-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Above the graph are representative images of cells lacking MepS or PBP4 in combination with a deletion of NlpI. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, dacB encodes PBP4, pbpG encode PBP7, mepM encodes MepM, meps encodes MepS.D. Inducible mepS expression system (pBAD30) strongly overproduces MepS. Strains were grown in LB at 30°C and cells were collected at OD600≈0.4. The level of MepS contained in the membrane fraction was detected using purified anti-MepS antibody.E. Visualization of the effect of MepS absence or its overexpression on cell width by phase contrast microscopy. Cultures were grown in LB at 30°C and aliquots of culture were taken at OD600≈0.1. The scale bar equals 5 μm.F. MepS level modulates cell width. The graph shows the distribution of mean cell width for each cell, with n corresponding to the number of cells measured for each strain and the median width for the population being indicated by a dotted line and referred to as w.G. Relative fitness of ΔnlpI, ΔmepS and ΔnlpIΔmepS mutants. Strains were arrayed using a Rotor HDA replicator on Lennox LB agar plates supplemented 10% sucrose, or LB agar plates containing 0 mM or 500 mM NaCl. Plates were incubated for 12 h at 37°C. Each plate contained 384 colonies, 96 from the wildtype, single mutants and double mutants.H. Cells of wildtype (WT), ΔnlpI, ΔmepS and ΔnlpIΔmepS containing multicopy plasmids with lacZ were grown onto CPRG indicator agar to assay envelope integrity. CPRG (yellow) cannot penetrate intact Gram-negative envelopes. Its conversion by intracellular β-galactosidase to CPR (red) indicates loss of envelope integrity.


Figure 4A . Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37 ° C and immunolabelled with specific antibodies against NlpI . Scale bar equals 2 µm . B . Genetic interactions of NlpI with PG machineries . An example of a 384 plate is shown . Bar plots shows the averaged values of 2 experiments ( i = 2 , n = 192 ) . Error bars denote the 95 % confidence interval of the mean . C . NlpI deletion exacerbates the morphological defects of the PBP1B / LpoB - mutant strains . The graph shows the cell width of single and double deletions strains ( 800 < n < 2000 cells ) . The box has a medium between 25 and 75 % . The whiskers with the upper and lower vertical line indicating the 95 and 5 % . The dots are individual points outside the 5 and 95 % range . Representative images of the strains are shown above the graph . The scale bar equals 2 µm . Gene encoding protein legend : nlpI encodes NlpI , mrcA encodes PBP1A , lpoA encodes LpoA , mrcB encodes PBP1B , lpoB encodes LpoB . D . Dissociation constants for interactions between PBP1A and LpoA with NlpI , MepM , MepS , PBP4 and PBP7 as determined by MST . The values are mean ± standard deviation of three independent experiments . 1PBP1A was used as fluorescently labelled protein in all assays . 2LpoA was used as unlabelled ligand in all combinations , except with MepM and PBP4 .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4A. Phase contrast image and corresponding fluorescence SIM image of BW25113 cells that have been grown in LB at 37°C and immunolabelled with specific antibodies against NlpI. Scale bar equals 2 µm.B. Genetic interactions of NlpI with PG machineries. An example of a 384 plate is shown. Bar plots shows the averaged values of 2 experiments (i=2, n=192). Error bars denote the 95% confidence interval of the mean.C. NlpI deletion exacerbates the morphological defects of the PBP1B/LpoB-mutant strains. The graph shows the cell width of single and double deletions strains (800 < n < 2000 cells). The box has a medium between 25 and 75%. The whiskers with the upper and lower vertical line indicating the 95 and 5%. The dots are individual points outside the 5 and 95% range. Representative images of the strains are shown above the graph. The scale bar equals 2 µm. Gene encoding protein legend: nlpI encodes NlpI, mrcA encodes PBP1A, lpoA encodes LpoA, mrcB encodes PBP1B, lpoB encodes LpoB.D. Dissociation constants for interactions between PBP1A and LpoA with NlpI, MepM, MepS, PBP4 and PBP7 as determined by MST. The values are mean ± standard deviation of three independent experiments. 1PBP1A was used as fluorescently labelled protein in all assays. 2LpoA was used as unlabelled ligand in all combinations, except with MepM and PBP4.


Figure 5A - B . PBP4 has different interaction sites for PBP1A / LpoA and NlpI as shown by a single concentration MST assay . Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4 , NlpI or PBP4 - NlpI . Values are mean ± standard deviation of three independent experiments .

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Figure 5A-B. PBP4 has different interaction sites for PBP1A/LpoA and NlpI as shown by a single concentration MST assay. Plots show the FNorm or fluorescence values of fluorescently labelled PBP1A or LpoA with or without PBP4, NlpI or PBP4-NlpI. Values are mean ± standard deviation of three independent experiments.


Figure 6A . Estimated number of molecules and molarity of PBP1a / LpoA and EPases . 1Numbers obtained by ribosomal profiling in rich growth medium ( Li et al . , 2014 ) . 2Concentration of monomer . 3Decreases in the presence of NlpI ( Singh et al . , 2015 ) . 4 " no change detected " . The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3 . 33 × 10 - 16 L , where 1 molecule corresponds to 5 nM .

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Figure 6A. Estimated number of molecules and molarity of PBP1a/LpoA and EPases. 1Numbers obtained by ribosomal profiling in rich growth medium (Li et al., 2014). 2Concentration of monomer. 3Decreases in the presence of NlpI (Singh et al., 2015). 4"no change detected". The periplasmic concentrations of proteins were estimated for a cell with periplasmic volume of 3.33 ×10-16 L, where 1 molecule corresponds to 5 nM.


Figure 1 Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α - V5 tagged . Cells were fixed and stained with anti - calnexin ( green ) or anti - V5 ( red ) . An overlay of the cells is presented in the third panel . Bar = 10 µm . Whole cell lysates from HEK293T cells , with or without stable expression of ATF6α ( as indicated ) were immunoisolated with mouse anti - V5 . RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3 . Immunoisolated material was separated by SDS - PAGE under reducing conditions followed by western blotting to determine the co - isolation of ER proteins with ATF6α . Ig - HC ( anti - BiP blot ) indicates immunoglobulin heavy chains . Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated ( - ) or treated ( + ) with 5 µM thapsigargin ( TG ) for 1 h . THBS4 was transiently transfected into cells in the experiment depicted in panel 2 . ATF6α was either immunoisolated with anti - V5 ( panel 1 and 3 ) or anti - HA ( panel 2 ) . For the experiment in panel 3 the cells were cross - linked prior to immunoisolation . Samples were separated by SDS - PAGE under reducing conditions followed by western blots to determine co - isolation of endogenous PDIR , ERp18 or exogenously expressed THBS4 with ATF6α . HC indicates immunoglobulin heavy chain .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1 Immunofluorescence microscopy of HEK293T exogenously expressing ATF6α-V5 tagged. Cells were fixed and stained with anti-calnexin (green) or anti-V5 (red). An overlay of the cells is presented in the third panel. Bar = 10 µm.  Whole cell lysates from HEK293T cells, with or without stable expression of ATF6α (as indicated) were immunoisolated with mouse anti-V5. RCN1 was transiently transfected into both cell lines in the experiment depicted in panel 3. Immunoisolated material was separated by SDS-PAGE under reducing conditions followed by western blotting to determine the co-isolation of ER proteins with ATF6α. Ig-HC (anti-BiP blot) indicates immunoglobulin heavy chains. Whole cell lysates from HEK293T cells stably expressing ATF6α were either untreated (-) or treated (+) with 5 µM thapsigargin (TG) for 1 h. THBS4 was transiently transfected into cells in the experiment depicted in panel 2. ATF6α was either immunoisolated with anti-V5 (panel 1 and 3) or anti-HA (panel 2). For the experiment in panel 3 the cells were cross-linked prior to immunoisolation. Samples were separated by SDS-PAGE under reducing conditions followed by western blots to determine co-isolation of endogenous PDIR, ERp18 or exogenously expressed THBS4 with ATF6α. HC indicates immunoglobulin heavy chain.


Figure 2HEK293T cells stably expressing ATF6α were either left untransfected ( UT ) or transfected with substrate - trapping mutants of PDI oxidoreductases . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots confirm mixed - disulfide complexes between ATF6α and ERp18 , ERp57 , and PDIR indicated with arrows ( lanes 2 , 4 , 5 ) . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain ( ATF6 DM ) were transfected with PDI substrate - trapping mutants . ATF6α was immunoisolated with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions , and ATF6α detected with rabbit anti - HA .

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Figure 2HEK293T cells stably expressing ATF6α were either left untransfected (UT) or transfected with substrate-trapping mutants of PDI oxidoreductases. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots confirm mixed-disulfide complexes between ATF6α and ERp18, ERp57, and PDIR indicated with arrows (lanes 2, 4, 5). M, D and O refer to ATF6α monomer, dimer and oligomer respectively. Whole cell lysates of HEK293T cells expressing an ATF6α mutant containing cysteine to alanine mutations in the lumenal domain (ATF6 DM) were transfected with PDI substrate-trapping mutants. ATF6α was immunoisolated with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions, and ATF6α detected with rabbit anti-HA.


Figure 3 HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µg / ml tunicamycin to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments . HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 20 µM MG132 to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . Blots indicate that mixed - disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress . M , D and O refer to ATF6α monomer , dimer and oligomer respectively . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes . * * indicates hypoglycosylated ATF6α . Data shown are representative of three independent experiments . HEK293T cells were co - transfected with ATF6α S1P and PDI substrate - trapping mutants and either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . Samples from ( A ) were rerun and probed with anti - sera raised against respective PDI enzymes as indicated ( D ) . * indicates positions of the ATF6α - PDI enzymes mixed - disulfide complexes .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3 HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.  HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µg/ml tunicamycin to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.  HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 20 µM MG132 to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. Blots indicate that mixed-disulfide complexes between ATF6α and PDI enzymes are regulated by ER stress. M, D and O refer to ATF6α monomer, dimer and oligomer respectively. *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes. ** indicates hypoglycosylated ATF6α. Data shown are representative of three independent experiments.  HEK293T cells were co-transfected with ATF6α S1P and PDI substrate-trapping mutants and either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. Samples from (A) were rerun and probed with anti-sera raised against respective PDI enzymes as indicated (D). *indicates positions of the ATF6α-PDI enzymes mixed-disulfide complexes.


Figure 4 HEK293T cells were co - transfected with the ERp18 substrate - trapping mutant and the indicated ATF6α constructs . Cell were allowed to recover for 24 h post - transfection before being either left untreated ( - ) or treated ( + ) for 1 h with 5 µM TG to induce ER stress . ATF6α was immunoisolated from cell lysates with mouse anti - ATF6α , separated by SDS - PAGE under non - reducing conditions and ATF6α detected by western blotting using rabbit anti - HA . The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress . M , D and O refer to ATF6 monomer , dimer and oligomer respectively . * indicates positions of the ATF6 - ERp18 mixed - disulfide complexes . HEK293T cells were co - transfected with GFP - ATF6α and ERp18 trapping mutant constructs as indicated . GFP - ATF6α complexes were immunoisolated using GFP - trap . Immunoisolated material was separated by SDS - PAGE under non - reducing conditions and silver stained to detect ATF6α disulfide - linked complexes . MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18 , whilst bands marked as monomer ( M ) and oligomer ( O ) contain ATF6α only .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4 HEK293T cells were co-transfected with the ERp18 substrate-trapping mutant and the indicated ATF6α constructs. Cell were allowed to recover for 24 h post-transfection before being either left untreated (-) or treated (+) for 1 h with 5 µM TG to induce ER stress. ATF6α was immunoisolated from cell lysates with mouse anti-ATF6α, separated by SDS-PAGE under non-reducing conditions and ATF6α detected by western blotting using rabbit anti-HA. The blot confirms that ATF6α C618A forms a stable complex with ERp18 trapping mutant following ER stress. M, D and O refer to ATF6 monomer, dimer and oligomer respectively. *indicates positions of the ATF6-ERp18 mixed-disulfide complexes.  HEK293T cells were co-transfected with GFP-ATF6α and ERp18 trapping mutant constructs as indicated. GFP-ATF6α complexes were immunoisolated using GFP-trap. Immunoisolated material was separated by SDS-PAGE under non-reducing conditions and silver stained to detect ATF6α disulfide-linked complexes. MS analyses confirmed that protein bands indicated by black arrows are a complex comprising ATF6α and ERp18, whilst bands marked as monomer (M) and oligomer (O) contain ATF6α only.


Figure 5CRISPR / Cas9 - based knockout of ERp18 in HEK293T cells expressing ATF6α . ERp18 levels in control ( WT ) and knockout ( KO ) cells were detected with an antibody to ERp18 . qPCR analysis of UPR target genes in the absence of ER stress . Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type . Error bars represent ± standard deviation for three independent experiments . qPCR analysis of UPR target genes following ER stress . Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis . mRNA levels for Grp78 , Grp94 , ATF6α , ATF4 , XBP1s ( spliced form of XBP1 ) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls . Error bars represent ± standard deviation for three independent experiments . Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised BiP levels from ( D ) were quantified . Error bars represent ± standard deviation for three independent experiments . Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin ( TG ) . Panel 1 compares wild type and ERp18 KO cells as indicated . Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 ( RV ) . Normalised ERp72 levels from ( F ) were quantified . Error bars represent ± standard deviation for three independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5CRISPR/Cas9-based knockout of ERp18 in HEK293T cells expressing ATF6α. ERp18 levels in control (WT) and knockout (KO) cells were detected with an antibody to ERp18. qPCR analysis of UPR target genes in the absence of ER stress. Wild type and ERp18 knockout HEK293T cells were untreated prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type. Error bars represent ± standard deviation for three independent experiments.   qPCR analysis of UPR target genes following ER stress. Wild type and ERp18 knockout HEK293T cells were treated with 1 µM TG for approximately 16 h prior to isolation of mRNA for qPCR analysis. mRNA levels for Grp78, Grp94, ATF6α, ATF4, XBP1s (spliced form of XBP1) and ERp72 were normalized to GAPDH and then ERp18 KO levels compared to wild type controls. Error bars represent ± standard deviation for three independent experiments.    Levels of BiP protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised BiP levels from (D ) were quantified. Error bars represent ± standard deviation for three independent experiments.    Levels of ERp72 protein in wild type or ERp18 KO cells before and after ER stress induction with thapsigargin (TG). Panel 1 compares wild type and ERp18 KO cells as indicated. Panel 2 compares ERp18 KO cells to ERp18 KO cells that have been transfected with ERp18 (RV).   Normalised ERp72 levels from (F) were quantified. Error bars represent ± standard deviation for three independent experiments.


Figure 6 A , B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times . Cells were then subject to differential centrifugation to obtain membrane ( A ) and nuclear fractions ( B ) which were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of full - length ATF6α ( ATF6 - FL ) , cleaved membrane - associated ATF6α ( ATF6 - P ) and nuclear translocated S2P cleaved ATF6α ( ATF6 - N ) are as indicated . HDAC2 was used as a nuclear marker . ( C ) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α . Samples were separated by SDS - PAGE and ATF6α was detected by western blotting . HC indicates immunoglobulin heavy chains . ( D , E ) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α , either untreated ( - ) or treated ( + ) with 30 µM PF429242 ( S1P inhibitor ) prior to and following induction of ER stress with 10 mM DTT . The positions of ATF6 - P , ATF6 - M and ATF6 - N are as indicated . The unprocessed ER - localised ATF6α ( designated ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( designated G ) are indicated . HDAC2 was included as a nuclear marker . The blots in ( D , E ) confirm that ATF6 - P is produced independently of S1P and also the absence of ATF6α processing to ATF6 - N in the presence of the S1P inhibitor .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6 A, B Wild type and ERp18 KO cells stably expressing ATF6α were treated with 5 µM TG for the indicated times. Cells were then subject to differential centrifugation to obtain membrane (A) and nuclear fractions (B) which were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of full-length ATF6α (ATF6-FL), cleaved membrane-associated ATF6α (ATF6-P) and nuclear translocated S2P cleaved ATF6α (ATF6-N) are as indicated. HDAC2 was used as a nuclear marker.  (C) ATF6α was immunoisolated from wild type or ERp18 KO cells expressing ATF6α. Samples were separated by SDS-PAGE and ATF6α was detected by western blotting. HC indicates immunoglobulin heavy chains.  (D, E) Blots of membrane and nuclear fractions respectively from ERp18 KO cells overexpressing ATF6α, either untreated (-) or treated (+) with 30 µM PF429242 (S1P inhibitor) prior to and following induction of ER stress with 10 mM DTT. The positions of ATF6-P, ATF6-M and ATF6-N are as indicated. The unprocessed ER-localised ATF6α (designated ER) and the O-linked glycan modified Golgi-translocated ATF6α (designated G) are indicated. HDAC2 was included as a nuclear marker. The blots in (D, E) confirm that ATF6-P is produced independently of S1P and also the absence of ATF6α processing to ATF6-N in the presence of the S1P inhibitor.


Figure 7 HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor . Cell lysates were prepared , separated by SDS - PAGE and then probed with anti - ATF6α . Blot reveals the presence of O - linked glycan modified Golgi - translocated ATF6 ( designated G ) , which is absent for the D564G mutant , confirming a lack of ER to Golgi trafficking . Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated ( - ) or treated ( + ) with 5 µg / ml brefeldin A for1 h prior to cell lysis . The anti - ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6 - M , only in the brefeldin A treated cells . Nuclear fractions confirm the presence of S2P cleavage product , ATF6 - N following brefeldin A treatment . Anti - HDAC2 was used as a loading control for the nuclear fraction .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 7 HEK293T cells stably overexpressing wild type ATF6α or a D564G mutant were treated with 10 mM DTT as indicated to induce ER stress in the presence of the S1P inhibitor. Cell lysates were prepared, separated by SDS-PAGE and then probed with anti-ATF6α. Blot reveals the presence of O-linked glycan modified Golgi-translocated ATF6 (designated G), which is absent for the D564G mutant, confirming a lack of ER to Golgi trafficking.  Wild type and ERp18 KO cell lines stably expressing ATF6α D564G were either left untreated (-) or treated (+) with 5 µg/ml brefeldin A for1 h prior to cell lysis. The anti-ATF6α western blot of membrane fractions reveals proteolytic processing by S1P to produce ATF6-M, only in the brefeldin A treated cells. Nuclear fractions confirm the presence of S2P cleavage product, ATF6-N following brefeldin A treatment. Anti-HDAC2 was used as a loading control for the nuclear fraction.


Figure 8 A - C Wild type cells stably expressing ATF6α ( A ) or ERp18 KO cells stably expressing ATF6α ( B ) , and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 ( RV cells ) ( C ) , were pre - treated with 30 µM PF429242 ( S1P inhibitor ) for 1 h prior to induction of ER stress with 10 mM DTT as indicated . D - F Wild type cells transiently expressing ATF6 C467A ( D ) , ATF6 C618A ( E ) , or ATF6 DM ( F ) were treated as above . Whole cell lysates were separated by SDS - PAGE under reducing conditions and analysed by western blotting for ATF6α . The positions of unprocessed ER - localised ATF6α ( ER ) and the O - linked glycan modified Golgi - translocated ATF6α ( Gol ) are indicated as is ATF6 - P . G The percentage of Golgi - ATF6α was quantified , the error bars represent ± standard deviation for at least three independent experiments .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 8 A-C Wild type cells stably expressing ATF6α (A) or ERp18 KO cells stably expressing ATF6α (B), and ERp18 KO cells stably expressing ATF6α transiently transfected with ERp18 (RV cells) (C), were pre-treated with 30 µM PF429242 (S1P inhibitor) for 1 h prior to induction of ER stress with 10 mM DTT as indicated.  D-F Wild type cells transiently expressing ATF6 C467A (D), ATF6 C618A (E), or ATF6 DM (F) were treated as above. Whole cell lysates were separated by SDS-PAGE under reducing conditions and analysed by western blotting for ATF6α. The positions of unprocessed ER-localised ATF6α (ER) and the O-linked glycan modified Golgi-translocated ATF6α (Gol) are indicated as is ATF6-P.  G The percentage of Golgi-ATF6α was quantified, the error bars represent ± standard deviation for at least three independent experiments.


Figure 1Relative expression of Rab39a in siRNA - treated cells after 48 hours of knockdown . Rab39a expression in I - Ab or Rab39a siRNA - treated cells was normalized to expression in cells treated with control β2m siRNA . Error bars show SD between three independent experiments . * p < 0 . 05 using two - way Student ' s t - test . XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments . XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - latex The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments . XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . RLU indicates relative luminescence units produced by reporter T cell stimulation . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experiments . DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 Cells were then fed with Ova - Fe and exposed to reporter T cells as in ( B ) . Error bars show SD of duplicate wells . * p < 0 . 05 using two - way ANOVA . Representative plot of 3 independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 1Relative expression of Rab39a in siRNA-treated cells after 48 hours of knockdown. Rab39a expression in I-Ab or Rab39a siRNA-treated cells was normalized to expression in cells treated with control β2m siRNA. Error bars show SD between three independent experiments. * p < 0.05 using two-way Student's t-test.XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-latex The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of unconjugated soluble Ova The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. RLU indicates relative luminescence units produced by reporter T cell stimulation. Error bars show SD of >3 replicate wells. * p < 0.05 vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experiments.DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 Cells were then fed with Ova-Fe and exposed to reporter T cells as in (B). Error bars show SD of duplicate wells. * p < 0.05 using two-way ANOVA. Representative plot of 3 independent experiments.


Figure 2MHC - II presentation of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of Ova - Fe . The cells were then exposed to MF2 . 2D9 - Luc Reporter CD4 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control β2m using two - way ANOVA . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m . Representative plot of 3 independent experimentsDC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with Ova - Fe and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . None of the bead concentrations are p < 0 . 05 for Rab39a siRNA vs β2m using two - way ANOVA . Representative plot of 3 independent experimentsMHC - I Presentation of SIINFEKL - H2 - Kb complexes on siRNA treated DC3 . 2 NS - Ova ( C ) or DC3 . 2 UbS8L ( D ) cells . 48 hours after siRNA transfection , the indicated amounts of dox was added to the cells to induce Ova expression . After 2 hours at 37oC , RF33 . 70 - Luc CD8 T cells were added along with Brefeldin A ( Golgiplug ) to a final concentration of 1 : 1000 . The cells were incubated overnight for reporter T cell luciferase expression . Error bars show SD of > 3 replicate wells . * p < 0 . 05 vs negative control I - Ab using two - way ANOVA . None of the dox concentrations are p < 0 . 05 for Rab39a siRNA vs I - Ab . Representative plot of 3 independent experimentsFlow cytometric analysis of surface H2 - Kb and I - Ab levels of siRNA treated DC3 . 2R cells . Shown are results after 48 hours of knockdown . Representative plot of 3 independent

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 2MHC-II presentation of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of Ova-Fe. The cells were then exposed to MF2.2D9-Luc Reporter CD4 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control β2m using two-way ANOVA. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m. Representative plot of 3 independent experimentsDC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with Ova-Fe and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. None of the bead concentrations are p < 0.05 for Rab39a siRNA vs β2m using two-way ANOVA. Representative plot of 3 independent experimentsMHC-I Presentation of SIINFEKL-H2-Kb complexes on siRNA treated DC3.2 NS-Ova (C) or DC3.2 UbS8L (D) cells. 48 hours after siRNA transfection, the indicated amounts of dox was added to the cells to induce Ova expression. After 2 hours at 37oC, RF33.70-Luc CD8 T cells were added along with Brefeldin A (Golgiplug) to a final concentration of 1:1000. The cells were incubated overnight for reporter T cell luciferase expression. Error bars show SD of >3 replicate wells. * p < 0.05 vs negative control I-Ab using two-way ANOVA. None of the dox concentrations are p < 0.05 for Rab39a siRNA vs I-Ab. Representative plot of 3 independent experimentsFlow cytometric analysis of surface H2-Kb and I-Ab levels of siRNA treated DC3.2R cells. Shown are results after 48 hours of knockdown. Representative plot of 3 independent experiments


Figure 3Phagocytosis assay after 48 hours of siRNA knockdown . DC3 . 2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads . The mixture was then placed in media at 37oC for 1 hour . Cells were harvested and incubated on ice with 1 mg / ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry . As a control , some cells were incubated with 10 µM Cytochalasin D during the feeding period . Shown are percentages of cells that have taken up beads . Representative plot of two independent experiments . Rab39a was induced in DC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) via overnight incubation with 1µg / ml dox . KO and rescued cells were then harvested and assayed as in ( A ) for phagocytosis . Representative plot of two independent experiments . DC3 . 2 - Rab39a cells were induced to express Rab39a overnight . Cells on coverslips were washed , fixed and stained according to the listed protocol and antibodies . Green = Rab39a ( HA tag ) , Magenta = Lamp2 . Arrows highlight punctate structures with colocalization . Scale bar = 10 µm . Representative image of two independent experiments . DC3 . 2 Rab39a KO cells were transduced with dox inducible mcherry - tagged Rab39a . Cells were plated in 35 mm glass bottom dishes and induced with 1 µg / ml dox . The next day , 1 µm latex beads conjugated with Alexa 488 - NHS ( 1 bead / cell ) were added . After 2 hours at 37oC , cells were imaged under a Leica TCS SP5 confocal microscope . Green = Alexa 488 labeled beads . Magenta = mcherry - Rab39a . Arrows highlight internalized beads surrounded by Rab39a . Scale bar = 10 µm . Representative image of two independent experiments . 5x105 of DC3 . 2 - Rab39a cells were induced to express Rab39a overnight with 1 µg / ml dox in a 6 well plate . Cells were fed at 1 bead / cell of biotinylated - 6µm magnetic beads for the indicated lengths of time . Magnetic - bead - phagosomes were isolated using the listed protocol . Phagosomes were washed and stained for Rab39a ( HA - tag ) . Representative data for 3 independent experiments . 1 . 25 x 105 of DC3 . 2 - Rab39a cells were incubated in a 12 well plate overnight ± dox ( 1µg / ml ) to induce Rab39a . Cells were fed at 4 beads / cell of biotinylated - ova - 6µm - magnetic - beads for 3 hours with or without 1 : 1000 BFA ( golgiplug , BD ) . Phagosomes were isolated using the listed protocol . Isolated phagosomes were washed , permeabilized and stained . Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes . Representative plot of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 3Phagocytosis assay after 48 hours of siRNA knockdown. DC3.2R cells treated with the indicated siRNAs were incubated on ice for 30 minutes with 4 beads per cell of Alexa Fluor 647 conjugated beads. The mixture was then placed in media at 37oC for 1 hour. Cells were harvested and incubated on ice with 1 mg/ml of Trypan Blue to quench uneaten beads before analysis through flow cytometry. As a control, some cells were incubated with 10 µM Cytochalasin D during the feeding period. Shown are percentages of cells that have taken up beads. Representative plot of two independent experiments.Rab39a was induced in DC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) via overnight incubation with 1µg / ml dox. KO and rescued cells were then harvested and assayed as in (A) for phagocytosis. Representative plot of two independent experiments.DC3.2-Rab39a cells were induced to express Rab39a overnight. Cells on coverslips were washed, fixed and stained according to the listed protocol and antibodies. Green = Rab39a (HA tag), Magenta = Lamp2. Arrows highlight punctate structures with colocalization. Scale bar = 10 µm. Representative image of two independent experiments.DC3.2 Rab39a KO cells were transduced with dox inducible mcherry-tagged Rab39a. Cells were plated in 35 mm glass bottom dishes and induced with 1 µg/ml dox. The next day, 1 µm latex beads conjugated with Alexa 488-NHS (1 bead/cell) were added. After 2 hours at 37oC, cells were imaged under a Leica TCS SP5 confocal microscope. Green = Alexa 488 labeled beads. Magenta = mcherry-Rab39a. Arrows highlight internalized beads surrounded by Rab39a. Scale bar = 10 µm. Representative image of two independent experiments.5x105 of DC3.2-Rab39a cells were induced to express Rab39a overnight with 1 µg/ml dox in a 6 well plate. Cells were fed at 1 bead/cell of biotinylated-6µm magnetic beads for the indicated lengths of time. Magnetic-bead-phagosomes were isolated using the listed protocol. Phagosomes were washed and stained for Rab39a (HA-tag). Representative data for 3 independent experiments.1.25 x 105 of DC3.2-Rab39a cells were incubated in a 12 well plate overnight ± dox (1µg/ml) to induce Rab39a. Cells were fed at 4 beads/cell of biotinylated-ova-6µm-magnetic-beads for 3 hours with or without 1:1000 BFA (golgiplug, BD). Phagosomes were isolated using the listed protocol. Isolated phagosomes were washed, permeabilized and stained. Shown are histograms of indicated stains as well as % of Rab39a positive phagosomes. Representative plot of two independent experiments.


Figure 4XPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experimentsXPT of siRNA treated DC3 . 2R cells after 48 hours of knockdown . Treated cells were fed with the indicated amounts o Ova ( B ) conjugated to iron oxide beads via a disulfide bond . The cells were then exposed to RF33 . 70 - Luc Reporter CD8 T cells overnight . Error bars show SD of > 3 replicate wells . * p < 0 . 05 for siRNA vs control I - Ab using two - way ANOVA . Representative plot of 3 independent experimentsDC3 . 2 - Rab39a cells ( Rab39a KO with dox - inducible Rab39a ) were incubated overnight with or without 1 µg / ml dox to induce Rab39a expression . 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in ( A ) . Error bars show SD of duplicate wells . * p < 0 . 05 between no dox and + dox using two - way ANOVA . Representative plot of 3 independent experimentsSame as in ( A ) but cells were fed with 8 µg of A9M peptide bead and exposed to 12 . 64 - Luc Reporter T cells . Experiments were combined by normalizing RLU values to the average of the negative control I - Ab . Error bars show SD of 3 independent experiments . * p < 0 . 05 for Rab39a or β2m siRNA vs negative control I - Ab using

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 4XPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts of C8L peptid conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experimentsXPT of siRNA treated DC3.2R cells after 48 hours of knockdown. Treated cells were fed with the indicated amounts o Ova (B) conjugated to iron oxide beads via a disulfide bond. The cells were then exposed to RF33.70-Luc Reporter CD8 T cells overnight. Error bars show SD of >3 replicate wells. * p < 0.05 for siRNA vs control I-Ab using two-way ANOVA. Representative plot of 3 independent experimentsDC3.2-Rab39a cells (Rab39a KO with dox-inducible Rab39a) were incubated overnight with or without 1 µg/ml dox to induce Rab39a expression. 1x105 cells were then fed with C8L peptide beads and exposed to reporter T cells as in (A). Error bars show SD of duplicate wells. * p < 0.05 between no dox and + dox using two-way ANOVA. Representative plot of 3 independent experimentsSame as in (A) but cells were fed with 8 µg of A9M peptide bead and exposed to 12.64-Luc Reporter T cells. Experiments were combined by normalizing RLU values to the average of the negative control I-Ab. Error bars show SD of 3 independent experiments. * p < 0.05 for Rab39a or β2m siRNA vs negative control I-Ab using ANOVA


Figure 5DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms shown in ( B ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2 - Ld ( dox / no dox ) was calculated . Three independent experiments were combined for analysis and error bars show the SD between experiments . Open H2 - Ld , but not Closed H2 - Ld was statistically significant ( p < 0 . 05 ) between KO and rescued cells using two - tailed ratio - paired Student ' s t - test . DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - magnetic - beads for 4 hours . Magnetic bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open ( 64 - 3 - 7 ) and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . H2 - Ld cell surface levels of KO and induced cells as in ( A ) . Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains . Phagosomes from KO and induced cells were isolated as in ( A ) . Permeabilized , unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer . Phagosomes were then washed and stained for H2 - Ld . Shown are histograms of indicated stains . Flow cytometry plots of ( D ) . Analysis of phagosomes shown in ( D ) and ( E ) . The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated . DC3 . 2 Rab39a - H2 - Ld cells were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours in the presence or absence of BFA . Isolated phagosomes were stained for H2 - Ld . Representative of two independent experiments .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 5DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. Representative histograms shown in (B). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of open or closed H2-Ld (dox/no dox) was calculated. Three independent experiments were combined for analysis and error bars show the SD between experiments. Open H2-Ld, but not Closed H2-Ld was statistically significant (p<0.05) between KO and rescued cells using two-tailed ratio-paired Student's t-test.DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-magnetic-beads for 4 hours. Magnetic bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open (64-3-7) and Closed (30-5-7s) forms of H2-Ld. H2-Ld cell surface levels of KO and induced cells as in (A).Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains.Phagosomes from KO and induced cells were isolated as in (A). Permeabilized, unfixed phagosomes were incubated with 10 µM of the indicated peptides for 3 hours at 25oC in Perm buffer. Phagosomes were then washed and stained for H2-Ld. Shown are histograms of indicated stains. Flow cytometry plots of (D).Analysis of phagosomes shown in (D) and (E). The background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of pulsed / unpulsed phagosomes was calculated.DC3.2 Rab39a-H2-Ld cells were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours in the presence or absence of BFA. Isolated phagosomes were stained for H2-Ld. Representative of two independent experiments.


Figure 6DC3 . 2 - Rab39a - H2 - Ld cells ( Rab39a KO with dox - inducible Rab39a ) were incubated with 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours with the indicated inhibitors . Magnetic - bead phagosomes were isolated using the listed protocol . Phagosomes were washed , fixed , permeabilized and stained for Open and Closed ( 30 - 5 - 7s ) forms of H2 - Ld . Representative histograms of two independent experiments shown . Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were fed at 4 beads / cell of biotinylated - Ova - magnetic - beads for 4 hours . Phagosomes were isolated and stained for Ova and Open H2 - Ld . Representative of 3 independent experiments . Quantification of Ova remaining in bead - phagosomes ( C ) and Nox2 staining ( D ) . Representative Histogram shown . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Cells as in ( A ) were incubated with or without 1 µg / ml dox overnight to induce Rab39a expression . 1x106 cells were incubated on ice with 4 beads / cell of biotinylated - Ova - beads for bead attachment . Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red . After 4 hours of incubation , unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence . For analysis , the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change ( dox / no dox ) was calculated . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Same as in C with Sec22b staining . Three independent experiments were combined . P < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Cells as in ( A ) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH . After 4 hours of incubation , whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads . For analysis , the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox / no dox was calculated . Three independent experiments were combined . p < 0 . 05 using two - tailed ratio - paired Student ' s t - test . Error bars show SD between experiments . Also shown are histograms of whole cells compared to background fluorescence of pH beads . DC3 . 2R cells were treated with the indicated siRNA . 48 hours after transfection , cells were fed with Ova - Fe ( 2 µg Ova ) with or without 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA . 5x104 DC3 . 2 - Rab39a - H2 - Ld cells were fed with Ova - Fe ( 1µg Ova ) with or without 1 µg dox and / or 100 µM leupeptin . RF33 . 70 - Luc reporter T cells were added . After overnight incubation , T cell luciferase was measured . Representative plot of two independent experiments . Error bars show SD between 3 replicate wells . p values were calculated using 2 - way ANOVA .

O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O B-PANEL_START O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Figure 6DC3.2-Rab39a-H2-Ld cells (Rab39a KO with dox-inducible Rab39a) were incubated with 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours with the indicated inhibitors. Magnetic-bead phagosomes were isolated using the listed protocol. Phagosomes were washed, fixed, permeabilized and stained for Open and Closed (30-5-7s) forms of H2-Ld. Representative histograms of two independent experiments shown.Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were fed at 4 beads/cell of biotinylated-Ova-magnetic-beads for 4 hours. Phagosomes were isolated and stained for Ova and Open H2-Ld. Representative of 3 independent experiments.Quantification of Ova remaining in bead-phagosomes (C) and Nox2 staining (D). Representative Histogram shown. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change of target protein (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.Cells as in (A) were incubated with or without 1 µg/ml dox overnight to induce Rab39a expression. 1x106 cells were incubated on ice with 4 beads/cell of biotinylated-Ova-beads for bead attachment. Cells were then transferred into 37oC media containing 5 µM CellROX Deep Red. After 4 hours of incubation, unfixed phagosomes were extracted and analyzed via flow cytometry for CellROX fluorescence. For analysis, the background fluorescence of unstained beads was subtracted from the resulting phagosome gMFI and the fold change (dox/no dox) was calculated. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.Same as in C with Sec22b staining. Three independent experiments were combined. P<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments.Cells as in (A) were fed at 1 bead per cell with pHrodo conjugated beads that increase fluorescence at low pH. After 4 hours of incubation, whole cells were harvested and run through flow cytometry to measure fluorescence of eaten beads. For analysis, the background fluorescence of uneaten beads was subtracted from the resulting gMFI and the fold change of dox/no dox was calculated. Three independent experiments were combined. p<0.05 using two-tailed ratio-paired Student's t-test. Error bars show SD between experiments. Also shown are histograms of whole cells compared to background fluorescence of pH beads.DC3.2R cells were treated with the indicated siRNA. 48 hours after transfection, cells were fed with Ova-Fe (2 µg Ova) with or without 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.5x104 DC3.2-Rab39a-H2-Ld cells were fed with Ova-Fe (1µg Ova) with or without 1 µg dox and/or 100 µM leupeptin. RF33.70-Luc reporter T cells were added. After overnight incubation, T cell luciferase was measured. Representative plot of two independent experiments. Error bars show SD between 3 replicate wells. p values were calculated using 2-way ANOVA.