Figure 4 ( A ) Five µL from wild - type , XccbphP , pXccBphP and pC13S bacterial cultures ( OD600 = 1 ) were plated onto PYM - glucose agar plates under light or dark conditions . Xanthan production was determined measuring colony diameters ( n = 4 replicates ) for 20 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 ) . ( B ) Wild - type , XccbphP , pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions . Extracellular xanthan was purified by KCl addition and ethanol precipitation , then dried and weighted ( n = 2 replicates ) for 4 independent experiments . ( A - B ) Values are expressed as mean ± s . e . m . Statistical analysis was performed by a Kruskal - Wallis test and Dunn ´ s multiple comparison test ( * P < 0 . 05 ) . ( C ) Representative confocal laser - scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions . The scale bar represents 30 µm .

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 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

Figure 4(A) Five µL from wild-type, XccbphP, pXccBphP and pC13S bacterial cultures (OD600 = 1) were plated onto PYM-glucose agar plates under light or dark conditions. Xanthan production was determined measuring colony diameters (n = 4 replicates) for 20 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).(B) Wild-type, XccbphP, pXccBphP and pC13S were grown in 20 ml PYM liquid medium under light or dark conditions. Extracellular xanthan was purified by KCl addition and ethanol precipitation, then dried and weighted (n = 2 replicates) for 4 independent experiments. (A-B) Values are expressed as mean ± s.e.m. Statistical analysis was performed by a Kruskal-Wallis test and Dunn´s multiple comparison test (*P < 0.05).(C) Representative confocal laser-scanning microscopy pictures of biofilm development of bacteria cultured for 4 d in minimal medium in chambered cover slides under light or dark conditions. The scale bar represents 30 µm.


Figure 1Northern blotting shows that Gasz depletion ( Gasz KD ) led to the accumulation of flam - piRNA precursors but reduced the level of mature idefix - piRNA arising from the precursors , as in the case of Zuc depletion ( Zuc KD ) . U6 snRNA was detected as a loading control . Western blotting showing the protein levels of endogenous Armi , Piwi , SoYb , Yb , Vret , and Gasz in total OSC lysate ( total ) , the mitochondrial fraction ( mito ) , and the cytoplasmic fraction after mitochondrial isolation ( cyto ) . β - Tubulin ( β - Tub ) and HSP60 were detected as markers for the " cyto " and " mito " fractions , respectively . Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Zuc KD ) Zuc knockdown in OSCs . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi signal ( green ) is strongly overlapped with the mitochondrial signal ( red ) in Zuc - depleted OSCs ( Zuc KD ) . Armi is localized to Yb bodies in control OSCs ( control ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei . Left : Western blotting comparing the abundances of Armi and Piwi in total OSC lysate ( total ) and the mitochondrial fraction ( mito ) before ( control ) and after ( Gasz KD ) Gasz depletion in Zuc - depleted OSCs ( Zuc KD ) . Relative mito - localization intensity ( * ) shows the amount of proteins in " mito " normalized by the amount of proteins in " total " and HSP60 . Right : Immunofluorescence shows that the Armi mitochondrial signal ( green ) found in Zuc - depleted cells ( Zuc KD / control ) mostly disappeared after additional Gasz depletion in the cells ( Zuc KD / Gasz KD ) , but was found at Yb bodies . Mitochondrial signal is shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei .

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 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 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

Figure 1Northern blotting shows that Gasz depletion (Gasz KD) led to the accumulation of flam-piRNA precursors but reduced the level of mature idefix-piRNA arising from the precursors, as in the case of Zuc depletion (Zuc KD). U6 snRNA was detected as a loading control.Western blotting showing the protein levels of endogenous Armi, Piwi, SoYb, Yb, Vret, and Gasz in total OSC lysate (total), the mitochondrial fraction (mito), and the cytoplasmic fraction after mitochondrial isolation (cyto). β-Tubulin (β-Tub) and HSP60 were detected as markers for the "cyto" and "mito" fractions, respectively.Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Zuc KD) Zuc knockdown in OSCs. Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi signal (green) is strongly overlapped with the mitochondrial signal (red) in Zuc-depleted OSCs (Zuc KD). Armi is localized to Yb bodies in control OSCs (control). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.Left: Western blotting comparing the abundances of Armi and Piwi in total OSC lysate (total) and the mitochondrial fraction (mito) before (control) and after (Gasz KD) Gasz depletion in Zuc-depleted OSCs (Zuc KD). Relative mito-localization intensity (*) shows the amount of proteins in "mito" normalized by the amount of proteins in "total" and HSP60. Right: Immunofluorescence shows that the Armi mitochondrial signal (green) found in Zuc-depleted cells (Zuc KD/control) mostly disappeared after additional Gasz depletion in the cells (Zuc KD/Gasz KD), but was found at Yb bodies. Mitochondrial signal is shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.


Figure 2Armi and Piwi co - immunoprecipitated with Flag - Gasz ( F - Gasz ) from the mitochondrial fraction ( mito fraction ) of OSCs ( input ) . F - Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation . * : Heavy chains of antibodies used for immunoprecipitation . n . i . : Non - immune IgG used as a negative control . Co - immunoprecipitation from whole - cell lysate of OSCs before ( control ) and after ( Zuc KD ) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag - Gasz ( F - Gasz ) complex increased upon Zuc depletion ( Zuc KD ) . Northern blotting shows that the flam - piRNA precursors in the Flag - Gasz complex ( Flag ) accumulated upon Zuc depletion ( Zuc KD ) . Upper : The flam - piRNA precursors associated with Piwi disappeared upon Armi depletion ( Armi KD ) in Zuc - lacking OSCs ( Zuc KD ) . Lower : The abundance of immunopurified Piwi is shown by western blotting .

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 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 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

Figure 2Armi and Piwi co-immunoprecipitated with Flag-Gasz (F-Gasz) from the mitochondrial fraction (mito fraction) of OSCs (input). F-Gasz was ectopically expressed in the cells by transfection prior to immunoprecipitation. *: Heavy chains of antibodies used for immunoprecipitation. n.i.: Non-immune IgG used as a negative control.Co-immunoprecipitation from whole-cell lysate of OSCs before (control) and after (Zuc KD) Zuc depletion and subsequent western blotting show that the levels of Piwi and Armi in the Flag-Gasz (F-Gasz) complex increased upon Zuc depletion (Zuc KD).Northern blotting shows that the flam-piRNA precursors in the Flag-Gasz complex (Flag) accumulated upon Zuc depletion (Zuc KD).Upper: The flam-piRNA precursors associated with Piwi disappeared upon Armi depletion (Armi KD) in Zuc-lacking OSCs (Zuc KD). Lower: The abundance of immunopurified Piwi is shown by western blotting.


Figure 3Depletion of endogenous Piwi ( Piwi KD ) in Zuc - lacking OSCs ( Zuc KD ) caused Armi ( green ) to relocate to Yb bodies . Localization of Armi at Yb bodies is shown in B . Mitochondrial ( upper panel ) and Yb ( lower panel ) signals are shown in red . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei . The Armi complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and Gasz . Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc - lacking OSCs . Co - IP index ( * ) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 0 . 05 . The Flag - Gasz ( F - Gasz ) complex was immunopurified from Zuc - lacking OSCs ( Zuc KD ) before ( control ) and after ( Piwi KD ) Piwi depletion and probed for detecting Armi and F - Gasz . F - Gasz was ectopically expressed prior to immunoprecipitation . Co - IP index ( * ) shows that the ratio of Armi amount in the F - Gasz complex isolated from Zuc - depleted cells ( Zuc KD / control ) and Zuc - and Piwi - depleted cells ( Zuc KD / Piwi KD ) is 1 : 0 . 31 . In vitro pull - down assays show that Armi - Flag ( Armi - F ) directly binds with GST - Gasz ∆ C113 but hardly with GST . Armi - F was immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F was detected by western blotting using anti - Flag antibodies .

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

Figure 3Depletion of endogenous Piwi (Piwi KD) in Zuc-lacking OSCs (Zuc KD) caused Armi (green) to relocate to Yb bodies. Localization of Armi at Yb bodies is shown in B. Mitochondrial (upper panel) and Yb (lower panel) signals are shown in red. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.The Armi complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and Gasz. Armi no longer stably associated with endogenous Gasz upon Piwi depletion in Zuc-lacking OSCs. Co-IP index (*) shows that the ratio of Gasz amount in the Armi complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.05.The Flag-Gasz (F-Gasz) complex was immunopurified from Zuc-lacking OSCs (Zuc KD) before (control) and after (Piwi KD) Piwi depletion and probed for detecting Armi and F-Gasz. F-Gasz was ectopically expressed prior to immunoprecipitation. Co-IP index (*) shows that the ratio of Armi amount in the F-Gasz complex isolated from Zuc-depleted cells (Zuc KD/control) and Zuc- and Piwi-depleted cells (Zuc KD/Piwi KD) is 1:0.31.In vitro pull-down assays show that Armi-Flag (Armi-F) directly binds with GST-Gasz∆C113 but hardly with GST. Armi-F was immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F was detected by western blotting using anti-Flag antibodies.


Figure 4Upper : Northern blotting shows that Flag - Piwi WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with flam - piRNA precursors in Zuc - depleted OSCs . Endogenous Piwi was depleted simultaneously with Zuc ( Zuc KD + Piwi KD ) . Zuc was depleted by RNAi and so residual Zuc may have remained in cells . Therefore , a tiny amount of piRNAs was loaded onto Piwi . F - Piwi WT , PAZmt , and MIDmt were made to be RNAi - resistant by mutagenesis ( see Materials and Methods ) . Lower : The abundance of immunopurified Flag - Piwi ( F - Piwi ) is shown by western blotting . Armi localization to Yb bodies observed in Zuc - and Piwi - depleted OSCs ( Zuc KD + Piwi KD ) ( Figs 3A and B ) was restored by ectopic expression of Flag - Piwi WT and PAZmt but not of MIDmt ( see also Fig EV4B ) . Armi , mitochondria , and F - Piwi are shown in green , red , and yellow , respectively . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei . Western blotting shows that Flag - Piwi ( F - Piwi ) WT ( WT ) and PAZ mutant ( PAZmt ) but not MID mutant ( MIDmt ) bound with Armi in Zuc / Piwi - depleted OSCs ( Zuc KD + Piwi KD ) .

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Figure 4Upper: Northern blotting shows that Flag-Piwi WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with flam-piRNA precursors in Zuc-depleted OSCs. Endogenous Piwi was depleted simultaneously with Zuc (Zuc KD + Piwi KD). Zuc was depleted by RNAi and so residual Zuc may have remained in cells. Therefore, a tiny amount of piRNAs was loaded onto Piwi. F-Piwi WT, PAZmt, and MIDmt were made to be RNAi-resistant by mutagenesis (see Materials and Methods). Lower: The abundance of immunopurified Flag-Piwi (F-Piwi) is shown by western blotting.Armi localization to Yb bodies observed in Zuc- and Piwi-depleted OSCs (Zuc KD + Piwi KD) (Figs 3A and B) was restored by ectopic expression of Flag-Piwi WT and PAZmt but not of MIDmt (see also Fig EV4B). Armi, mitochondria, and F-Piwi are shown in green, red, and yellow, respectively. The scale bar represents 5 μm. DAPI (blue) shows the nuclei.Western blotting shows that Flag-Piwi (F-Piwi) WT (WT) and PAZ mutant (PAZmt) but not MID mutant (MIDmt) bound with Armi in Zuc/Piwi-depleted OSCs (Zuc KD + Piwi KD).


Figure 5Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT bound with Piwi and Gasz . The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz . Luciferase - Flag ( Luc - F ) was used as a negative control . Armi - Flag ( Armi - F ) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F N756A mutant ( green ) localized to Yb bodies ( see also Fig EV5D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei . In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and N756A mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and N756A mutant were detected by western blotting using anti - Flag antibodies .

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 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 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

Figure 5Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT bound with Piwi and Gasz. The Armi N756A mutant bound with Piwi strongly but only weakly with Gasz. Luciferase-Flag (Luc-F) was used as a negative control.Armi-Flag (Armi-F) WT and the N756A mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F N756A mutant (green) localized to Yb bodies (see also Fig EV5D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and N756A mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and N756A mutant were detected by western blotting using anti-Flag antibodies.


Figure 6Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Immunoprecipitation and subsequent western blotting show that Armi - F WT but not Armi ∆ N34 mutant bound with Gasz in the cells . Both Armi WT and ∆ N34 mutant interacted with Piwi to similar extents . Luciferase - Flag ( Luc - F ) was used as a negative control . Armi - Flag ( Armi - F ) WT and the ∆ N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi ( Zuc KD + Armi KD ) . Armi - F WT ( green ) localized onto mitochondria ( red ) , whereas Armi - F ∆ N34 mutant ( green ) localized to Yb bodies ( see also Fig EV6D ) . The scale bar represents 5 μm . DAPI ( blue ) shows the nuclei . In vitro pull - down assays show that both Armi - Flag ( Armi - F ) WT and ∆ N34 mutant directly bind with GST - Gasz ∆ C113 but not with GST . Armi - F WT and mutant were immunopurified from Schneider 2 ( S2 ) cells under harsh conditions . GST and GST - Gasz ∆ C113 were visualized by CBB staining , while Armi - F WT and mutant were detected by western blotting using anti - Flag antibodies . Immunoprecipitation and subsequent western blotting shows that Armi - Flag ( Armi - F ) but not Flag - Armi ( F - Armi ) strongly bound with Gasz in Zuc / Armi - depleted OSCs ( Zuc KD + Armi KD ) . Piwi bound with both Armi ( F - Armi and Armi - F ) . Luciferase - Flag ( Luc - F ) was used as a negative control .

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 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 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 6Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Immunoprecipitation and subsequent western blotting show that Armi-F WT but not Armi ∆N34 mutant bound with Gasz in the cells. Both Armi WT and ∆N34 mutant interacted with Piwi to similar extents. Luciferase-Flag (Luc-F) was used as a negative control.Armi-Flag (Armi-F) WT and the ∆N34 mutant were expressed in OSCs where endogenous Armi and Zuc had been depleted by RNAi (Zuc KD + Armi KD). Armi-F WT (green) localized onto mitochondria (red), whereas Armi-F ∆N34 mutant (green) localized to Yb bodies (see also Fig EV6D). The scale bar represents 5 μm. DAPI (blue) shows the nuclei.In vitro pull-down assays show that both Armi-Flag (Armi-F) WT and ∆N34 mutant directly bind with GST-Gasz∆C113 but not with GST. Armi-F WT and mutant were immunopurified from Schneider 2 (S2) cells under harsh conditions. GST and GST-Gasz∆C113 were visualized by CBB staining, while Armi-F WT and mutant were detected by western blotting using anti-Flag antibodies.Immunoprecipitation and subsequent western blotting shows that Armi-Flag (Armi-F) but not Flag-Armi (F-Armi) strongly bound with Gasz in Zuc/Armi-depleted OSCs (Zuc KD + Armi KD). Piwi bound with both Armi (F-Armi and Armi-F). Luciferase-Flag (Luc-F) was used as a negative control.


Figure 4 ( A ) Representative images of immunofluorescence analysis ( with anti - PlxnB2 antibodies ) of the phenotype of COS7 cells transfected with wild - type or mutated PlxnB2 , compared to the collapsed phenotype induced by the cognate ligand Sema4C ( 1 μg / ml ) in cells expressing the WT receptor . ( B ) The stacked bar graph shows the fraction of collapsed PlxnB2 - expressing cells , identified as having a surface minor of 2000 μm2 ( measured by ImageJ software ) , out of 50 - 100 cells counted per each condition , from different fields and n > 3 experiments ) . The statistical analysis was performed comparing WT untreated cells with all other groups , by Fisher ' s Exact Test , which indicated a * * * * * p value < 0 . 00001 for Sema4C - treated and for G842C - PlxnB2 - expressing cells ( confirmed by ANOVA analysis on individual cell values , shown in Appendix Fig . S2 ) . By Fisher ' s test , the only other mutants significantly different from WT were R820H ( p < 0 . 001 ) and R843Q ( p < 0 . 01 ) , although ANOVA analysis on individual cell values shown in Appendix Fig . S2 didn ' t confirm such significance for other PlxnB2 variants .

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Figure 4(A) Representative images of immunofluorescence analysis (with anti-PlxnB2 antibodies) of the phenotype of COS7 cells transfected with wild-type or mutated PlxnB2, compared to the collapsed phenotype induced by the cognate ligand Sema4C (1 μg/ml) in cells expressing the WT receptor. (B) The stacked bar graph shows the fraction of collapsed PlxnB2-expressing cells, identified as having a surface minor of 2000 μm2 (measured by ImageJ software), out of 50-100 cells counted per each condition, from different fields and n>3 experiments). The statistical analysis was performed comparing WT untreated cells with all other groups, by Fisher's Exact Test, which indicated a *****p value < 0.00001 for Sema4C-treated and for G842C-PlxnB2-expressing cells (confirmed by ANOVA analysis on individual cell values, shown in Appendix Fig. S2). By Fisher's test, the only other mutants significantly different from WT were R820H (p< 0.001) and R843Q (p<0.01), although ANOVA analysis on individual cell values shown in Appendix Fig. S2 didn't confirm such significance for other PlxnB2 variants.


Figure 5 ( A ) The graphs show the analysis of cellular viability in shPlxnB2 - tranduced agnospheres , compared to their respective scramble - transfected controls , after 24 , 48 , 72 , and 96 hours of growth in culture . Values are mean ± SD of n ≥ 3 independent experiments ( 6 technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons ( Graph Pad Prism 8 . 0 . 0 ) : * * p = 0 . 0053 ; * * * * p < 0 . 0001 . ( C ) After transplantation in immunodeficient mice of CUP cells described above , the volume of ADX43 - and ( D ) ADX901 - shPlxnB2 tumors was assessed , in comparison with the respective controls . Plotted values indicate the mean ± SD ( n = 8 each group ) . The statistical significance was assessed by two - way ANOVA with Sidak ' s correction for multiple comparisons : * * p = 0 . 0060 ; * * * * p < 0 . 0001 .

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 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

Figure 5(A) The graphs show the analysis of cellular viability in shPlxnB2-tranduced agnospheres, compared to their respective scramble-transfected controls, after 24, 48, 72, and 96 hours of growth in culture. Values are mean ± SD of n≥3 independent experiments (6 technical replicates for each). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons (Graph Pad Prism 8.0.0): **p = 0.0053; ****p < 0.0001.(C) After transplantation in immunodeficient mice of CUP cells described above, the volume of ADX43- and (D) ADX901-shPlxnB2 tumors was assessed, in comparison with the respective controls. Plotted values indicate the mean ± SD (n = 8 each group). The statistical significance was assessed by two-way ANOVA with Sidak's correction for multiple comparisons: **p = 0.0060; ****p < 0.0001.


Figure 6 ( A ) Time course analysis of the cellular viability of AS901 , stably overexpressing either wild - type or G842C - mutated PlxnB2 , or controls transduced with an empty vector . Plotted values are the mean ± SD of n = 3 independent experiments ( six technical replicates for each ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs . controls ( CTRL ) : at 24h * * p = 0 . 0097 , 48h * * p = 0 . 0026 , 72h * * p = 0 . 0079 , 96h * p = 0 . 0226 . PlxnB2 - G842C vs . PlxnB2 - WT : at 48h $ p = 0 . 0418 , 72h $ p = 0 . 0318 . ( B ) Tumor growth curves of ADX 901 over - expressing either PlxnB2 - G842C or PlxnB2 - WT , or mock control . Plotted values are the mean ± SD ( n = 8 each groups ) . The statistical significance was assessed by two - way ANOVA with Tukey ' s correction for multiple comparisons . PlxnB2 - G842C vs control ( CTRL ) : at 25 days * * p = 0 . 0029 , 28 days * * p = 0 . 0028 , 32 days * * p = 0 . 0033 . PlxnB2 - G842C vs PlxnB2 - WT : at 25 days $ p = 0 . 0221 , 28 days $ p = 0 . 0234 , 32 days $ $ p = 0 . 0024 .

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 O O O O O O O O O O O O O O O O O O O O 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) Time course analysis of the cellular viability of AS901, stably overexpressing either wild-type or G842C-mutated PlxnB2, or controls transduced with an empty vector. Plotted values are the mean ± SD of n =3 independent experiments (six technical replicates for each). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs. controls (CTRL): at 24h **p = 0.0097, 48h **p = 0.0026, 72h **p = 0.0079, 96h *p = 0.0226. PlxnB2-G842C vs. PlxnB2-WT: at 48h $p = 0.0418, 72h $p = 0.0318. (B) Tumor growth curves of ADX 901 over-expressing either PlxnB2-G842C or PlxnB2-WT, or mock control. Plotted values are the mean ± SD (n = 8 each groups). The statistical significance was assessed by two-way ANOVA with Tukey's correction for multiple comparisons. PlxnB2-G842C vs control (CTRL): at 25 days **p = 0.0029, 28 days **p = 0.0028, 32 days **p = 0.0033. PlxnB2-G842C vs PlxnB2-WT: at 25 days $p = 0.0221, 28 days $p = 0.0234, 32 days $$p = 0.0024.


Figure 7 ( A ) Western blotting showing basal endogenous or overexpressed PlxnB2 , either WT or G842C - mutated isoforms , in MCF7 cells . In this wide view of the hybridized membrane , it is appreciable the presence of full length molecules at 250 kDa , as well as the processed 80 kDa fragments ( reported in ref . 64 ) . Vinculin staining provided protein loading controls . ( B ) The graph shows the mean ± SD of relative increase in 24 - hour spontaneous migration across Transwell inserts of MCF7 cells ( analysed above ) over - expressing either wild - type or G842C - mutated PlxnB2 , versus mock - transfected controls ( n = 4 biological replicates ) . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : PlxnB2 - WT vs . mock * p = 0 . 0145 ; PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0277 ; PlxnB2 - G842C vs . mock * * * p = 0 . 0002 . ( C ) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( with at least three technical replicates for each ) . The statistical significance was verified by one - way ANOVA with Tukey ' s correction for multiple comparisons : * p = 0 . 0350 ; * * p = 0 . 0029 ; * * * p = 0 . 0002 . Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B . ( D ) Western blotting analysis of endogenous and overexpressed PlxnB2 ( either WT or G842C - mutated isoforms ) in CUP cells AS906 ( both full length molecules at 250 kDa and processed 80 kDa fragments ) . Vinculin staining provided protein loading controls . ( E ) The invasive capacity of CUP cells AS906 ( shown in previous panel ) , overexpressing WT or G842 - mutated PlxnB2 ( or mock control ) , was assessed across Matrigel - coated transwell inserts . The graph shows the mean ± SD of values obtained in N = 3 independent experiments ( including technical duplicates , in two cases ) . Below the graph , are shown representative fields of the semipermeable membranes ( additional larger size images can be found in appendix Figure S4C ) ; scale bar : 100μm . The statistical significance was verified by one - way ANOVA with Bonferroni correction for multiple comparisons : * p = 0 . 0406 ; * * p = 0 . 0045 ; * * * p = 0 . 0003 .

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 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 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 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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(A) Western blotting showing basal endogenous or overexpressed PlxnB2, either WT or G842C-mutated isoforms, in MCF7 cells. In this wide view of the hybridized membrane, it is appreciable the presence of full length molecules at 250 kDa, as well as the processed 80 kDa fragments (reported in ref. 64). Vinculin staining provided protein loading controls.(B) The graph shows the mean ± SD of relative increase in 24-hour spontaneous migration across Transwell inserts of MCF7 cells (analysed above) over-expressing either wild-type or G842C-mutated PlxnB2, versus mock-transfected controls (n=4 biological replicates). The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: PlxnB2-WT vs. mock *p=0.0145; PlxnB2-G842C vs. PlxnB2-WT *p=0.0277; PlxnB2-G842C vs. mock ***p=0.0002.(C) Wound healing scratch assays on monolayers of MCF7 cells engineered as described above. The graph shows the mean ± SD of values obtained in N=3 independent experiments (with at least three technical replicates for each). The statistical significance was verified by one-way ANOVA with Tukey's correction for multiple comparisons: *p = 0.0350; **p = 0.0029; ***p = 0.0002. Representative phase contrast images of the wounded monolayers are further shown in larger size in Appendix Figure S4B.(D) Western blotting analysis of endogenous and overexpressed PlxnB2 (either WT or G842C-mutated isoforms) in CUP cells AS906 (both full length molecules at 250 kDa and processed 80 kDa fragments). Vinculin staining provided protein loading controls.(E) The invasive capacity of CUP cells AS906 (shown in previous panel), overexpressing WT or G842-mutated PlxnB2 (or mock control), was assessed across Matrigel-coated transwell inserts. The graph shows the mean ± SD of values obtained in N=3 independent experiments (including technical duplicates, in two cases). Below the graph, are shown representative fields of the semipermeable membranes (additional larger size images can be found in appendix Figure S4C); scale bar: 100μm. The statistical significance was verified by one-way ANOVA with Bonferroni correction for multiple comparisons: *p=0.0406; **p=0.0045; ***p=0.0003.


Figure 8 ( A ) Phospho - EGFR analysis by western blotting in control and PlxnB2 - silenced AS43 cells . Images show representative results of n = 3 independent experiments , while the graph at the bottom shows the mean ± SD of normalized band quantification . The statistical significance across replicates was verified by unpaired t - test : * * * * p < 0 . 0001 . ( B ) Phospho - EGFR analysis by western blotting in AS901 and AS906 cells either mock , or overexpressing WT or G842C - mutated PlxnB2 . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by one - way ANOVA followed by Tukey test : AS901 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0434 , PlxnB2 - G842C vs . mock * p = 0 . 0211 ; AS906 PlxnB2 - G842C vs . PlxnB2 - WT * p = 0 . 0436 , PlxnB2 - G842C vs . mock * p = 0 . 0481 . ( C ) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres , after 6 - hour treatment with the EGFR inhibitor cetuximab ( 50μg / ml ) . Images show representative results of n = 3 independent experiments , while the graphs at the bottom show the mean ± SD of normalized band quantification . The statistical significance across independent replicas was verified by Sidak ' s multiple comparison test between cetuximab - treated and untreated conditions , per each group : AS43 * p = 0 . 0153 , * * * p = 0 . 0001 , * * * * p < 0 . 0001 ; AS901 * p = 0 . 0201 .

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 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

Figure 8(A) Phospho-EGFR analysis by western blotting in control and PlxnB2-silenced AS43 cells. Images show representative results of n=3 independent experiments, while the graph at the bottom shows the mean ± SD of normalized band quantification. The statistical significance across replicates was verified by unpaired t-test: ****p<0.0001. (B) Phospho-EGFR analysis by western blotting in AS901 and AS906 cells either mock, or overexpressing WT or G842C-mutated PlxnB2. Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by one-way ANOVA followed by Tukey test: AS901 PlxnB2-G842C vs. PlxnB2-WT *p=0.0434, PlxnB2-G842C vs. mock *p=0.0211; AS906 PlxnB2-G842C vs. PlxnB2-WT *p=0.0436, PlxnB2-G842C vs. mock *p=0.0481.(C) Western blotting analysis of pERK and pS6 intracellular signal transducers in the indicated agnospheres, after 6-hour treatment with the EGFR inhibitor cetuximab (50μg/ml). Images show representative results of n=3 independent experiments, while the graphs at the bottom show the mean ± SD of normalized band quantification. The statistical significance across independent replicas was verified by Sidak's multiple comparison test between cetuximab-treated and untreated conditions, per each group: AS43 *p=0.0153, ***p=0.0001, ****p<0.0001; AS901 *p=0.0201.


Figure 9 ( A ) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab ( 50μg / ml ) . In the plot , each box represents values from 25th to 75th percentiles , the central band indicates the mean , and the whiskers expand from maximum to minimum values of individual cetuximab - treated experimental points ( n ≥ 9 ) , normalized to the average value of untreated cells within each group . The statistical significance across three independent experiments , each comprising ≥ 3 technical replicates , was verified by unpaired t - test multiple comparisons of cetuximab - treated and untreated conditions , per each group : * * * p = 0 . 0002 ; * * * * p < 0 . 0001 . ( B ) The viability of AS43 , in presence ( or absence ) of cetuximab ( 50μg / ml ) , was assessed over the indicated time course , compared to values at T0 . The graph shows the mean ± SD of two independent experiments ( with quadruplicate technical replicates each ) . The statistical significance was verified through multiple comparisons by two - way ANOVA with Bonferroni correction of cetuximab - treated and untreated samples , per each time point : * * * * p < 0 . 0001 . ( D ) The invasive capacity of AS906 cells ( overexpressing WT or G842C - mutated PlxnB2 , or mock transfected ) was assessed as in Fig . 7E , in the presence or absence of cetuximab 50μg / ml . Images show representative results ( scale bar : 100μm ) , while the graph on the right shows the mean ± SD of n = 3 independent experiments . The statistical significance across replicates was verified by one - way ANOVA with Bonferroni correction : * * p = 0 . 0010 ; * * * p = 0 . 0005 ; * * * * p < 0 . 0001 .

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 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 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 9(A) The cellular viability of the indicated agnospheres was assessed after six days in presence or absence of the EGFR inhibitor cetuximab (50μg/ml). In the plot, each box represents values from 25th to 75th percentiles, the central band indicates the mean, and the whiskers expand from maximum to minimum values of individual cetuximab-treated experimental points (n≥9), normalized to the average value of untreated cells within each group. The statistical significance across three independent experiments, each comprising ≥3 technical replicates, was verified by unpaired t-test multiple comparisons of cetuximab-treated and untreated conditions, per each group: ***p=0.0002; ****p<0.0001.(B) The viability of AS43, in presence (or absence) of cetuximab (50μg/ml), was assessed over the indicated time course, compared to values at T0. The graph shows the mean ± SD of two independent experiments (with quadruplicate technical replicates each). The statistical significance was verified through multiple comparisons by two-way ANOVA with Bonferroni correction of cetuximab-treated and untreated samples, per each time point: ****p<0.0001.(D) The invasive capacity of AS906 cells (overexpressing WT or G842C-mutated PlxnB2, or mock transfected) was assessed as in Fig. 7E, in the presence or absence of cetuximab 50μg/ml. Images show representative results (scale bar: 100μm), while the graph on the right shows the mean ± SD of n=3 independent experiments. The statistical significance across replicates was verified by one-way ANOVA with Bonferroni correction: **p= 0.0010; ***p=0.0005; ****p<0.0001.


Figure 1 ( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) produced with lopinavir ( LPV , 0 - 100 nM ) detecting p24 antibody . Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated THP - 1 shSAMHD1 cells . Mean ± SD , n = 3 . Titration of LPV - treated HIV - 1 GFP viruses on PMA - treated U87 cells . Mean ± SD , n = 3 . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . ISG qPCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with LPV - treated HIV - 1 GFP viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . G ) CXCL - 10 protein in cell supernatants from D - F ( ELISA ) . ( H ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 2 U RT / ml 30 nM LPV - treated HIV - 1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib . A control was stimulated with 1 ng / ml IFNβ . ( I ) RT products from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with 0 . 3 U RT / ml LPV - treated HIV - 1 GFP viruses . ( J ) Immunoblot of HIV - 1 R9 BaL virus particles ( 2 × 1011 genomes ) produced with LPV ( 0 - 100 nM ) detecting p24 . ( K , L ) ISG qRT - PCR ( K ) and infection data ( L ) from primary monocyte - derived macrophages ( MDM ) infected for 24 h with LPV - treated HIV - 1 R9 BaL viruses ( 0 . 2 U RT / ml ) . Data are collated from two donors ( represented by circles and squares ) , n = 3 . Horizontal line represents the median . For experiments in which the virus dose used was normalised by RT activity , the number of genome copies was also measured by qPCR of virus . This gave dose equivalents of within 2 - 3 fold of RT equivalents .

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 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 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 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 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 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 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 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

Figure 1(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) produced with lopinavir (LPV, 0-100 nM) detecting p24 antibody.Titration of LPV-treated HIV-1 GFP viruses on PMA-treated THP-1 shSAMHD1 cells. Mean ± SD, n=3.Titration of LPV-treated HIV-1 GFP viruses on PMA-treated U87 cells. Mean ± SD, n=3. ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).  ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).  ISG qPCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with LPV-treated HIV-1 GFP viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). G) CXCL-10 protein in cell supernatants from D-F (ELISA).(H) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.2 U RT/ml 30 nM LPV-treated HIV-1 GFP in the presence of DMSO vehicle or 2 μM ruxolitinib. A control was stimulated with 1 ng/ml IFNβ.(I) RT products from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with 0.3 U RT/ml LPV-treated HIV-1 GFP viruses.(J) Immunoblot of HIV-1 R9 BaL virus particles (2×1011 genomes) produced with LPV (0-100 nM) detecting p24.(K, L) ISG qRT-PCR (K) and infection data (L) from primary monocyte-derived macrophages (MDM) infected for 24 h with LPV-treated HIV-1 R9 BaL viruses (0.2 U RT/ml). Data are collated from two donors (represented by circles and squares), n=3. Horizontal line represents the median. For experiments in which the virus dose used was normalised by RT activity, the number of genome copies was also measured by qPCR of virus. This gave dose equivalents of within 2-3 fold of RT equivalents.


Figure 2 ( A ) Immunoblot of HIV - 1 GFP virus particles ( 2 × 1011 genomes ) with varying proportions of ΔCA - SP1 protease cleavage mutation detecting p24 . ( B ) Titration of HIV - 1 GFP ΔCA - SP1 viruses on U87 cells . Mean ± SD , n = 3 . ( C ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . D ) ISG qRT - PCR from PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . PMA - treated THP - 1 shSAMHD1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 1 U RT / ml red line , 0 . 5 U RT / ml blue line ) . E ) CXCL - 10 protein in supernatants from ( C , D ) ( ELISA ) . ( F ) RT products from THP - 1 cells transduced for 24 h with 6 × 109 genomes / ml ( approx . 0 . 5 U RT / ml ) HIV - 1 GFP ΔCA - SP1 viruses . ( G ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ΔCA - SP1 viruses ( 0 . 016 - 0 . 2 U RT / ml ) . Data are shown as individual measurements , representative of 2 repeats . ( H ) IFIT - 1 reporter activity from monocytic THP - 1 - IFIT - 1 cells transduced with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 mutant , or stimulated with 4 μg / ml cGAMP as a control , in the presence of DMSO vehicle or 2 μM ruxolitinib . ( I ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 3 × 108 genomes / ml or 1 . 5 × 109 genomes / ml , equivalent to 0 . 1 U RT / ml and 0 . 5 U RT / ml ) . ( J ) ISG qRT - PCR from primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . primary MDM transduced for 24 h with WT HIV - 1 GFP or 75 % ΔCA - SP1 mutant ( 1 . 5 × 109 genomes / ml ) , or stimulated with 1 ng / ml IFNβ , in the presence of DMSO vehicle or 2 μM ruxolitinib . K ) CXCL - 10 protein in supernatants from ( J ) ( ELISA ) .

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 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 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 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 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 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 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 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 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

Figure 2(A) Immunoblot of HIV-1 GFP virus particles (2×1011 genomes) with varying proportions of ΔCA-SP1 protease cleavage mutation detecting p24.(B) Titration of HIV-1 GFP ΔCA-SP1 viruses on U87 cells. Mean ± SD, n=3.(C ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).D) ISG qRT-PCR from PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line).PMA-treated THP-1 shSAMHD1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.1 U RT/ml red line, 0.5 U RT/ml blue line). E) CXCL-10 protein in supernatants from (C, D) (ELISA).(F) RT products from THP-1 cells transduced for 24 h with 6×109 genomes/ml (approx. 0.5 U RT/ml) HIV-1 GFP ΔCA-SP1 viruses.(G) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP ΔCA-SP1 viruses (0.016 - 0.2 U RT/ml). Data are shown as individual measurements, representative of 2 repeats.(H) IFIT-1 reporter activity from monocytic THP-1-IFIT-1 cells transduced with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 mutant, or stimulated with 4 μg/ml cGAMP as a control, in the presence of DMSO vehicle or 2 μM ruxolitinib.(I) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (3×108 genomes/ml or 1.5×109 genomes/ml, equivalent to 0.1 U RT/ml and 0.5 U RT/ml).(J) ISG qRT-PCR from primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib.primary MDM transduced for 24 h with WT HIV-1 GFP or 75 % ΔCA-SP1 mutant (1.5×109 genomes/ml), or stimulated with 1 ng/ml IFNβ, in the presence of DMSO vehicle or 2 μM ruxolitinib. K) CXCL-10 protein in supernatants from (J) (ELISA).


Figure 3 ( A ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 1 U RT / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . B ) CXCL - 10 protein in supernatants from ( A ) ( ELISA ) . ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . ISG qRT - PCR from THP - 1 - IFIT - 1 cells transduced for 24 h with 0 % ( WT ) or 75 % ΔCA - SP1 mutant ( 109 and 3 × 109 genomes / ml ) in the presence of DMSO vehicle , 5 μM neviripine or 10 μM raltegravir . ( E ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP containing 0 % ΔCA - SP1 ( WT ) , 75 % ΔCA - SP1 , 75 % ΔCA - SP1 carrying a mutation in reverse transcriptase ( 75 % ΔCA - SP1 RT D185E ) or 75 % ΔCA - SP1 carrying a mutation in integrase ( 75 % ΔCA - SP1 INT D116N ) ( 3 . 75 × 109 , 7 . 5 × 109 and 1 . 5 × 1010 genomes / ml ) . ( F ) CXCL - 10 protein in supernatants from ( E ) ( ELISA ) . Triangles indicate CXCL - 10 not detected .

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 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 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 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 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 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

Figure 3(A) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % (WT) or 75 % ΔCA-SP1 mutant (1 U RT/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir. B) CXCL-10 protein in supernatants from (A) (ELISA).ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.ISG qRT-PCR from THP-1-IFIT-1 cells transduced for 24 h with 0 % (WT) or 75 % ΔCA-SP1 mutant (109 and 3×109 genomes/ml) in the presence of DMSO vehicle, 5 μM neviripine or 10 μM raltegravir.(E) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP containing 0 % ΔCA-SP1 (WT), 75 % ΔCA-SP1, 75 % ΔCA-SP1 carrying a mutation in reverse transcriptase (75 % ΔCA-SP1 RT D185E) or 75 % ΔCA-SP1 carrying a mutation in integrase (75 % ΔCA-SP1 INT D116N) (3.75×109, 7.5×109 and 1.5×1010 genomes/ml).(F) CXCL-10 protein in supernatants from (E) (ELISA). Triangles indicate CXCL-10 not detected.


Figure 4 ( A ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING or MAVS , or a gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP 75 % ΔCA - SP1 ( 0 . 4 U RT / ml ) or stimulated by transfection with either HT - DNA ( 0 . 1 μg / ml ) or poly I : C ( 0 . 5 μg / ml ) . IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line stimulated for 24 h with poly I : C ( transfection , 0 . 5 μg / ml ) , HT - DNA ( transfection , 0 . 1 μg / ml ) , LPS ( 50 ng / ml ) or cGAMP ( transfection , 0 . 5 μg / ml ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP containing either 0 % ( WT ) or 75 % ΔCA - SP1 ( 1 × 1010 and 2 × 1010 genomes / ml ) ( C ) . D ) CXCL - 10 protein in supernatants from ( C ) ( ELISA ) . Triangles indicate CXCL - 10 not detected . ( E ) IFIT - 1 reporter activity from PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( F ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . PMA - treated THP - 1 - IFIT - 1 shSAMHD1 cells lacking STING , MAVS or matching gRNA control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . G ) CXCL - 10 protein in supernatants from ( E ) . PMA - treated THP - 1 Dual shSAMHD1 cells lacking cGAS or matching control ( Ctrl ) cell line transduced for 24 h with DRV - treated HIV - 1 GFP ( 1 × 1010 genomes / ml ) . ( H ) CXCL - 10 protein in supernatants from ( F ) ( ELISA ) . ( I ) IRF reporter activity from PMA - treated THP - 1 Dual shSAMHD1 control cells transduced for 48 h with WT , DRV - treated ( DRV , 12 . 5 nM ) or HIV - 1 GFP containing 90 % ΔCA - SP1 ( 1 × 1010 genomes / ml ) in the presence of DMSO vehicle , 2 μM ruxolitinib or 0 . 5 μg / ml H151 .

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 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 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 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 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 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

Figure 4(A) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING or MAVS, or a gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP 75 % ΔCA-SP1 (0.4 U RT/ml) or stimulated by transfection with either HT-DNA (0.1 μg/ml) or poly I:C (0.5 μg/ml).IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line stimulated for 24 h with poly I:C (transfection, 0.5 μg/ml), HT-DNA (transfection, 0.1 μg/ml), LPS (50 ng/ml) or cGAMP (transfection, 0.5 μg/ml)IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml)PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP containing either 0 % (WT) or 75 % ΔCA-SP1 (1×1010 and 2×1010 genomes/ml) (C). D) CXCL-10 protein in supernatants from (C) (ELISA). Triangles indicate CXCL-10 not detected.(E) IFIT-1 reporter activity from PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).(F) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml).PMA-treated THP-1-IFIT-1 shSAMHD1 cells lacking STING, MAVS or matching gRNA control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). G) CXCL-10 protein in supernatants from (E).PMA-treated THP-1 Dual shSAMHD1 cells lacking cGAS or matching control (Ctrl) cell line transduced for 24 h with DRV-treated HIV-1 GFP (1×1010 genomes/ml). (H) CXCL-10 protein in supernatants from (F) (ELISA).(I) IRF reporter activity from PMA-treated THP-1 Dual shSAMHD1 control cells transduced for 48 h with WT, DRV-treated (DRV, 12.5 nM) or HIV-1 GFP containing 90 % ΔCA-SP1 (1×1010 genomes/ml) in the presence of DMSO vehicle, 2 μM ruxolitinib or 0.5 μg/ml H151.


Figure 5Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with a fixed dose of HIV - 1 GFP ( 5 × 107 genomes / ml ) and increasing doses of HIV - LUC ΔCA - SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 0 . 001 . Abrogation - of - restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5 . FRhK4 cells were co - transduced with LPV - treated HIV - LUC viruses Rescue of GFP infectivity was assessed by flow cytometry . Data are singlet % GFP values and two repeats of the experiment are shown . See also Appendix figure S3 . Statistical analyses were performed using 2 - way ANOVA with multiple comparisons . * P < 0 . 05 , * * P < 0 . 01 , * * * P < 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 O O O O O O O O O O O O O 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

Figure 5Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with a fixed dose of HIV-1 GFP (5×107 genomes/ml) and increasing doses of HIV-LUC ΔCA-SP1 mutants Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.Abrogation-of-restriction assay in FRhK4 cells expressing restrictive rhesus TRIM5. FRhK4 cells were co-transduced with LPV-treated HIV-LUC viruses Rescue of GFP infectivity was assessed by flow cytometry. Data are singlet % GFP values and two repeats of the experiment are shown. See also Appendix figure S3. Statistical analyses were performed using 2-way ANOVA with multiple comparisons. * P<0.05, ** P<0.01, *** P<0.001.


Figure 6 ( A ) Infection data for THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 1 - 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . B ) IFIT - 1 reporter activity in supernatant from ( A ) . ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ISG qRT - PCR from monocytic THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 0 . 25 - 1 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( E ) CXCL - 10 protein in supernatants of THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( F ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells transduced for 24 h with HIV - 1 GFP ( 3 U / ml RT ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) and ruxolitinib ( Rux , 2 μM ) as indicated . ( G ) IRF reporter activity from THP - 1 Dual shSAMHD1 cells lacking cGAS or a matching control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) . ( H ) IFIT - 1 reporter activity from THP - 1 - IFIT - 1 cells lacking MAVS or a matching gRNA control ( Ctrl ) cell line transduced for 24 h with HIV - 1 GFP ( 3 U / ml and 6 U / ml ) in the presence of DMSO vehicle or PF - 74 ( 10 μM ) .

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 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 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 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 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 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 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 6(A) Infection data for THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.1-3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM). B) IFIT-1 reporter activity in supernatant from (A).ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).ISG qRT-PCR from monocytic THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (0.25-1 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM).(E) CXCL-10 protein in supernatants of THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).(F) IFIT-1 reporter activity from THP-1-IFIT-1 cells transduced for 24 h with HIV-1 GFP (3 U/ml RT) in the presence of DMSO vehicle or PF-74 (10 μM) and ruxolitinib (Rux, 2 μM) as indicated.(G) IRF reporter activity from THP-1 Dual shSAMHD1 cells lacking cGAS or a matching control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).(H) IFIT-1 reporter activity from THP-1-IFIT-1 cells lacking MAVS or a matching gRNA control (Ctrl) cell line transduced for 24 h with HIV-1 GFP (3 U/ml and 6 U/ml) in the presence of DMSO vehicle or PF-74 (10 μM).


Figure 1Substrate was synthesized in reticulocyte lysate in the presence of 35S - methionine and mixed with SecA and SecYEG proteoliposomes . After incubation with ATP , the samples were treated with proteinase K to degrade any non - translocated material . Where indicated , the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X - 100 ( TX - 100 ) . Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT , generating protease - protected full - length ( FL ) substrate or a translocation intermediate ( IM ) , respectively . All samples were analyzed by SDS PAGE followed by autoradiography . Lane 1 shows 10 % of the input sample . The gel is representative of three replicates . Molecular weight markers are estimated from Coomassie - stained gels run in parallel . Translocation intermediates ( IM ) were incubated in the presence of different nucleotides ( Nuc . ) for different time periods and then treated with proteinase K ( Prot . K ) . One sample received hexokinase and glucose ( HKG ) to generate ADP , other samples ADP • BeFx or ATPγS . The gel is representative of two replicates . Quantification of ( C ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show the average of replicates . Colors correspond to different nucleotides as shown in the inset box . As in ( C ) , but DTT was added concurrently with HKG or nucleotides . The gel is representative of two replicates . Quantification of ( E ) as in ( D ) , except intensities were calculated for the FL bands .

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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Substrate was synthesized in reticulocyte lysate in the presence of 35S-methionine and mixed with SecA and SecYEG proteoliposomes. After incubation with ATP, the samples were treated with proteinase K to degrade any non-translocated material. Where indicated, the reaction was performed in the absence of ATP and proteolysis in the presence of Triton X-100 (TX-100). Translocation reactions were performed in the presence of the reducing agent DTT or oxidizing agent NaTT, generating protease-protected full-length (FL) substrate or a translocation intermediate (IM), respectively. All samples were analyzed by SDS PAGE followed by autoradiography. Lane 1 shows 10% of the input sample. The gel is representative of three replicates. Molecular weight markers are estimated from Coomassie-stained gels run in parallel.Translocation intermediates (IM) were incubated in the presence of different nucleotides (Nuc.) for different time periods and then treated with proteinase K (Prot. K). One sample received hexokinase and glucose (HKG) to generate ADP, other samples ADP•BeFx or ATPγS. The gel is representative of two replicates.   Quantification of (C). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show the average of replicates. Colors correspond to different nucleotides as shown in the inset box.   As in (C), but DTT was added concurrently with HKG or nucleotides. The gel is representative of two replicates.   Quantification of (E) as in (D), except intensities were calculated for the FL bands.


Figure 2WB SecA with additional mutations ( WT , Y794G , R342E ) was added to preformed intermediates ( IM ) and incubated for the indicated times before proteolysis . The gel is representative of two replicates . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to WB SecA carrying additional mutations , as shown in the inset box .

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

Figure 2WB SecA with additional mutations (WT, Y794G, R342E) was added to preformed intermediates (IM) and incubated for the indicated times before proteolysis. The gel is representative of two replicates.   Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to WB SecA carrying additional mutations, as shown in the inset box.


Figure 3Imidazole ( imi ) and either WT or mutant SecA were added to translocation intermediates ( IM ) pre - formed with SecA dN20 : : H6 . The samples were incubated for the indicated times before proteolysis with proteinase K ( Prot . K ) . The gel is representative of two replicates , except for the experiment with the R792G / K797G mutant and the 30 - min time points . Quantification of ( B ) . Points show IM band intensities normalized to the 0 - min time point for each replicate . Lines show average of replicates . Colors correspond to different SecA mutants as shown in the inset box . As in ( B ) , but ATPγS was added together with imidazole . The gel is representative of two replicates . Quantification of ( E ) as in ( C ) . As in ( D ) , but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA . The gel is representative of two replicates . Quantification of ( G ) as in ( F ) , except that both the intensities of the IM ( darker colors ) and FL ( lighter colors ) bands were measured .

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

Figure 3Imidazole (imi) and either WT or mutant SecA were added to translocation intermediates (IM) pre-formed with SecA dN20::H6. The samples were incubated for the indicated times before proteolysis with proteinase K (Prot. K). The gel is representative of two replicates, except for the experiment with the R792G/K797G mutant and the 30-min time points. Quantification of (B). Points show IM band intensities normalized to the 0-min time point for each replicate. Lines show average of replicates. Colors correspond to different SecA mutants as shown in the inset box. As in (B), but ATPγS was added together with imidazole. The gel is representative of two replicates. Quantification of (E) as in (C). As in (D), but DTT was added with ATPγS ten minutes after the addition of imidazole and WT or mutant SecA. The gel is representative of two replicates.   Quantification of (G) as in (F), except that both the intensities of the IM (darker colors) and FL (lighter colors) bands were measured.


Figure 1Time - lapse imaging of an oocyte expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF - dependent MII arrest . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown after anaphase I onset ( min : sec ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 1Time-lapse imaging of an oocyte expressing CENP-C-EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints of four stages from anaphase I onset to CSF-dependent MII arrest. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown after anaphase I onset (min:sec). Scale bars, 5 μm.


Figure 2Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1 . The black arrow on the scheme above the chart indicates chromosome - centre distance . Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . Changes in the speed of all chromosomes observed for the oocyte from Fig 1 . Chromosome parameters are shown in A - C on the vertical axis , and time after anaphase I onset is shown in min on the horizontal axis . Thin grey lines in A - C represent individual chromosomes ; the distantly observed chromosome is highlighted by dashed line ; the mean value for each timepoint is indicated by thick black line . The stages between anaphase I and metaphase II are color - coded and labelled on the top of the charts . On the schemes above the charts in ( A ) and ( C ) , chromosomes are red with green centromeres . ( C ) Changes in inter - centromere distance ( indicated on a scheme by the black arrow ) , observed for the oocyte from Fig 1 . Kinetochore - MT attachments at the prometaphase II stage were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maximum intensity projection through all z - planes containing MTs . Representative chromosomes displaying syntelic , amphitelic or merotelic / lateral attachments are enclosed in white frames and their enlarged single z - plane images are shown to the right . PB : polar body . Scale bar , 10μm . Chromosomes with syntelic ( red ) , amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . Data shown for 156 chromosomes taken from 8 prometaphase II oocytes .

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 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 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 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

Figure 2Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. Changes in the distance to the centre for all chromosomes in a representative oocyte undergoing transition from anaphase I to metaphase II from Fig 1. The black arrow on the scheme above the chart indicates chromosome-centre distance.Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. Changes in the speed of all chromosomes observed for the oocyte from Fig 1.Chromosome parameters are shown in A-C on the vertical axis, and time after anaphase I onset is shown in min on the horizontal axis. Thin grey lines in A-C represent individual chromosomes; the distantly observed chromosome is highlighted by dashed line; the mean value for each timepoint is indicated by thick black line. The stages between anaphase I and metaphase II are color-coded and labelled on the top of the charts. On the schemes above the charts in (A) and (C), chromosomes are red with green centromeres. (C) Changes in inter-centromere distance (indicated on a scheme by the black arrow), observed for the oocyte from Fig 1.Kinetochore-MT attachments at the prometaphase II stage were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maximum intensity projection through all z-planes containing MTs. Representative chromosomes displaying syntelic, amphitelic or merotelic/lateral attachments are enclosed in white frames and their enlarged single z-plane images are shown to the right. PB: polar body. Scale bar, 10μm. Chromosomes with syntelic (red), amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). Data shown for 156 chromosomes taken from 8 prometaphase II oocytes.


Figure 3Time - lapse imaging of a representative MII oocyte with a normal segregation pattern ( i . e . all 20 chromosomes show synchronous equational chromatid separation at anaphase onset , leaving no chromatids behind at the midzone ) , expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles . The 3D positions of centromeres are shown as black dots in a side view ( along the spindle equator , middle row ) and top view ( perpendicular to the equator plane , bottom row ) . Tracks of individual centromeres are color - coded according to their speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 3Time-lapse imaging of a representative MII oocyte with a normal segregation pattern (i.e. all 20 chromosomes show synchronous equational chromatid separation at anaphase onset, leaving no chromatids behind at the midzone), expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. Number next to the circles enclosing segregating chromatids denotes amount of chromatids segregated to each of the poles. The 3D positions of centromeres are shown as black dots in a side view (along the spindle equator, middle row) and top view (perpendicular to the equator plane, bottom row). Tracks of individual centromeres are color-coded according to their speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Figure 4Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . On the schemes near the charts chromosomes are red with green centromeres ; spindle equator in A and spindle axis in B - C are indicated by dashed lines . Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern , shown in Fig 3 . The analysed distance is indicated by a black arrowed line on the scheme above the chart . Changes in distance to the spindle axis ( shown on the scheme by a black arrowed line ) for all chromosomes in oocyte from Fig 3 . Changes in chromosome orientation for all chromosomes in oocyte from Fig 3 . The orientation is characterized by the angle ( highlighted black ) between the spindle axis and the line connecting the sister centromeres ( black solid line on the scheme ) . Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A - D on the vertical axis , and time in min on the horizontal axis relative to the anaphase II onset . Thin grey lines in A - D represent individual chromosomes ; thick black line in C - D shows the mean value for each timepoint . ( D ) Changes in inter - centromere distance , shown on the scheme by black arrowed line , for all chromosomes in oocyte from Fig 3 . Kinetochore - MT attachments in oocytes released from the CSF - dependent metaphase arrest were visualized using an anti - tubulin antibody ( white ) , an anti - centromeric ACA antibody ( green ) and DAPI to label chromatin ( red ) . The image represents maxiumum intensity projection through all z - planes containing MTs . Single z - planes of representative chromosomes displaying amphitelic or merotelic / lateral attachments are shown to the right . Scale bar , 10μm . Chromosomes with amphitelic ( green ) and merotelic / lateral ( grey ) attachments were plotted according to their inter - centromere distance ( on the horizontal axis ) and distance to spindle equator ( vertical axis ) . The inter - centromere distance in fixed oocytes is 1 . 2 ± 0 . 2 μm for amphitelic and 1 . 0 ± 0 . 2 μm for merotelic attachments ( Mean ± SD ) . Data shown for 95 chromosomes taken from 5 oocytes released from the CSF - dependent metaphase II arrest .

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 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 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

Figure 4Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by dashed lines. Changes in distance to the equator plane for all chromosomes in the representative oocyte with normal segregation pattern, shown in Fig 3. The analysed distance is indicated by a black arrowed line on the scheme above the chart. Changes in distance to the spindle axis (shown on the scheme by a black arrowed line) for all chromosomes in oocyte from Fig 3. Changes in chromosome orientation for all chromosomes in oocyte from Fig 3. The orientation is characterized by the angle (highlighted black) between the spindle axis and the line connecting the sister centromeres (black solid line on the scheme).Chromosome parameters obtained for a representative oocyte with normal segregation pattern are shown in A-D on the vertical axis, and time in min on the horizontal axis relative to the anaphase II onset. Thin grey lines in A-D represent individual chromosomes; thick black line in C-D shows the mean value for each timepoint. (D) Changes in inter-centromere distance, shown on the scheme by black arrowed line, for all chromosomes in oocyte from Fig 3.Kinetochore-MT attachments in oocytes released from the CSF-dependent metaphase arrest were visualized using an anti-tubulin antibody (white), an anti-centromeric ACA antibody (green) and DAPI to label chromatin (red). The image represents maxiumum intensity projection through all z-planes containing MTs. Single z-planes of representative chromosomes displaying amphitelic or merotelic/lateral attachments are shown to the right. Scale bar, 10μm. Chromosomes with amphitelic (green) and merotelic/lateral (grey) attachments were plotted according to their inter-centromere distance (on the horizontal axis) and distance to spindle equator (vertical axis). The inter-centromere distance in fixed oocytes is 1.2±0.2 μm for amphitelic and 1.0±0.2 μm for merotelic attachments (Mean±SD). Data shown for 95 chromosomes taken from 5 oocytes released from the CSF-dependent metaphase II arrest.


Figure 5A time - lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP - C - EGFP ( centromeres , green ) and H2B - mCherry ( chromatin , red ) . Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II . A lagging chromatid is indicated by a blue arrowhead . Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole . The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots , and centromeres of laggard - producing chromosome as red dots in a side view along the spindle equator . One of the centromeres is left behind at anaphase ( blue arrowhead ) , but will segregate equationally . Tracks of individual centromeres are color - coded according to the speed , as indicated by the colour bar . Time is shown relative to anaphase II onset ( min : sec ) . Scale bars , 5 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 O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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 5A time-lapse imaging of a representative MII oocyte with visible lagging chromatid at midzone during anaphase II expressing CENP-C- EGFP (centromeres, green) and H2B-mCherry (chromatin, red). Upper row shows maximum intensity z projection images from representative timepoints at metaphase II and anaphase II. A lagging chromatid is indicated by a blue arrowhead. Number next to the circles enclosing the segregating chromatids denotes number of chromatids at each pole. The bottom row depicts 3D positions for centromeres of normally segregating chromosomes as black dots, and centromeres of laggard-producing chromosome as red dots in a side view along the spindle equator. One of the centromeres is left behind at anaphase (blue arrowhead), but will segregate equationally. Tracks of individual centromeres are color-coded according to the speed, as indicated by the colour bar. Time is shown relative to anaphase II onset (min:sec). Scale bars, 5 μm.


Figure 6Chromosome parameters obtained for laggard - producing chromosomes are shown in A - D on the vertical axis , time in min on the horizontal axis relative to the anaphase II onset . Light blue lines indicate 12 laggard - producing chromosomes with correct ( equational ) chromatid segregation ; darker line highlights one chromosome with chromatid non - disjunction . On the schemes near the charts chromosomes are red with green centromeres ; spindle equator in A and spindle axis in B - C are indicated by black dashed lines . Changes in the distance to the equator plane ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . All laggard - producing chromosomes are positioned above the equator at the start of analysis period . Black dotted lines indicate the average thickness of the metaphase plate . Changes in the distance to spindle axis ( shown by a black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Black dotted line indicates the average radius of the metaphase plate . Changes in the angle to the spindle axis ( highlighted in black on the scheme ) for 13 laggard - producing chromosomes . Dashed line on the chart indicates the mean angle for normally segregating chromosomes . Changes in the inter - centromere distances ( shown by black arrowed line on the scheme ) for 13 laggard - producing chromosomes . Dashed line indicates the mean inter - centromere distance for normally segregating chromosomes .

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

Figure 6Chromosome parameters obtained for laggard-producing chromosomes are shown in A-D on the vertical axis, time in min on the horizontal axis relative to the anaphase II onset. Light blue lines indicate 12 laggard-producing chromosomes with correct (equational) chromatid segregation; darker line highlights one chromosome with chromatid non-disjunction. On the schemes near the charts chromosomes are red with green centromeres; spindle equator in A and spindle axis in B-C are indicated by black dashed lines. Changes in the distance to the equator plane (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. All laggard-producing chromosomes are positioned above the equator at the start of analysis period. Black dotted lines indicate the average thickness of the metaphase plate. Changes in the distance to spindle axis (shown by a black arrowed line on the scheme) for 13 laggard-producing chromosomes. Black dotted line indicates the average radius of the metaphase plate. Changes in the angle to the spindle axis (highlighted in black on the scheme) for 13 laggard-producing chromosomes. Dashed line on the chart indicates the mean angle for normally segregating chromosomes. Changes in the inter-centromere distances (shown by black arrowed line on the scheme) for 13 laggard-producing chromosomes. Dashed line indicates the mean inter-centromere distance for normally segregating chromosomes.