Phase - contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists ( Wnt3a / RSPO1 ) over 2 passages ( Scale bar : 500µm ) . Differences in cell number were confirmed by the respective cell viability assays ( performed in technical triplicates ) . Data represent the mean ± SEM for 5 different OC organoid lines ( n = 5 ) . * * * p < 0 . 001 , two - sided Student ` s t - test .

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Phase-contrast images of cancer organoids which entered growth arrest upon treatment with Wnt agonists (Wnt3a/RSPO1) over 2 passages (Scale bar: 500µm). Differences in cell number were confirmed by the respective cell viability assays (performed in technical triplicates). Data represent the mean ±SEM for 5 different OC organoid lines (n=5). *** p < 0.001, two-sided Student`s t-test.


The number of CD133 + cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a / RSPO1 and with or without Noggin over 2 passages , confirms a sharp drop in stemness in the presence of Wnt agonists .

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The number of CD133+ cells determined by FACS for OC7 and OC4 organoids grown in different media with or without Wnt3a/RSPO1 and with or without Noggin over 2 passages, confirms a sharp drop in stemness in the presence of Wnt agonists.


Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids , proving that the growth inhibitory effect is due to the presence of Wnt agonists . The bar plot represents the mean ± SEM of triplicates performed with a luminescent cell viability assay . * p < 0 . 05 , * * * p < 0 . 001 , two - sided Student ` s t - test .

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Addition of conditioned medium collected from the parental Wnt3a cell line did not suppress growth of cancer organoids, proving that the growth inhibitory effect is due to the presence of Wnt agonists. The bar plot represents the mean ±SEM of triplicates performed with a luminescent cell viability assay. * p < 0.05, *** p < 0.001, two-sided Student`s t-test.


Comparative analysis of microarray data revealed consistent up - regulation of MYCN , the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1 , AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids . Differential expression was determined either by single - color microarray for the cancer samples ( 8 replicates ) or dual - color microarray for the knockdowns ( 2 replicates ) and is significant for all genes with p < 0 . 05 .

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Comparative analysis of microarray data revealed consistent up-regulation of MYCN, the canonical Wnt inhibitors KREMEN2 and DKK3 as well as differentiation marker FOXJ1, AR and PGR in HGSOC cancer organoids as well as triple KD FT organoids. Differential expression was determined either by single-color microarray for the cancer samples (8 replicates) or dual-color microarray for the knockdowns (2 replicates) and is significant for all genes with p < 0.05.


Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs . FTM conditions . Depicted is the mean ± SEM of the normalized MYCN mRNA expression level of three biological replicates ( n = 3 ) . * * p < 0 . 01 , * * * p < 0 . 001 , two - sided Student ` s t - test .

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Transcriptional induction of MYCN is confirmed by qPCR for three patients between cancer organoids and normal FT organoids as well as triple KD organoids grown in OCM vs. FTM conditions. Depicted is the mean ±SEM of the normalized MYCN mRNA expression level of three biological replicates (n=3). ** p < 0.01, *** p < 0.001, two-sided Student`s t-test.


Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids . Differential expression determined by dual - color microarray for 2 biological replicates was significant for all genes with p ≤ 0 . 005 .

Microarray data revealed that expression levels of Wnt target genes are downregulated in triple KD compared to WT control FT organoids. Differential expression determined by dual-color microarray for 2 biological replicates was significant for all genes with p ≤ 0.005.


FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples . Data represent the mean ± SEM of technical triplicates .

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FOXJ1 expression was upregulated in HGSOC organoids in FTM compared to OCM as shown by qPCR analysis of 3 different patient samples. Data represent the mean ±SEM of technical triplicates.


A ) Perturbation in the free energy of PKD2L1 opening ( ∆ G ° ) and difference in closure rates ( ∆ τclose ) inducted by substitutions within the pore module . Error bars = S . D as estimated by the propagation of error equation , as descried in statistics subsection .

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A) Perturbation in the free energy of PKD2L1 opening (∆G°) and difference in closure rates (∆τclose) inducted by substitutions within the pore module. Error bars = S.D as estimated by the propagation of error equation, as descried in statistics subsection.


B ) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure - mutations which cause slower closure ( + ∆ τclose ) while also increasing the free energy opening ( + ∆ G ° ) .

B) Exemplar tail currents from mutant channels where the energetics of opening are disassociated with the rate of channel closure- mutations which cause slower closure (+∆τclose) while also increasing the free energy opening (+∆G°).


D ) Comparing voltage - dependent opening based on normalized tail currents measured from WT , single and double mutant channels . Top , single and double mutant channels including mutations to the outer and inner gate . Middle and bottom , control groups comparing tail currents from non - gate S4 - S5 linker mutations ( D476A and G479A ) and those from the outer and inner gates . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group .

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D) Comparing voltage-dependent opening based on normalized tail currents measured from WT, single and double mutant channels. Top, single and double mutant channels including mutations to the outer and inner gate. Middle and bottom, control groups comparing tail currents from non-gate S4-S5 linker mutations (D476A and G479A) and those from the outer and inner gates. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.


E ) The coupling energy ( ∆ ∆ G ° ) resulting from the mutant cycle analysis resulting from the data shown in D . Error bars indicate standard deviation for as estimated by the propagation of error equation , as descried in statistics subsection . Number cells tested ( N ) are indicated in parentheses for each group .

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E) The coupling energy (∆∆G°) resulting from the mutant cycle analysis resulting from the data shown in D. Error bars indicate standard deviation for as estimated by the propagation of error equation, as descried in statistics subsection. Number cells tested (N) are indicated in parentheses for each group.


B ) Summary of the change in voltage - dependent inactivation ( ∆ V1 / 2 Inact . ) of pore mutant channels . ∆ V1 / 2 Inact . , are plotted relative to the pore module topology of the channel .

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B) Summary of the change in voltage-dependent inactivation (∆V1/2 Inact.) of pore mutant channels. ∆V1/2 Inact., are plotted relative to the pore module topology of the channel.


C ) Voltage - dependent inactivation of test pulse tail currents recorded from WT , single and double mutant channels .

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C) Voltage-dependent inactivation of test pulse tail currents recorded from WT, single and double mutant channels.


C ) Exemplar whole cell currents ( Top ) at the zero , three and seven minute time points . Current records ( Bottom ) from cells expressing the F514C : P538C and treated with extracellular 100 μM 1 , 2 - methanediyl bismethanethiosulfonate ( M2M ) .

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C) Exemplar whole cell currents (Top) at the zero, three and seven minute time points. Current records (Bottom) from cells expressing the F514C:P538C and treated with extracellular 100 μM 1,2-methanediyl bismethanethiosulfonate (M2M).


D ) Box ( S . E . M . ) and whisker ( S . D . ) plots comparing tail currents normalized to their initial amplitude at 3 minute ( Left ) and 7 - minute time points ( Right ) . N = 6 cells per group . Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line . P - values results from a one - way ANOVA statistical analysis is shown above the plots . Dashed lines the level normalcy , no change in Itail relative to time point zero .

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D) Box (S.E.M.) and whisker (S.D.) plots comparing tail currents normalized to their initial amplitude at 3 minute (Left) and 7-minute time points (Right). N = 6 cells per group. Results from each individual cells are indicated by grey open circles and the average for the channel is indicated by a horizontal line. P-values results from a one-way ANOVA statistical analysis is shown above the plots. Dashed lines the level normalcy, no change in Itail relative to time point zero.


Left : Fluorescence changes of IRDye700 labelled dsDNA ( at 125 nM ) when incubated at increasing ID2 ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FANCD2 ( D2Ub ) concentrations ( ranging from 1 . 3 nM to 5 . 9 µM ) . Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from model fitting . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 23 - 24 data points each ) .

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Left: Fluorescence changes of IRDye700 labelled dsDNA (at 125 nM) when incubated at increasing ID2 (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FANCD2 (D2Ub) concentrations (ranging from 1.3 nM to 5.9 µM). Measurement of fluorescence enhancement for each ID2 complex was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from model fitting. Error bars: Asymmetric 95% confidence intervals from non-linear regression (23-24 data points each).


Assessment of protein - DNA interactions using Electro - mobility shift assays ( EMSA ) . IRDye700 labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of non / single / double - ubiquitinated ID2 ( His6 - TEV - V5 - FANCI and FLAG - FANCD2 ) protein complexes ( I + D2 , IUb + D2Ub , I + D2Ub ) or ubiquitinated FLAG - FANCD2 ( D2Ub ) . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . EMSA gels of ID2 complexes are representative of 3 replicate experiments .

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Assessment of protein-DNA interactions using Electro-mobility shift assays (EMSA). IRDye700 labelled dsDNA (at 2 nM) was incubated with indicated amounts of non/single/double-ubiquitinated ID2 (His6-TEV-V5-FANCI and FLAG-FANCD2) protein complexes (I+D2, IUb+D2Ub, I+D2Ub) or ubiquitinated FLAG-FANCD2 (D2Ub). Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. EMSA gels of ID2 complexes are representative of 3 replicate experiments.


Left : CryoEM 2D classes of ID2Ub and IUbD2Ub - dsDNA . Right : 3D reconstructions of ID2Ub ( EMD - 10843 ) and IUbD2Ub - DNA ( EMD - 10844 ) . Both structures exhibit a torus - like shape .

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Left: CryoEM 2D classes of ID2Ub and IUbD2Ub-dsDNA. Right: 3D reconstructions of ID2Ub (EMD-10843) and IUbD2Ub-DNA (EMD-10844). Both structures exhibit a torus-like shape.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI can be efficiently ubiquitinated in the presence of DNA . Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI can be efficiently ubiquitinated in the presence of DNA. Reactions were performed at 2 μM FANCI substrate for 60 min in the absence or presence of 4 µM ssDNA.


Both wild - type ( IWT ) and R1285Q mutant ( IR1285Q ) FANCI efficiently associate with FANCD2 . Fluorescence changes occurring when RED - tris - NTA - labelled FANCI ( IWT or IR1285Q ; both at 60 nM ) is incubated at increasing concentrations of FANCD2 ( ranging from 2 . 48 nM to 5 . 08 µM ) . Titrations were conducted twice ( two technical replicates ) and all data points for each protein - combination were used in fitting of a one - site binding model . Apparent Kd values ( mean ± SEM ) derived from fitting of a one - site binding model to the 24 data points of the two technical replicates are shown .

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Both wild-type (IWT) and R1285Q mutant (IR1285Q) FANCI efficiently associate with FANCD2. Fluorescence changes occurring when RED-tris-NTA-labelled FANCI (IWT or IR1285Q; both at 60 nM) is incubated at increasing concentrations of FANCD2 (ranging from 2.48 nM to 5.08 µM). Titrations were conducted twice (two technical replicates) and all data points for each protein-combination were used in fitting of a one-site binding model. Apparent Kd values (mean ± SEM) derived from fitting of a one-site binding model to the 24 data points of the two technical replicates are shown.


FANCD2 within an IR1285QD2 complex is resistant to ubiquitination . Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA . Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS - PAGE .

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FANCD2 within an IR1285QD2 complex is resistant to ubiquitination. Reactions were performed at 4 μM ID2 substrate and 16 μM dsDNA. Progress of FANCD2 and FANCI ubiquitination was monitored by western blotting following SDS-PAGE.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by PIFE . Left : Fluorescence changes when IRD700 - labelled dsDNA ( at 125 nM ) is incubated at increasing concentrations of IR1285Q + D2 or IR1285Q + D2Ub ( complex concentrations ranging from 1 . 54 nM to 3 . 8 µM ) . Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes ( two technical repeats ) and all data points for each protein - combination were used in fitting of a one - site binding model . Right : Bar graph showing mean apparent Kd values calculated from the one - site binding model . Error bars : Asymmetric 95 % confidence intervals from non - linear regression ( 22 data points each ) . IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values ( from data points shown in Figure 1A ) are also shown for comparison .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by PIFE. Left: Fluorescence changes when IRD700-labelled dsDNA (at 125 nM) is incubated at increasing concentrations of IR1285Q +D2 or IR1285Q +D2Ub (complex concentrations ranging from 1.54 nM to 3.8 µM). Measurement of fluorescence enhancement for each protein combination was conducted for two separately prepared complexes (two technical repeats) and all data points for each protein-combination were used in fitting of a one-site binding model. Right: Bar graph showing mean apparent Kd values calculated from the one-site binding model. Error bars: Asymmetric 95% confidence intervals from non-linear regression (22 data points each). IWT + D2 and IWT + D2Ub previously calculated curves and corresponding Kd values (from data points shown in Figure 1A) are also shown for comparison.


FANCD2 ubiquitination cannot robustly enhance ID2 - DNA binding when complexed with IR1285Q , as determined by EMSAs . IRDye700 - labelled dsDNA ( at 2 nM ) was incubated with indicated amounts of ID2 ( His6 - FANCI or His6 - FANCIR1285Q mixed with non - ubiquitinated or ubiquitinated FLAG - FANCD2 . Mixes were ran on non - denaturing gels , and the resolved free - and protein - bound DNA bands were visualized using an infrared scanner . Gels shown are representative of two replicate experiments .

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FANCD2 ubiquitination cannot robustly enhance ID2-DNA binding when complexed with IR1285Q, as determined by EMSAs. IRDye700-labelled dsDNA (at 2 nM) was incubated with indicated amounts of ID2 (His6-FANCI or His6-FANCIR1285Q mixed with non-ubiquitinated or ubiquitinated FLAG-FANCD2. Mixes were ran on non-denaturing gels, and the resolved free- and protein-bound DNA bands were visualized using an infrared scanner. Gels shown are representative of two replicate experiments.


USP1 - UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI ( I ) , but not in the presence of ubiquitinated FANCI ( IUb ) . Ubiquitinated FANCD2 ( D2Ub ) was mixed with a 50 base pair dsDNA and either His6 - TEV - V5 - FANCI ( I ) , ubiquitinated His6 - TEV - V5 - FANCI ( IUb ) or no protein ; protein - DNA mixes were subsequently incubated with USP1 - UAF1 ( at 25 nM or 100 nM ) for indicated time periods . Deubiquitination of D2Ub and IUb was assessed , following SDS - PAGE , by Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody .

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USP1-UAF1 can efficiently deubiquitinate D2Ub in the presence or absence of FANCI (I), but not in the presence of ubiquitinated FANCI (IUb). Ubiquitinated FANCD2 (D2Ub) was mixed with a 50 base pair dsDNA and either His6-TEV-V5-FANCI (I), ubiquitinated His6-TEV-V5-FANCI (IUb) or no protein; protein-DNA mixes were subsequently incubated with USP1-UAF1 (at 25 nM or 100 nM) for indicated time periods. Deubiquitination of D2Ub and IUb was assessed, following SDS-PAGE, by Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody.


USP7 and USP2 can deubiquitinate D2Ub , but their activity towards D2Ub is greatly reduced in the presence of FANCI ( I ) or ubiquitinated FANCI ( IUb ) . Reactions were set - up as in A , but with 100 nM USP7 or USP2 . Deubiquitination was assessed , following SDS - PAGE , by Coomassie staining .

USP7 and USP2 can deubiquitinate D2Ub, but their activity towards D2Ub is greatly reduced in the presence of FANCI (I) or ubiquitinated FANCI (IUb). Reactions were set-up as in A, but with 100 nM USP7 or USP2. Deubiquitination was assessed, following SDS-PAGE, by Coomassie staining.


Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity / resistance to USP1 - UAF1 mediated deubiquitination of resulting products . Deubiquitination reactions in the presence or absence of USP1 - UAF1 ( 100 nM ) incubated for 30 minutes .

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Time course of FANCI ubiquitination with various ubiquitin mutants and corresponding sensitivity/resistance to USP1-UAF1 mediated deubiquitination of resulting products. Deubiquitination reactions in the presence or absence of USP1-UAF1 (100 nM) incubated for 30 minutes.


Time course of USP1 - UAF1 mediated deubiquitination of IUbD2Ub - DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild - type ( WT ) ubiquitin . Progress of deubiquitination reaction was assessed following SDS - PAGE , by both Coomassie staining of the gels , as well as by western blotting of transferred blots with a specific FANCD2 antibody . FANCI ubiquitination with indicated ubiquitin - mutants ( or WT ) and subsequent deubiquitination of resulting IUbD2Ub - DNA complexes were conducted twice ; the residual FANCD2 ubiquitination , calculated from the FANCD2 blots for each time - point , was plotted for each type of ubiquitin in the protein complex , and corresponding deubiquitination progress curves were fitted using a one - phase decay model .

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Time course of USP1-UAF1 mediated deubiquitination of IUbD2Ub-DNA complexes consisting of D2Ub a 50 base pair dsDNA and IUb produced with various ubiquitin mutants or wild-type (WT) ubiquitin. Progress of deubiquitination reaction was assessed following SDS-PAGE, by both Coomassie staining of the gels, as well as by western blotting of transferred blots with a specific FANCD2 antibody. FANCI ubiquitination with indicated ubiquitin-mutants (or WT) and subsequent deubiquitination of resulting IUbD2Ub-DNA complexes were conducted twice; the residual FANCD2 ubiquitination, calculated from the FANCD2 blots for each time-point, was plotted for each type of ubiquitin in the protein complex, and corresponding deubiquitination progress curves were fitted using a one-phase decay model.


( B ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP encoding IFITM3 variants containing N - terminal FLAG ( pQCXIP - FLAG - IFITM3 ) and immunostained with anti - FLAG M2 antibody . Scale bar , 5 μm .

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(B) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP encoding IFITM3 variants containing N-terminal FLAG (pQCXIP-FLAG-IFITM3) and immunostained with anti-FLAG M2 antibody. Scale bar, 5 μm.


( C ) Flow cytometry analysis of transfected cells in ( B ) immunostained with anti - FLAG M2 antibody . Corresponding mean flourescence intensity values are shown .

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(C) Flow cytometry analysis of transfected cells in (B) immunostained with anti-FLAG M2 antibody. Corresponding mean flourescence intensity values are shown.


( D ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( B ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies .

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(D) SDS-PAGE of cell lysates derived from 293T cells transfected in (B) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( E ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 variants , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag ( 183 - H12 - 5C ) .

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(E) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 variants, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag (183-H12-5C).


( F ) Opti - prep purified supernatants from ( E ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . Mean + SD of 5 - 10 experiments ( each performed with virus produced from an independent transfection ) is shown . ( G ) Virus used in ( E ) were used to infect SupT1 cells and productive infection was scored at 48 - 60 hours by immunostaining with the KC57 antibody and flow cytometry . Expression of pCMV - MxA served as a negative control . The mean + SD of 5 - 10 experiments is shown .

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(F) Opti-prep purified supernatants from (E) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. Mean + SD of 5-10 experiments (each performed with virus produced from an independent transfection) is shown. (G) Virus used in (E) were used to infect SupT1 cells and productive infection was scored at 48-60 hours by immunostaining with the KC57 antibody and flow cytometry. Expression of pCMV-MxA served as a negative control. The mean + SD of 5-10 experiments is shown.


( H ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg pQCXIP - FLAG - IFITM3 variants and immunostained with anti - FLAG M2

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(H) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg pQCXIP-FLAG-IFITM3 variants and immunostained with anti-FLAG M2 antibody


( I ) SDS - PAGE of cell lysates derived from 293T cells transfected in ( H ) followed by immunoblotting with anti - FLAG M2 and anti - actin antibodies .

(I) SDS-PAGE of cell lysates derived from 293T cells transfected in (H) followed by immunoblotting with anti-FLAG M2 and anti-actin antibodies.


( J ) SDS - PAGE of Opti - prep purified supernatants ( 25 ng p24 equivalent ) derived from 293T cells transfected with pQCXIP - FLAG - IFITM3 constructs , HIV - 1 pNL4 - 3 , and Vpr - Blam plasmid followed by immunoblotting with anti - FLAG M2 and anti - p24 Gag . Scale bar , 5 μm .

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(J) SDS-PAGE of Opti-prep purified supernatants (25 ng p24 equivalent) derived from 293T cells transfected with pQCXIP-FLAG-IFITM3 constructs, HIV-1 pNL4-3, and Vpr-Blam plasmid followed by immunoblotting with anti-FLAG M2 and anti-p24 Gag. Scale bar, 5 μm.


( K ) Opti - prep purified supernatants from ( J ) were incubated with SupT1 target cells for 4 hours and virus - cell fusion was scored by flow cytometry . The mean + SD of 3 experiments is shown .

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(K) Opti-prep purified supernatants from (J) were incubated with SupT1 target cells for 4 hours and virus-cell fusion was scored by flow cytometry. The mean + SD of 3 experiments is shown.


( A ) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet - inducible pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - IFITM3 Δ1 - 21 ( Tet - ON ) immunostained with anti - FLAG M2 antibody following overnight treatment with 500 ng / mL doxycycline . Scale bar , 10 μm .

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(A) Confocal fluorescent microscopy of SupT1 T cells transduced with Tet-inducible pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-IFITM3 Δ1-21 (Tet-ON) immunostained with anti-FLAG M2 antibody following overnight treatment with 500 ng/mL doxycycline. Scale bar, 10 μm.


( B ) Tet - ON SupT1 cell lines were treated or not with 500 ng / mL doxycycline overnight and induction of IFITM3 protein was assessed anti - FLAG M2 immunostaining and flow cytometry .

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(B) Tet-ON SupT1 cell lines were treated or not with 500 ng/mL doxycycline overnight and induction of IFITM3 protein was assessed anti-FLAG M2 immunostaining and flow cytometry.


( C ) Tet - ON SupT1 cell lines were productively infected with NL4 - 3 VSV - G and then treated with 500 ng / mL doxycycline overnight to produce virus from IFITM3 - and IFITM3 + cells . 25 ng p24 equivalents of purified were used to infect fresh Tet - ON SupT1 cells cell targets , which were previously treated with 500 ng / mL doxycycline or not . Infection was scored at 72h post - infection by immunostaining with KC57 and flow cytometry . Mean + SD of 3 experiments are shown .

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(C) Tet-ON SupT1 cell lines were productively infected with NL4-3 VSV-G and then treated with 500 ng/mL doxycycline overnight to produce virus from IFITM3- and IFITM3+ cells. 25 ng p24 equivalents of purified were used to infect fresh Tet-ON SupT1 cells cell targets, which were previously treated with 500 ng/mL doxycycline or not. Infection was scored at 72h post-infection by immunostaining with KC57 and flow cytometry. Mean + SD of 3 experiments are shown.


( D ) 25 ng p24 equivalents of virus used in ( C ) was subjected to SDS - PAGE . Immunoblotting was performed using the anti - FLAG M2 , anti - p24 Gag , and anti - gp120 Env ( NIH # 288 ) antibodies .

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(D) 25 ng p24 equivalents of virus used in (C) was subjected to SDS-PAGE. Immunoblotting was performed using the anti-FLAG M2, anti-p24 Gag, and anti-gp120 Env (NIH #288) antibodies.


( E ) 293T cells were transfected with the indicated amount of pQCXIP - FLAG - IFITM3 or pQCXIP - FLAG - Δ1 - 21 or pCMV - MxA and 1 . 0 μg of HIV - 1 pNL4 - 3 . SDS - PAGE was performed on whole cell lysates followed by immunoblotting with anti - FLAG M2 , anti - p24 Gag , anti - Env gp120 ( NIH # 288 ) , and anti - tubulin antibodies . Western blot images are representative of 3 independent experiments . Unpaired t - test , * p < 0 . 05 , ♯ p = 0 . 12 , non statistically significant . Comparisons were made between the condition indicated and the correponding condition in Tet - ON IFITM3 cells .

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(E) 293T cells were transfected with the indicated amount of pQCXIP-FLAG-IFITM3 or pQCXIP-FLAG-Δ1-21 or pCMV-MxA and 1.0 μg of HIV-1 pNL4-3. SDS-PAGE was performed on whole cell lysates followed by immunoblotting with anti-FLAG M2, anti-p24 Gag, anti-Env gp120 (NIH #288), and anti-tubulin antibodies. Western blot images are representative of 3 independent experiments. Unpaired t-test, *p<0.05, ♯p=0.12, non statistically significant. Comparisons were made between the condition indicated and the correponding condition in Tet-ON IFITM3 cells.


( A ) Confocal fluorescence microscopy of 293T cells transfected with 0 . 5 μg of pQCXIP - FLAG - Human IFITM3 mutated at indicated residues and immunostained with anti - FLAG M2 antibody . Scale bars , 5 μm . The P17L , P18H , Δ22 - 24 , P18L / L23P and P18H / Δ19 - 23 mutations were introduced into the human IFITM3 background .

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(A) Confocal fluorescence microscopy of 293T cells transfected with 0.5 μg of pQCXIP-FLAG-Human IFITM3 mutated at indicated residues and immunostained with anti-FLAG M2 antibody. Scale bars, 5 μm. The P17L, P18H, Δ22-24, P18L/L23P and P18H/Δ19-23 mutations were introduced into the human IFITM3 background.


( B ) SDS - PAGE of FLAG - immunoprecipitated fractions from 293T cells following transfection of 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants and 0 . 5 μg of pCI - HA - NEDD4 or pCI - HA - NEDD4 C867A . Immunoblotting was performed with anti - FLAG M2 , anti - IFITM3 ( Abcam , EPR5242 ) , and an anti - ubiquitin antibody ( Enzo Life Sciences , recognizing K29 - , K48 - , and K63 - linked mono - and polyubiquitinylated proteins ) . ' L ' indicates the presence of light chain antibody derived from the FLAG antibody - coupled beads used for immunoprecipitation . * , * * , and * * * indicate mono - , di - , and tri - ubiquitinated forms of IFITM3 .

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(B) SDS-PAGE of FLAG-immunoprecipitated fractions from 293T cells following transfection of 0.5 μg of pQCXIP-FLAG-IFITM3 variants and 0.5 μg of pCI-HA-NEDD4 or pCI-HA-NEDD4 C867A. Immunoblotting was performed with anti-FLAG M2, anti-IFITM3 (Abcam, EPR5242), and an anti-ubiquitin antibody (Enzo Life Sciences, recognizing K29-, K48-, and K63-linked mono- and polyubiquitinylated proteins). 'L' indicates the presence of light chain antibody derived from the FLAG antibody-coupled beads used for immunoprecipitation. *, **, and *** indicate mono-, di-, and tri-ubiquitinated forms of IFITM3.


( C ) SDS - PAGE of whole cell lysate input fractions from 293T cells used in ( B ) . Immunoblotting was performed with anti - FLAG M2 , anti - NEDD4 , and anti - actin .

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(C) SDS-PAGE of whole cell lysate input fractions from 293T cells used in (B). Immunoblotting was performed with anti-FLAG M2, anti-NEDD4, and anti-actin.


( D ) 293T cells were transfected with 1 . 0 μg of pNL4 - 3 and 0 . 5 μg of pQCXIP - FLAG - IFITM3 variants . Virus - containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells . Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post - infection . The means + SD of 3 experiments are shown .

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(D) 293T cells were transfected with 1.0 μg of pNL4-3 and 0.5 μg of pQCXIP-FLAG-IFITM3 variants. Virus-containing supernatants were harvested and 10 ng p24 equivalents were used to reinfect fresh SupT1 T cells. Productive infection was scored by immunostaining with the KC57 antibody and flow cytometry at 48 hours post-infection. The means + SD of 3 experiments are shown.


( E ) 2 . 0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV - 1NL4 - 3 in the presence of 2 μg / mL DEAE - Dextran . Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post - infection . The ' Colobus native ' sequence represents the full IFITM3 sequence identified in the colobus monkey . ( F ) As in ( E ) , except cells were challenged with Influenza A virus H1N1 PR / 8 / 34 , ( Charles River Laboratories ) , at a dose that resulted in ˜ 50 % infection of control ( Empty ) cells ( equivalent to 103 TCID50 / 0 . 2 mL ) . Infection was scored by immunostaining with an anti - IAV NP antibody and flow cytometry at 18 hours post - infection . Results are presented in a logarithmic scale . Means + SD of 3 - 5 experiments are shown . Unpaired t - test , * p < 0 . 05 , * * p < 0 . 005 .

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(E) 2.0 x 10ˆ5 cells were seeded and challenged with 10 ng of HIV-1NL4-3 in the presence of 2 μg/mL DEAE-Dextran. Productive infection was scored by immunostaining with KC57 and flow cytometry at 48 hours post-infection. The 'Colobus native' sequence represents the full IFITM3 sequence identified in the colobus monkey. (F) As in (E), except cells were challenged with Influenza A virus H1N1 PR/8/34, (Charles River Laboratories), at a dose that resulted in ˜50% infection of control (Empty) cells (equivalent to 103 TCID50 / 0.2 mL). Infection was scored by immunostaining with an anti-IAV NP antibody and flow cytometry at 18 hours post-infection. Results are presented in a logarithmic scale. Means + SD of 3-5 experiments are shown. Unpaired t-test, *p<0.05, **p<0.005.


Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK - p25 animal mice . Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte .

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Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from CK-p25 animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.


Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice . Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte .

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Heatmaps of enriched hippocampus phosphotyrosine peptides in hippocampus tissue from 5XFAD animal mice. Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte.


Colors indicate fold change relative to control animals on a log2 - scale . Row colors ( left ) indicate peptides from predicted cell - type specific proteins . Green = Astrocyte , Orange = Neuron , Blue = Microglia , Purple = Oligodendrocyte . ( E ) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals .

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Colors indicate fold change relative to control animals on a log2-scale. Row colors (left) indicate peptides from predicted cell-type specific proteins. Green = Astrocyte, Orange = Neuron, Blue = Microglia, Purple = Oligodendrocyte. (E) Heatmap showing enriched hippocampus phosphotyrosine peptides from 6 month old Tau P301S animals.


( F - G ) Relative abundances across all tissues for phosphotyrosine peptides : ( F ) SVyTEIK ( Siglec - F pY561 ) ; ( G ) LyDFVK ( Inpp5d pY868 ) . Bars indicate mean ± 95 % confidence interval ( CI ) ; n = 3 - 8 samples ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(F-G) Relative abundances across all tissues for phosphotyrosine peptides: (F) SVyTEIK (Siglec-F pY561); (G) LyDFVK (Inpp5d pY868). Bars indicate mean ± 95% confidence interval (CI); n = 3-8 samples; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( A ) Immunofluorescence ( IF ) staining showing Siglec - F and Iba1 localization in hippocampus CA3 region of 3wk induced CK ( top ) or CK - p25 ( bottom ) mice . Scale bars = 50 μm . Colors are : Magenta = Iba1 , Cyan = Siglec - F . Images are 60x super - resolution max z - stack projections taken from coronal slices . ( B - C ) Percent area coverage of ( B ) Iba1 and ( C ) Iba1 - proximal Siglec - F between CK and CK - p25 mice across hippocampal regions . Bars indicate mean ± 95 % CI ; n = 3 - 4 animals ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(A) Immunofluorescence (IF) staining showing Siglec-F and Iba1 localization in hippocampus CA3 region of 3wk induced CK (top) or CK-p25 (bottom) mice. Scale bars = 50 μm. Colors are: Magenta = Iba1, Cyan = Siglec-F. Images are 60x super-resolution max z-stack projections taken from coronal slices. (B-C) Percent area coverage of (B) Iba1 and (C) Iba1-proximal Siglec-F between CK and CK-p25 mice across hippocampal regions. Bars indicate mean ± 95% CI; n = 3-4 animals; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( D - E ) IF staining of Siglec - F and Iba1 in ( D ) 5XFAD 2 month hypothalamus ( HY ) and ( E ) 5XFAD 6 month dentate gyrus ( DG ) . Yellow = Aβ from methoxy X04 ( D ) or D54D2 ( E ) . Scale bars = 50 μm .

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(D-E) IF staining of Siglec-F and Iba1 in (D) 5XFAD 2 month hypothalamus (HY) and (E) 5XFAD 6 month dentate gyrus (DG). Yellow = Aβ from methoxy X04 (D) or D54D2 (E). Scale bars = 50 μm.


( F ) Percent area coverage of Iba1 - proximal Siglec - F relative to total Iba1 across all analyzed thalamic , cortical , and hippocampal regions of 6 month WT ( non - Tg ) and 5XFAD mice . Box indicates quartiles and whiskers indicate the last datum within 1 . 5 inter - quartile range ; n = 15 - 40 images ( 3 - 5 animals ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . ( G ) Percent area coverage of Siglec - F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non - Tg control mice . Linear regression R2 value is shown for images with ≥ 10 μm2 total amyloid volume .

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(F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. (G) Percent area coverage of Siglec-F localized to Iba1 compared to total Aβ plaque volume in each field of view across all analyzed images from 5XFAD and non-Tg control mice. Linear regression R2 value is shown for images with ≥10 μm2 total amyloid volume.


( F ) Percent area coverage of Iba1 - proximal Siglec - F relative to total Iba1 across all analyzed thalamic , cortical , and hippocampal regions of 6 month WT ( non - Tg ) and 5XFAD mice . Box indicates quartiles and whiskers indicate the last datum within 1 . 5 inter - quartile range ; n = 15 - 40 images ( 3 - 5 animals ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(F) Percent area coverage of Iba1-proximal Siglec-F relative to total Iba1 across all analyzed thalamic, cortical, and hippocampal regions of 6 month WT (non-Tg) and 5XFAD mice. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 15-40 images (3-5 animals); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( H ) Cumulative distribution function ( CDF ) of the distance between Siglec - F + and Iba1 + mask voxels and nearest Aβ plaques in 6 month 5XFAD mice . Each value in the CDF represents the mean distance calculated from a single z - stack of a ROI containing ≥ 1 plaque of size ≥ 10 μm2 . n = 55 images ( 4 animals ) .

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(H) Cumulative distribution function (CDF) of the distance between Siglec-F+ and Iba1+ mask voxels and nearest Aβ plaques in 6 month 5XFAD mice. Each value in the CDF represents the mean distance calculated from a single z-stack of a ROI containing ≥ 1 plaque of size ≥10 μm2. n = 55 images (4 animals).


( I - J ) Relative RNA abundances for Siglec - 8 in ( H ) hippocampus and ( I ) posterior cingulate cortex post - mortem patient tissue . Linear regression R2 values are shown on plots .

(I-J) Relative RNA abundances for Siglec-8 in (H) hippocampus and (I) posterior cingulate cortex post-mortem patient tissue. Linear regression R2 values are shown on plots.


( K ) IF staining showing Siglec - 8 and Iba1 , and MHC - II in cortical white matter tissue from a patient with late - onset AD ( 20x wide - field ; Case ID : 01 - 43 ) . Scale bars = 50 μm . Images are 60x super - resolution max z - stack projections .

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(K) IF staining showing Siglec-8 and Iba1, and MHC-II in cortical white matter tissue from a patient with late-onset AD (20x wide-field; Case ID: 01-43). Scale bars = 50 μm. Images are 60x super-resolution max z-stack projections.


( L - M ) Percent area coverage of Siglec - 8 localized to Iba1 or MHC in individuals with non - AD or AD . In ( K ) each point represents one WM or GM region average from each individual . In ( L ) values and statistics are shown for each image analyzed . Boolean mask operators indicate : ° = mask dilation , ⋂ = intersection , ⋃ = union , NOT = inverse mask . Box indicates quartiles and whiskers indicate the last datum within 1 . 5 inter - quartile range ; n = 8 - 43 images ( n = 1 ND , 3 early - onset AD , 4 late - onset AD ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(L-M) Percent area coverage of Siglec-8 localized to Iba1 or MHC in individuals with non-AD or AD. In (K) each point represents one WM or GM region average from each individual. In (L) values and statistics are shown for each image analyzed. Boolean mask operators indicate: ° = mask dilation, ⋂ = intersection, ⋃ = union, NOT = inverse mask. Box indicates quartiles and whiskers indicate the last datum within 1.5 inter-quartile range; n = 8-43 images (n = 1 ND, 3 early-onset AD, 4 late-onset AD); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( A ) Flow cytometry quantification of BV - 2 Siglec - F expression levels . Cells were cotreated with 0 - 10 ng / mL IFNγ and 0 - 10 μM tofacitinib . Values are geometric mean fluorescence intensities ( gMFI ) . Bars indicate mean ± 95 % CI ; n = 2 - 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group .

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(A) Flow cytometry quantification of BV-2 Siglec-F expression levels. Cells were cotreated with 0-10 ng/mL IFNγ and 0-10 μM tofacitinib. Values are geometric mean fluorescence intensities (gMFI). Bars indicate mean ± 95% CI; n = 2-4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( B ) IF staining of iMGLs treated with IFNγ . Siglec - 8 fluorescence is only shown on non - nuclear ( Hoechst 33342 - ) regions . Images are 60x super - resolution max z - stack projections . Colors are : Red = Iba1 , Green = Siglec - 8 , Blue = 33342 . Scale bars = 20 μm .

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(B) IF staining of iMGLs treated with IFNγ. Siglec-8 fluorescence is only shown on non-nuclear (Hoechst 33342-) regions. Images are 60x super-resolution max z-stack projections. Colors are: Red = Iba1, Green = Siglec-8, Blue = 33342. Scale bars = 20 μm.


( C ) Quantification of Siglec - 8 on iMGLs treated with PBS or 25 ng / mL IFNα , IFNβ , or IFNγ . Values are mean Siglec - 8 intensity values from Iba1 + ; 33342 - regions . Bars indicate mean ± 95 % CI ; n = 8 images ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group .

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(C) Quantification of Siglec-8 on iMGLs treated with PBS or 25 ng/mL IFNα, IFNβ, or IFNγ. Values are mean Siglec-8 intensity values from Iba1+;33342- regions. Bars indicate mean ± 95% CI; n = 8 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( D ) Quantification of Siglec - 8 on iMGLs treated ± 25 ng / mL IFNγ and 5 μM tofacitinib . Legend is same as ( C ) . Bars indicate mean ± 95 % CI ; n = 5 - 20 images ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated group .

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(D) Quantification of Siglec-8 on iMGLs treated ± 25 ng/mL IFNγ and 5 μM tofacitinib. Legend is same as (C). Bars indicate mean ± 95% CI; n = 5-20 images (2 replicates); *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated group.


( A ) Confluency doubling times quantified from BV - 2 cells with stable retroviral expression of Siglec - F and related human Siglec constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field images . Cells grown in media alone or + 50 mU sialidase ( + SA ) . Bars indicate mean ± 95 % CI ; n = 3 - 12 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F groups .

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(A) Confluency doubling times quantified from BV-2 cells with stable retroviral expression of Siglec-F and related human Siglec constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Cells grown in media alone or + 50 mU sialidase (+SA). Bars indicate mean ± 95% CI; n = 3-12 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F groups.


( B ) Flow cytometry quantification of cell live / dead markers Annexin - V and propidium iodide on BV - 2 cells with stable retrovirus expression of Siglec - F constructs . % parent values are shown for early apoptotic ( Annexin - V + ; PI ‑ ) , late apoptotic ( Annexin - V + ; PI ‑ ) , and necrotic ( Annexin - V - ; PI + ) populations . Bars indicate mean ± SD ; n = 8 replicates .

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(B) Flow cytometry quantification of cell live / dead markers Annexin-V and propidium iodide on BV-2 cells with stable retrovirus expression of Siglec-F constructs. % parent values are shown for early apoptotic (Annexin-V+;PI‑), late apoptotic (Annexin-V+;PI‑), and necrotic (Annexin-V-;PI+) populations. Bars indicate mean ± SD; n = 8 replicates.


( C ) Confluency doubling times quantified from BV - 2 cells with dox - inducible expression of Siglec - F constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field images . Bars indicate mean ± SD ; n = 8 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

(C) Confluency doubling times quantified from BV-2 cells with dox-inducible expression of Siglec-F constructs. Plots indicating doubling time (normalized to 2xY->F constructs) estimated from Incucyte bright-field images. Bars indicate mean ± SD; n = 8 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( D ) Selected gene expression changes ( top ) and enriched gene sets ( bottom ) taken from RNA - seq analysis of BV - 2 cells with stable retroviral expression of Siglec constructs , 2xY - > F , and empty vector controls . Heatmaps share a colormap range of 0 . 25 to 4 for z - score values ( top ) , normalized enrichment scores ( NES , bottom ) .

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(D) Selected gene expression changes (top) and enriched gene sets (bottom) taken from RNA-seq analysis of BV-2 cells with stable retroviral expression of Siglec constructs, 2xY->F, and empty vector controls. Heatmaps share a colormap range of 0.25 to 4 for z-score values (top), normalized enrichment scores (NES, bottom).


( F ) Expression levels of C1qa , IL - 1β , and β - actin by qPCR analysis in BV - 2 cells with inducible Siglec - F expression after 48 hours of treatment with 500 ng / mL doxycycline . Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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(F) Expression levels of C1qa, IL-1β, and β-actin by qPCR analysis in BV-2 cells with inducible Siglec-F expression after 48 hours of treatment with 500 ng/mL doxycycline. Fold Change values are equal to 2ΔΔCq normalized relative to GADPH mRNA levels. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) Western blot showing pro - IL - 1β and β - tubulin in BV - 2 cells induced to express Siglec - F at 0 and 48 hours .

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(G) Western blot showing pro-IL-1β and β-tubulin in BV-2 cells induced to express Siglec-F at 0 and 48 hours.


( B - C ) Volcano plots showing phosphotyrosine peptides that were ( B ) increased or ( C ) decreased after Siglec - F expression . Plots show Siglec - F compared to 2xY - > F mutant data integrated across all three runs . Protein names are shown for altered peptides . Labels are only shown for peptides with maximum directional change from each protein . Labels colored according to GO terms : magenta = signal transduction , red = endosome , cyan = cell adhesion , yellow = cytoskeleton , green = metabolism .

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(B-C) Volcano plots showing phosphotyrosine peptides that were (B) increased or (C) decreased after Siglec-F expression. Plots show Siglec-F compared to 2xY->F mutant data integrated across all three runs. Protein names are shown for altered peptides. Labels are only shown for peptides with maximum directional change from each protein. Labels colored according to GO terms: magenta = signal transduction, red = endosome, cyan = cell adhesion, yellow = cytoskeleton, green = metabolism.


( D ) Heatmap showing phosphosites in CK - p25 which overlapped in their directional change with BV - 2 Siglec - F - associated sites . Colors indicate fold change relative to 2xY - > F controls .

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(D) Heatmap showing phosphosites in CK-p25 which overlapped in their directional change with BV-2 Siglec-F-associated sites. Colors indicate fold change relative to 2xY->F controls.


Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were increased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) .

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Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were increased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


Heatmap showing phosphosites from CD33 , Siglec - 5 , and Siglec - 8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec - F . Siglec - F column indicates average of all untreated Siglec - F replicates . Color scale is same as ( D ) .

Heatmap showing phosphosites from CD33, Siglec-5, and Siglec-8 overexpression that were decreased with Siglec expression and shared their directional change with Siglec-F. Siglec-F column indicates average of all untreated Siglec-F replicates. Color scale is same as (D).


( G ) Western blot quantification of myc , SHP - 2 , and SHP - 1 from myc co - IP eluates . Co - IP lysates were prepared from BV - 2 cells stably expressing Siglec - F and optionally treated with 1 μM SHP099 .

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(G) Western blot quantification of myc, SHP-2, and SHP-1 from myc co-IP eluates. Co-IP lysates were prepared from BV-2 cells stably expressing Siglec-F and optionally treated with 1 μM SHP099.


( H ) Heatmap showing phosphosites that were perturbed by SHP099 treatment . Color scale is same as ( D ) .

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(H) Heatmap showing phosphosites that were perturbed by SHP099 treatment. Color scale is same as (D).


( B - C ) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec - F expression for ( B ) Siglec - F and ( C ) Siglec - F 2xY - > F . Siglec - expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates .

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(B-C) Flow cytometry quantification of fluorescent monomeric Aβ uptake and Siglec-F expression for (B) Siglec-F and (C) Siglec-F 2xY->F. Siglec-expressing cells were plated with empty vector control cells in the same well alongside fluorescent substrates.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ , Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of monomeric Aβ, Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of 10 , 000 MW Dextran Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 10,000 MW Dextran Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


Quantification of relative mean fluorescence intensities ( MFI ) between empty vector and Siglec - expressing populations . Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of 2xY - > F group .

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Quantification of relative mean fluorescence intensities (MFI) between empty vector and Siglec-expressing populations. Relative values are shown for each mouse and human Siglec construct for uptake of 1 μm FluoSpheres. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of 2xY->F group.


( G ) pHrodo Dextran uptake in BV - 2 cells with stable expression of Siglec - F estimated by Incucyte measurements . Bars indicate mean ± 95 % CI ; n = 6 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided .

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(G) pHrodo Dextran uptake in BV-2 cells with stable expression of Siglec-F estimated by Incucyte measurements. Bars indicate mean ± 95% CI; n = 6 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided.


( H ) pHrodo dextran uptake in BV - 2 cells stably expressing Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from the same experiment as ( G ) .

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(H) pHrodo dextran uptake in BV-2 cells stably expressing Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G).


( I ) Confluency doubling times for BV - 2 cells with stable expression of Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from the same experiment as ( G ) . Bars indicate mean ± 95 % CI ; n = 4 replicates ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - test , two - sided . Dotted line indicates mean of untreated 2xY - > F group .

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(I) Confluency doubling times for BV-2 cells with stable expression of Siglec-F with 0 - 2.5 μM SHP099 treatment. Data is from the same experiment as (G). Bars indicate mean ± 95% CI; n = 4 replicates; *: p < 5e-2, **: p < 1e-2, ***: p < 1e-3, ****: p < 1e-4; ns: not significant, using unpaired Student's t-test, two-sided. Dotted line indicates mean of untreated 2xY->F group.


B . Flow cytometry of cells infected with the lentiCRISPR v2 . 0 library where the expression of GFP indicates latency reactivation . Target genes were enriched through two - round sorting . Continuously cultured C11 latent cells infected with the lentiCRISPRv2 . 0 served as control . Blue dots and the encircled area represent the GFP - positive cells for flow cytometry analysis .

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B. Flow cytometry of cells infected with the lentiCRISPR v2.0 library where the expression of GFP indicates latency reactivation. Target genes were enriched through two-round sorting. Continuously cultured C11 latent cells infected with the lentiCRISPRv2.0 served as control. Blue dots and the encircled area represent the GFP-positive cells for flow cytometry analysis.


C . Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2 . 0 library infection .

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C. Validation of sgRNAs in unsorted and sorted C11 cells by PCR after lentiCRISPR v2.0 library infection.


D . Before next generation sequencing , the GFP expression in two - round sorted C11 cells was confirmed by fluorescence microscopy . Scale bar , 100 μm .

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D. Before next generation sequencing, the GFP expression in two-round sorted C11 cells was confirmed by fluorescence microscopy. Scale bar, 100 μm.


E . Fold change ( Log2 ) of the abundance of target genes in sorted C11 cells . Enriched sgRNA genes are highlighted .

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E. Fold change (Log2) of the abundance of target genes in sorted C11 cells. Enriched sgRNA genes are highlighted.


A . Validation of the top candidate genes in the C11 cell line . C11 cells were infected by lentiCRISPR v2 . 0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg / ml puromycin . The percentage of GFP - positive cells was measured by flow cytometry to determine the level of HIV - 1 reactivation .

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A. Validation of the top candidate genes in the C11 cell line. C11 cells were infected by lentiCRISPR v2.0 packaged lentiviruses with sgRNA following by screening for 14 days with 2 µg/ml puromycin. The percentage of GFP-positive cells was measured by flow cytometry to determine the level of HIV-1 reactivation.


The effect of candidate genes on HIV latency was further verified in J - Lat 10 . 6 ( B ) models of HIV latency . GFP expression in J - Lat 10 . 6 cells was analyzed by flow

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The effect of candidate genes on HIV latency was further verified in J-Lat 10.6 (B) models of HIV latency. GFP expression in J-Lat 10.6 cells was analyzed by flow cytometry


The effect of candidate genes on HIV latency was further verified in ACH2 ( C ) models of HIV latency . p24 in ACH2 cells was analyzed by ELISA , respectively .

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The effect of candidate genes on HIV latency was further verified in ACH2 (C) models of HIV latency. p24 in ACH2 cells was analyzed by ELISA, respectively.


E . PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1 - sg1 . Mock C11 cells served as control .

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E. PEBP1 protein levels were measured by Western blot after knock out in C11 cells by LvPEBP1-sg1. Mock C11 cells served as control.


A . PEBP1 expression was measured by qPCR ( left panel ) and Western blot ( right panel ) in the latently infected C11 cells and the parental Jurkat cells .

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A. PEBP1 expression was measured by qPCR (left panel) and Western blot (right panel) in the latently infected C11 cells and the parental Jurkat cells.


B . The immunoprecipitation assay was performed in C11 cell lysates with anti - PEBP1 antibody followed by Western blot with anti - Raf1 or anti - IKK antibodies .

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B. The immunoprecipitation assay was performed in C11 cell lysates with anti-PEBP1 antibody followed by Western blot with anti-Raf1 or anti-IKK antibodies.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . The levels of indicated proteins in total protein lysates ( C ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. The levels of indicated proteins in total protein lysates (C) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


Effect of PEBP1 on the Raf1 / ERK / IκB and IKK / IκB / NF - κB signaling pathways . the levels of NF - κB / p65 protein in nucleus ( D ) were analysed by Western blot in C11 - PEBP1 - KO cells and mock C11 cells .

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Effect of PEBP1 on the Raf1/ERK/IκB and IKK/IκB/NF-κB signaling pathways. the levels of NF-κB/p65 protein in nucleus (D) were analysed by Western blot in C11-PEBP1-KO cells and mock C11 cells.


E . NF - κB / p65 protein recruitment into HIV LTR was analyzed by ChIP - qPCR with specific primers targeting the HIV LTR after normalization to the input .

E. NF-κB/p65 protein recruitment into HIV LTR was analyzed by ChIP-qPCR with specific primers targeting the HIV LTR after normalization to the input.


F . The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells . 293T cells were co - transfected with PEBP1 - sg1 alone or PEBP1 - sg1 with HIV - 1 LTR - empty plasmids , HIV - 1 wild type LTR - luciferase plasmids , HIV - 1 LTR - Δsp1 - luciferase , HIV - 1 LTR - ΔNF - κB - luciferase , HIV - 1 LTR - ΔAp1 - luciferase or HIV - 1 LTR - ΔYY1 - luciferase . Transcription of HIV - 1 was determined by luciferase reporter assay .

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F. The impact of PEBP1 on the activation of the HIV LTR was explored by luciferase reporter assay in 293T cells. 293T cells were co-transfected with PEBP1-sg1 alone or PEBP1-sg1 with HIV-1 LTR-empty plasmids, HIV-1 wild type LTR-luciferase plasmids, HIV-1 LTR-Δsp1-luciferase, HIV-1 LTR-ΔNF-κB-luciferase, HIV-1 LTR-ΔAp1-luciferase or HIV-1 LTR-ΔYY1-luciferase. Transcription of HIV-1 was determined by luciferase reporter assay.


A . The expression of PEBP1 was analysed by Western blot in TZM - bl cells infected with mock , Lv - PCDH - empty or Lv - PCDH - PEBP1 plasmids .

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A. The expression of PEBP1 was analysed by Western blot in TZM-bl cells infected with mock, Lv-PCDH-empty or Lv-PCDH-PEBP1 plasmids.


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . The transcription of HIV - 1 was evaluated 72 h post - infection by luciferase

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. The transcription of HIV-1 was evaluated 72 h post-infection by luciferase activity


Overexpression of PEBP1 suppressed HIV - 1 replication . TZM - bl cells were transfected with Lv - PCDH - empty or Lv - PCDH - PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies / ml . levels of p24 in the supernatants ( C ) .

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Overexpression of PEBP1 suppressed HIV-1 replication. TZM-bl cells were transfected with Lv-PCDH-empty or Lv-PCDH-PEBP1 followed by infection of HIV derived from patient plasma whose viral load is 129 copies/ml. levels of p24 in the supernatants (C).


D . The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h .

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D. The expression level of PEBP1 was detected by Western blot using the whole cell lysate of C11 cells treated with 10 μM EGCG or DHA for 24 h.


E . Induction of PEBP1 by EGCG or DHA in HIV - 1 infected primary CD4 + T cells . Primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . Seventy - two hours post - treatment with 10 µM EGCG or DHA , the expression of PEBP1 was detected by qPCR .

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E. Induction of PEBP1 by EGCG or DHA in HIV-1 infected primary CD4+ T cells. Primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. Seventy-two hours post-treatment with 10 µM EGCG or DHA, the expression of PEBP1 was detected by qPCR.


F . The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4 + T cells . The primary CD4 + T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV - 1 infection . Similar to Panel B and E , HIV - 1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies / ml . The supernatants from HIV - 1 - infected CD4 + T cell were collected 1 , 2 , 3 or 4 days post - infection . Replication of HIV - 1 was analyzed by p24 ELISA .

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F. The induction of PEBP1 by EGCG or DHA suppressed HIV replication in the primary CD4+ T cells. The primary CD4+ T cells from healthy donors were treated with 10 μM EGCG or DHA during HIV-1 infection. Similar to Panel B and E, HIV-1 was isolated from the blood supernatants of patients receiving ART with a viral load of 129 copies/ml. The supernatants from HIV-1-infected CD4+ T cell were collected 1, 2, 3 or 4 days post-infection. Replication of HIV-1 was analyzed by p24 ELISA.


A . EGCG inhibited α - CD3 / CD28 reactivation of latent HIV - 1 . Primary CD4 + T cells were isolated from HIV - 1 positive patients on antiretroviral therapy ( n = 5 , P1 - P5 ) . Primary CD4 + T cells were treated with α - CD3 / CD28 alone or α - CD3 / CD28 and 10 µM EGCG . Cell - associated RNA was extracted 7 days post - treatment . The transcription of HIV - 1 was determined by real - time qPCR .

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A. EGCG inhibited α-CD3/CD28 reactivation of latent HIV-1. Primary CD4+ T cells were isolated from HIV-1 positive patients on antiretroviral therapy (n=5, P1-P5). Primary CD4+ T cells were treated with α-CD3/CD28 alone or α-CD3/CD28 and 10 µM EGCG. Cell-associated RNA was extracted 7 days post-treatment. The transcription of HIV-1 was determined by real-time qPCR.


B . PEBP1 mRNA was induced by EGCG in patient primary CD4 + T cells . Primary CD4 + T cells were treated with 10 μM EGCG . The expression of PEBP1 in the cells was measured by qPCR .

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B. PEBP1 mRNA was induced by EGCG in patient primary CD4+ T cells. Primary CD4+ T cells were treated with 10 μM EGCG. The expression of PEBP1 in the cells was measured by qPCR.


C . PEBP1 protein was induced by EGCG in patient primary CD4 + T cells , which was determined by Western blot of whole cell lysate from primary CD4 + T cells .

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C. PEBP1 protein was induced by EGCG in patient primary CD4+ T cells, which was determined by Western blot of whole cell lysate from primary CD4+ T cells.


D . EGCG - induced PEBP1 suppressed nuclear entry of NF - κB / p65 . This was measured by Western blot of nuclear protein of primary CD4 + T cells treated with α - CD3 / CD28 or α - CD3 / CD28 plus 10 μM EGCG .

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D. EGCG-induced PEBP1 suppressed nuclear entry of NF-κB/p65. This was measured by Western blot of nuclear protein of primary CD4+ T cells treated with α-CD3/CD28 or α-CD3/CD28 plus 10 μM EGCG.


B . Transcription of HIV - 1 in the primary CD4 + T cells was determined by nanoluc luciferase assays during HIV - 1 infection .

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B. Transcription of HIV-1 in the primary CD4+ T cells was determined by nanoluc luciferase assays during HIV-1 infection.


C . After infection with VSV - G pseudotyped HIV - 1 NL4 . 3 - nanoluc , the mRNA expression of PEBP1 was measured by qPCR .

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C. After infection with VSV-G pseudotyped HIV-1 NL4.3-nanoluc, the mRNA expression of PEBP1 was measured by qPCR.


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The mRNA expression levels of PEBP1 were measured by

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The mRNA expression levels of PEBP1 were measured by qPCR


Jurkat CD4 + T cells were treated with IFN - α , IFN - β and IFN - γ for 24 h . The protein expression levels of PEBP1 were measured by Western blot .

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Jurkat CD4+ T cells were treated with IFN-α, IFN-β and IFN-γ for 24 h. The protein expression levels of PEBP1 were measured by Western blot.


F . PEBP1 knockout enhanced HIV - 1 transcription in the primary CD4 + T cell model of latency . The expression of HIV - 1 was measured by nanoluc after gene knockout where α - CD3 / CD28 stimulation served as a positive control .

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F. PEBP1 knockout enhanced HIV-1 transcription in the primary CD4+ T cell model of latency. The expression of HIV-1 was measured by nanoluc after gene knockout where α-CD3/CD28 stimulation served as a positive control.


( A ) Neural precursors derived from human iPS cells ( iNPCs ) were mock or ZIKV - infected and co - stained for Centrin ( green ) , Sox2 ( blue ) and flavivirus envelope ( red ) . All cells were fixed 16 hours post infection ( hpi ) . ( B ) Mock and ZIKV - infected U87 and H4 cells in S phase co - stained for Centrin ( green ) , flavivirus envelope ( red ) and DNA ( blue ) . ( C ) Quantification of the percentage of mock or ZIKV - infected iNPC , U87 or H4 cells in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 100nm .

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(A) Neural precursors derived from human iPS cells (iNPCs) were mock or ZIKV-infected and co-stained for Centrin (green), Sox2 (blue) and flavivirus envelope (red). All cells were fixed 16 hours post infection (hpi). (B) Mock and ZIKV-infected U87 and H4 cells in S phase co-stained for Centrin (green), flavivirus envelope (red) and DNA (blue). (C) Quantification of the percentage of mock or ZIKV-infected iNPC, U87 or H4 cells in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 100nm.


( A ) Mock or ZIKV - infected U87 cells in S phase co - stained for Centrin ( red ) and the centrosomal microcephaly - associated protein , CEP63 ( green ) . ( B ) The fluorescence intensity ± s . d . of CEP63 at the centrosome ( 0 . 5μm around the Centrin signal ) and around the centrosome ( 1μm surrounding the Centrin signal minus the centrosomal CEP63 ) were quantified in mock and ZIKV infected U87 cells . For fluorescence quantifications 8 cells were analyzed per experiment ( n = 3 ) . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

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(A) Mock or ZIKV-infected U87 cells in S phase co-stained for Centrin (red) and the centrosomal microcephaly-associated protein, CEP63 (green). (B) The fluorescence intensity ± s.d. of CEP63 at the centrosome (0.5μm around the Centrin signal) and around the centrosome (1μm surrounding the Centrin signal minus the centrosomal CEP63) were quantified in mock and ZIKV infected U87 cells. For fluorescence quantifications 8 cells were analyzed per experiment (n=3). Asterisk denotes p<0.005 (Student's t test). Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( D ) Immunoblot analysis of lysate from mock or ZIKV - infected U87 cells probed for CEP63 and ZIKV NS3 . Actin served as a loading control . Asterisk denotes specific band for CEP63 .

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(D) Immunoblot analysis of lysate from mock or ZIKV-infected U87 cells probed for CEP63 and ZIKV NS3. Actin served as a loading control. Asterisk denotes specific band for CEP63.


( E ) S - phase mock and ZIKV infected control and Cep63gt / gt MEFs co - stained for Centrin ( green ) and flavivirus envelope ( red ) . ( F ) Quantification of the percentage of mock or ZIKV - infected Het and Cep63gt / gt MEFs in S phase with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented . Data information : Scale bars indicate 5μm for all images . All cells were fixed 16 hours post infection ( hpi ) ,

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(E) S-phase mock and ZIKV infected control and Cep63gt/gt MEFs co-stained for Centrin (green) and flavivirus envelope (red). (F) Quantification of the percentage of mock or ZIKV-infected Het and Cep63gt/gt MEFs in S phase with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented. Data information: Scale bars indicate 5μm for all images. All cells were fixed 16 hours post infection (hpi),


( B ) Mock or ZIKV - infected U87 cell lysates were immunoprecipitated for CEP63 or c - Myc , which served as a negative control for this and other immunoprecipitations ( Control ) . Precipitated proteins were immunoblotted for CEP63 and NS3 . Asterisk denotes specific band .

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(B) Mock or ZIKV-infected U87 cell lysates were immunoprecipitated for CEP63 or c-Myc, which served as a negative control for this and other immunoprecipitations (Control). Precipitated proteins were immunoblotted for CEP63 and NS3. Asterisk denotes specific band.


( C ) We expressed Myc - tagged Brazilian Zika NS3 in 293T / 17 cells and immunoprecipitated c - Myc . Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63 . The centriole distal end component , CP110 served as a negative control . Asterisk denotes specific band for CEP63 .

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(C) We expressed Myc-tagged Brazilian Zika NS3 in 293T/17 cells and immunoprecipitated c-Myc. Precipitated proteins were probed for the Myc tag of NS3 and endogenous CEP63. The centriole distal end component, CP110 served as a negative control. Asterisk denotes specific band for CEP63.


( G ) HeLa cells transfected with Control or Myc - tagged Brazilian ZIKV NS3 ( Br NS3 ) co - stained for Myc ( red ) and Centrin ( green ) . Data information : Scale bars indicate 5μm for all images .

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(G) HeLa cells transfected with Control or Myc-tagged Brazilian ZIKV NS3 (Br NS3) co-stained for Myc (red) and Centrin (green). Data information: Scale bars indicate 5μm for all images.


( H ) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci . Bars represent the mean ± SD normalized to the control . For all quantifications at least 100 cells were counted per experiment ( biological replicates = 3 ) . p < 0 . 005 ( paired Student ' s t test ) for the comparison of NS3 to mock transfected cells was denoted with asterisk .

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(H) Quantification of the percentage of control and NS3 expressing HeLa cells with greater than four Centrin foci. Bars represent the mean ± SD normalized to the control. For all quantifications at least 100 cells were counted per experiment (biological replicates=3). p<0.005 (paired Student's t test) for the comparison of NS3 to mock transfected cells was denoted with asterisk.


( A ) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1 . Precipitated proteins were immunoblotted for CEP63 and TBK1 .

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(A) HeLa cell lysates were immunoprecipitated with antibodies to CEP63 and TBK1. Precipitated proteins were immunoblotted for CEP63 and TBK1.


( B ) Mock and ZIKV - infected U87 cells were co - stained for γ - tubulin ( red ) and p - TBK1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(B) Mock and ZIKV-infected U87 cells were co-stained for γ-tubulin (red) and p-TBK1 (green). Data information: Scale bars indicate 5μm for all images.


( C ) Lysate from mock or ZIKV - infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3 , TBK1 , and p - TBK1 . Actin served as a loading control .

(C) Lysate from mock or ZIKV-infected U87 cells at 16 hpi were immunoblotted with antibodies to NS3, TBK1, and p-TBK1. Actin served as a loading control.


( D ) RT - ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV - infected H4 cells . Bars represent the mean ± SD normalized to the control . Asterisk denotes p < 0 . 005 ( Student ' s t test ) . Three biological replicates are represented .

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(D) RT-ddPCR of IFNβ transcript at the indicated time points in mock and ZIKV-infected H4 cells. Bars represent the mean ± SD normalized to the control. Asterisk denotes p<0.005 (Student's t test). Three biological replicates are represented.


( E ) Control and Cep63gt / gt MEFs were co - stained with antibodies to γ - tubulin ( red ) and p - Tbk1 ( green ) . Data information : Scale bars indicate 5μm for all images .

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(E) Control and Cep63gt/gt MEFs were co-stained with antibodies to γ-tubulin (red) and p-Tbk1 (green). Data information: Scale bars indicate 5μm for all images.


( F ) Immunoblot analysis of control and Cep63gt / gt MEFs probed with antibodies to Tbk1 and p - Tbk1 . Actin served as a loading control .

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(F) Immunoblot analysis of control and Cep63gt/gt MEFs probed with antibodies to Tbk1 and p-Tbk1. Actin served as a loading control.


( G ) HeLa cell lysate was immunoprecipitated with antibodies to CEP63 , DTX4 or c - Myc ( negative control ) . Precipitated proteins were blotted with antibodies to CEP63 and DTX4 .

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(G) HeLa cell lysate was immunoprecipitated with antibodies to CEP63, DTX4 or c-Myc (negative control). Precipitated proteins were blotted with antibodies to CEP63 and DTX4.


( H ) Mock and ZIKV - infected U87 cells co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

(H) Mock and ZIKV-infected U87 cells co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( I ) Total cell lysate from mock and ZIKV - transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4 . Actin served as a loading control .

(I) Total cell lysate from mock and ZIKV-transfected U87 cells 16 hpi were analyzed by western blot using antibodies to ZIKV NS3 and DTX4. Actin served as a loading control.


( J ) SC and CEP63 siRNA treated HeLa cells were co - stained for Centrin ( red ) and DTX4 ( green ) . Data information : Scale bars indicate 5μm for all images .

(J) SC and CEP63 siRNA treated HeLa cells were co-stained for Centrin (red) and DTX4 (green). Data information: Scale bars indicate 5μm for all images.


( K ) TBK1 or c - Myc ( negative control ) were immunoprecipitated from Control or Cep63 mutant MEFs . Precipitating proteins were probed with antibodies to TBK1 and K48 - linked ubiquitin .

(K) TBK1 or c-Myc (negative control) were immunoprecipitated from Control or Cep63 mutant MEFs. Precipitating proteins were probed with antibodies to TBK1 and K48-linked ubiquitin.


( L ) 16hpi Mock and ZIKV - infected U87 cell lysate was immunoprecipitated for TBK1 or c - Myc . The immunoprecipitations were probed using antibodies to TBK1 and K - 48 - linked ubiquitin .

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(L) 16hpi Mock and ZIKV-infected U87 cell lysate was immunoprecipitated for TBK1 or c-Myc. The immunoprecipitations were probed using antibodies to TBK1 and K-48-linked ubiquitin.