Comparisons of serum triglyceride ( TG ) at indicated time points in WT and VR3iΔLEC mice ( n = 6 mice / group ) .

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Comparisons of serum triglyceride (TG) at indicated time points in WT and VR3iΔLEC mice (n = 6 mice/group).


Comparison of protein levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

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Comparison of protein levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from SPF , GF , and CONV mice ( n = 3 - 5 mice / group ) .

Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from SPF, GF, and CONV mice (n = 3-5 mice/group).


Comparison of mRNA levels of VEGF - C in sorted CD45 - stromal cells and CD45 + MHCII + F4 / 80 + macrophages from jejunum and ileum of vehicle - treated mice . The mean of transcription levels of VEGF - C in CD45 - stromal cells was normalized to 1 , and the relative levels in macrophages were presented as fold change ( n = 4 mice / group ) .

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Comparison of mRNA levels of VEGF-C in sorted CD45- stromal cells and CD45+ MHCII+ F4/80+ macrophages from jejunum and ileum of vehicle-treated mice. The mean of transcription levels of VEGF-C in CD45- stromal cells was normalized to 1, and the relative levels in macrophages were presented as fold change (n = 4 mice/group).


Comparisons of VEGF - C transcription level in CD45 - stromal cells ( left ) and CD45 + MHCII + F4 / 80 + macrophages ( right ) in jejunum and ileum of vehicle - and ABX - treated mice . The mean of transcription levels of VEGF - C in vehicle - treated mice was normalized to 1 , and the relative levels in ABX - treated mice were presented as fold change ( n = 5 - 6 mice / group ) .

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Comparisons of VEGF-C transcription level in CD45- stromal cells (left) and CD45+ MHCII+ F4/80+ macrophages (right) in jejunum and ileum of vehicle- and ABX-treated mice. The mean of transcription levels of VEGF-C in vehicle-treated mice was normalized to 1, and the relative levels in ABX-treated mice were presented as fold change (n = 5 -6 mice/group).


Images and comparison of F4 / 80 + macrophages in the villi of duodenum ( DD ) , jejunum ( JJ ) , and ileum ( IL ) from vehicle - or ABX - treated mice . Each dot indicates mean value of 5 sites in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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Images and comparison of F4/80+ macrophages in the villi of duodenum (DD), jejunum (JJ), and ileum (IL) from vehicle- or ABX-treated mice. Each dot indicates mean value of 5 sites in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells ( E ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

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Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells (E) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of CX3CR1 + F4 / 80 + macrophage gated on DAPI - CD45 + MHCII + cells ( F ) macrophage gated on DAPI - CD45 + cells from jejunum and ileum of vehicle - and ABX - treated mice ( n = 5 mice / group ) .

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Representative flow cytometric analysis and comparison of CX3CR1+ F4/80+ macrophage gated on DAPI- CD45+ MHCII+ cells (F) macrophage gated on DAPI- CD45+ cells from jejunum and ileum of vehicle- and ABX-treated mice (n = 5 mice/group).


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and DTR mice ( n = 5 mice / group ) .

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Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and DTR mice (n = 5 mice/group).


Comparison of mRNA levels of VEGF - C in the whole tissue of jejunum and ileum from WT and DTR mice ( n = 5 mice / group ) .

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Comparison of mRNA levels of VEGF-C in the whole tissue of jejunum and ileum from WT and DTR mice (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and DTR mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and DTR mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and DTR mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and DTR mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative flow cytometric analysis and comparison of MHCII + F4 / 80 + macrophage gated on DAPI - CD45 + cells from jejunum and ileum of WT and MyD88ΔMP mice ( n = 5 mice / group ) .

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Representative flow cytometric analysis and comparison of MHCII+ F4/80+ macrophage gated on DAPI- CD45+ cells from jejunum and ileum of WT and MyD88ΔMP mice (n = 5 mice/group).


Comparisons of mRNA levels of VEGF - C in sorted MHCII + F4 / 80 + macrophages from jejunum and ileum of WT and MyD88ΔMP mice . The mean of transcription levels of VEGF - C in WT mice was normalized to 1 , and the relative level in MyD88ΔMP mice were presented as fold change ( n = 5 mice / group ) .

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Comparisons of mRNA levels of VEGF-C in sorted MHCII+ F4/80+ macrophages from jejunum and ileum of WT and MyD88ΔMP mice. The mean of transcription levels of VEGF-C in WT mice was normalized to 1, and the relative level in MyD88ΔMP mice were presented as fold change (n = 5 mice/group).


Images and comparisons of absolute and relative lacteal lengths in duodenum ( DD ) , jejunum ( JJ ) and ileum ( IL ) of WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . Scale bars , 100 μm .

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Images and comparisons of absolute and relative lacteal lengths in duodenum (DD), jejunum (JJ) and ileum (IL) of WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). Scale bars, 100 μm.


Images and comparisons of number of Prox1 + LECs within initial 100 μm portion ( upper ) and VEGFR3 expression ( lower , presented as relative fluorescent intensity ( FI ) ) in CD31 + / LYVE - 1 + lacteals of jejunum from WT and MyD88ΔMP mice . Each dot indicates mean value of 5 - 10 villi . Each dot indicates mean value of 5 - 10 villi in a mouse ( n = 6 mice / group ) . AU , arbitrary unit . Scale bars , 100 μm .

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Images and comparisons of number of Prox1+ LECs within initial 100 μm portion (upper) and VEGFR3 expression (lower, presented as relative fluorescent intensity (FI)) in CD31+/LYVE-1+ lacteals of jejunum from WT and MyD88ΔMP mice. Each dot indicates mean value of 5-10 villi. Each dot indicates mean value of 5-10 villi in a mouse (n = 6 mice/group). AU, arbitrary unit. Scale bars, 100 μm.


Images and comparison of proportions of each VE - cadherin LEC junctions in CD31 + / LYVE - 1 + lacteals in jejunum between WT and MyD88ΔMP mice . Button - like ( red arrowheads ) and zipper - like ( open arrowheads ) junctions are indicated in each magnified box in below ( n = 6 mice / group , 5 - 10 villi / mouse ) . Scale bars , 25 μm .

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Images and comparison of proportions of each VE-cadherin LEC junctions in CD31+/LYVE-1+ lacteals in jejunum between WT and MyD88ΔMP mice. Button-like (red arrowheads) and zipper-like (open arrowheads) junctions are indicated in each magnified box in below (n = 6 mice/group, 5-10 villi/mouse). Scale bars, 25 μm.


Representative confocal images of cell - type - specific promoter : : GFP : AGO1 translational fusions .

Representative confocal images of cell-type-specific promoter::GFP:AGO1 translational fusions.


Immunoblot analysis of FLAG : AGO1 IPed in each root - tip layer . Scale bars : 50μM , NT : Non - transformed . CB : Coomassie blue .

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Immunoblot analysis of FLAG:AGO1 IPed in each root-tip layer. Scale bars: 50μM, NT: Non-transformed. CB: Coomassie blue.


Left : input and IPed AGO1 following post - lysis addition of a radiolabeled siRNA duplex ( siGFP ) . Right : northern analysis of AGO1 - bound sRNAs .

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Left: input and IPed AGO1 following post-lysis addition of a radiolabeled siRNA duplex (siGFP). Right: northern analysis of AGO1-bound sRNAs.


Layer - specific distribution of all AGO1 - loaded miRNAs .

Layer-specific distribution of all AGO1-loaded miRNAs.


Northern analysis of layer - specific AGO1 - loaded miRNAs using sRNAs IPed independently from that used for deep - sequencing . Bar - plots underneath each panel display the deep - sequencing results as normalized reads counts .

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Northern analysis of layer-specific AGO1-loaded miRNAs using sRNAs IPed independently from that used for deep-sequencing. Bar-plots underneath each panel display the deep-sequencing results as normalized reads counts.


Representative confocal images of cell - type - specific promoter : : GFP transcriptional fusions in hyl1 vs WT roots .

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Representative confocal images of cell-type-specific promoter::GFP transcriptional fusions in hyl1 vs WT roots.


Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots .

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Hierarchical clustering of validated miRNA targets between different cell layers in either WT or hyl1 roots.


qRT - PCR validation of three distinct classes of direct miRNA target transcripts from total root - , total root polysome - or epidermis - specific polysome - associated RNA . The AT2G37050 kinase is positively regulated by LAC17 . Scale bars : 50μM . All qPCR values were normalized to the internal control ACTIN2 .

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qRT-PCR validation of three distinct classes of direct miRNA target transcripts from total root-, total root polysome- or epidermis-specific polysome-associated RNA. The AT2G37050 kinase is positively regulated by LAC17. Scale bars: 50μM. All qPCR values were normalized to the internal control ACTIN2.


miRoot snapshots of whole - root ( left ) and layer - specific ( right ) LAC17 mRNA accumulation in WT versus hyl1 , as assessed by polysome profiling .

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miRoot snapshots of whole-root (left) and layer-specific (right) LAC17 mRNA accumulation in WT versus hyl1, as assessed by polysome profiling.


miRoot snapshot of cell - type specific miRNAs showing , in this case , a sum group of the miR156 family . The right panel shows the gradient of expression revealed upon removal of the stele ( blue box ) .

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miRoot snapshot of cell-type specific miRNAs showing, in this case, a sum group of the miR156 family. The right panel shows the gradient of expression revealed upon removal of the stele (blue box).


Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds . Removal of the stele ( blue box ) reveals subtleties of miR156 - mediated gene regulation in the other layers , resulting in a near - mirror image of ( A ) .

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Snapshot of the SPL6 miR156 translatome in WT and hyl1 backgrounds. Removal of the stele (blue box) reveals subtleties of miR156-mediated gene regulation in the other layers, resulting in a near-mirror image of (A).


Confocal image of pmiR396a : : H2B : GFP . Normalized read counts from AGO1 - IP - Seq and independent northern analysis of AGO1 - loaded miR396 .

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Confocal image of pmiR396a::H2B:GFP. Normalized read counts from AGO1-IP-Seq and independent northern analysis of AGO1-loaded miR396.


Representative confocal image of the pGRF2 : : H2B : GFP translational fusion .

Representative confocal image of the pGRF2::H2B:GFP translational fusion.


Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella . Graph : GRF2 normalized read counts .

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Focused miRoot analysis of the GRF2 translatome signal in WT versus hyl1 columella. Graph: GRF2 normalized read counts.


Representative confocal images of the pGRF2 : : GRF2 : GFP translational fusion in WT or hyl1 backgrounds .

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Representative confocal images of the pGRF2::GRF2:GFP translational fusion in WT or hyl1 backgrounds.


Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella .

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Focused miRoot analysis of the PLT1 translatome signal in WT versus hyl1 columella.


Layer - specific normalized read counts of miR169 paralogs ( separated into 4 different groups based on sequence identity : a , b - c , d - g , h - n ) from AGO1 - IP - deep - Seq and independent northern analysis , as in Fig . 1F .

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Layer-specific normalized read counts of miR169 paralogs (separated into 4 different groups based on sequence identity: a, b-c, d-g, h-n) from AGO1-IP-deep-Seq and independent northern analysis, as in Fig.1F.


Cell - type specific AGO1 - IP signals for each miR169 sequence - group with ( upper ) or without ( lower panel ) the miRoot scale normalization option .

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Cell-type specific AGO1-IP signals for each miR169 sequence-group with (upper) or without (lower panel) the miRoot scale normalization option.


Predicted base - pairing of indicated miR169 sequence - group to four NF - YA target transcripts . Pairing with expectation value greater than 3 was not considered . The color gradient ( pale : low , dark : strong ) qualitatively approximates the pairing contribution of each miR169 sequence - group according to the upper panel of ( B ) . miRoot snapshots of layer - specific translatome signals of four out - of - seven miR169 NF - YA targets in WT versus hyl1 backgrounds .

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Predicted base-pairing of indicated miR169 sequence-group to four NF-YA target transcripts. Pairing with expectation value greater than 3 was not considered. The color gradient (pale: low, dark: strong) qualitatively approximates the pairing contribution of each miR169 sequence-group according to the upper panel of (B). miRoot snapshots of layer-specific translatome signals of four out-of-seven miR169 NF-YA targets in WT versus hyl1 backgrounds.


Browser representation and independent northern analysis and AGO1 - loaded miR165 / 166 .

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Browser representation and independent northern analysis and AGO1-loaded miR165/166.


miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds .

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miRoot snapshot of PHB translatome signals in WT versus hyl1 backgrounds.


Representative confocal images of pmiR395 ( a / c / e ) : : H2B : GFP transcriptional fusions under SO4 - - deficient or SO4 - - proficient growth conditions . Northern analysis of AGO1 - loaded miR395 under the conditions in ( C ) .

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Representative confocal images of pmiR395(a/c/e)::H2B:GFP transcriptional fusions under SO4--deficient or SO4--proficient growth conditions. Northern analysis of AGO1-loaded miR395 under the conditions in (C).


Representative confocal images of the pAPS4 : : APS4 : GFP translational fusion under the conditions in ( C ) .

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Representative confocal images of the pAPS4::APS4:GFP translational fusion under the conditions in (C).


Representative confocal image of the pSHR : : GFP : P19 translational fusion and ( B ) principle of P19 - based miRNA duplex sequestration .

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Representative confocal image of the pSHR::GFP:P19 translational fusion and (B) principle of P19-based miRNA duplex sequestration.


Binding of miR395 by SHR : : HA : P19 under SO4 - - sufficient ( + ) and - deficient ( - ) growth conditions .

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Binding of miR395 by SHR::HA:P19 under SO4--sufficient (+) and -deficient (-) growth conditions.


qRT - PCR analysis of polysome - associated APS4 in the cortex or epidermis in the presence ( + ) or absence ( - ) of SHR : : HA : P19 . Signals are relative to SHR : : HA : P19 ( - ) lines given a value of 1 .

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qRT-PCR analysis of polysome-associated APS4 in the cortex or epidermis in the presence (+) or absence (-) of SHR::HA:P19. Signals are relative to SHR::HA:P19 (-) lines given a value of 1.


Representative confocal image of the pmiR397b : : H2B : GFP transcriptional fusion ( left ) and the browser AGO1 - loaded signal for miR397b ( right ) .

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Representative confocal image of the pmiR397b::H2B:GFP transcriptional fusion (left) and the browser AGO1-loaded signal for miR397b (right).


Cross section of Arabidopsis leaf showing cell - types isolated via Meselect .

Cross section of Arabidopsis leaf showing cell-types isolated via Meselect.


miR160 northern analysis in whole - leaf , vasculature and epidermis . U6 : loading control .

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miR160 northern analysis in whole-leaf, vasculature and epidermis. U6: loading control.


Western analysis of the promoter : : H2B : GFP translational fusions shown in ( 5C ) in the cell types isolated in ( G ) .

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Western analysis of the promoter::H2B:GFP translational fusions shown in (5C) in the cell types isolated in (G).


qRT - PCR analysis of ARF17 in whole - leaves or in separated cell types in WT versus hyl1 backgrounds .

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qRT-PCR analysis of ARF17 in whole-leaves or in separated cell types in WT versus hyl1 backgrounds.


( A ) Size exclusion chromatography profile of NS1 pull - down experiments showing a clear shift after incubation in complete or heat - inactivated human serum ( solid and dotted black lines , respectively ) from the same healthy donor compared to the NS1 protein alone ( blue line ) . NS1 protein interaction partners were identified by SDS - PAGE and N - terminal sequencing as the Apolipoprotein B scaffold of the low - density lipoproteins ( LDL ) in the first SEC elution peak , and the ApoA - I protein scaffold of the high - density lipoproteins ( HDL ) in the second elution peak .

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(A) Size exclusion chromatography profile of NS1 pull-down experiments showing a clear shift after incubation in complete or heat-inactivated human serum (solid and dotted black lines, respectively) from the same healthy donor compared to the NS1 protein alone (blue line). NS1 protein interaction partners were identified by SDS-PAGE and N-terminal sequencing as the Apolipoprotein B scaffold of the low-density lipoproteins (LDL) in the first SEC elution peak, and the ApoA-I protein scaffold of the high-density lipoproteins (HDL) in the second elution peak.


( B ) Biolayer interferometry ( BLI ) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL ( left panel ) and human LDL ( central panel ) particles . The concentration - dependence of the steady - state signal corresponding to the binding of NS1 to HDL ( black dots ) and LDL ( white dots ) is shown on the right - hand side panel . The measurements were replicated at least three times using novel biosensors and samples . Data points and error bars correspond to the mean ± SD .

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(B) Biolayer interferometry (BLI) profiles corresponding to the binding of NS1 at various concentrations respective to human HDL (left panel) and human LDL (central panel) particles. The concentration-dependence of the steady-state signal corresponding to the binding of NS1 to HDL (black dots) and LDL (white dots) is shown on the right-hand side panel. The measurements were replicated at least three times using novel biosensors and samples. Data points and error bars correspond to the mean ± SD.


( C ) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre - incubated together ( mixture of NS1 and HDL at a 1 : 1 or 5 : 1 mass ratio ) monitored using an interferometric detector . Peaks were integrated for all the detectors ( interference and absorbance at 280nm ) . The calculated stoichiometries are indicated for each peak . Solutions were equilibrated at 20 ° C for 2h before sedimentation velocity analysis .

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(C) Typical sedimentation coefficient distribution of NS1 or human HDL alone or pre-incubated together (mixture of NS1 and HDL at a 1:1 or 5:1 mass ratio) monitored using an interferometric detector. Peaks were integrated for all the detectors (interference and absorbance at 280nm). The calculated stoichiometries are indicated for each peak. Solutions were equilibrated at 20°C for 2h before sedimentation velocity analysis.


( A , B ) Electron microscopy observations from left to right : a representative image , followed by the three most representative classes of ( A ) purified HDL particles and ( B ) NS1 - HDL complexes . White bar : 50 nm , Black bar : 20 nm .

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(A, B) Electron microscopy observations from left to right: a representative image, followed by the three most representative classes of (A) purified HDL particles and (B) NS1-HDL complexes. White bar: 50 nm, Black bar: 20 nm.


( D ) Differential scanning calorimetry ( DSC ) of NS1 alone ( blue line ) or of an NS1 - HDL mixture at a 2 : 1 molar ratio ( orange ) . Of note , the HDL particles alone did not generate any signal in the temperature range tested .

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(D) Differential scanning calorimetry (DSC) of NS1 alone (blue line) or of an NS1-HDL mixture at a 2:1 molar ratio (orange). Of note, the HDL particles alone did not generate any signal in the temperature range tested.


( E ) Binding inhibition of hexameric NS1 to HDL with anti - ApoA - I polyclonal antibodies ( anti - ApoA - I Ab ) measured by BLI .

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(E) Binding inhibition of hexameric NS1 to HDL with anti-ApoA-I polyclonal antibodies (anti-ApoA-I Ab) measured by BLI.


( A - D ) Human primary macrophages were incubated for 24h with the different potential effectors ( NS1 , HDL , mix NS1 - HDL ) or with control suspensions ( PBS buffer , LPS , mix LPS - HDL ) . LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments . Phosphate buffer used in the SEC purification step was used as a negative control . Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of ( A ) TNF - α , ( B ) Il - 6 , ( C ) Il - 1ß and ( D ) Il - 10 released in the extracellular medium . Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors ( n = 9 for TNF - α , Il - 6 and Il - 1ß , n = 5 for Il - 10 ) . Data represent mean + / - SEM . A Mann - Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants . Not significant : ns , * p < 0 . 05 , * * * p < 0 . 001 .

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(A-D) Human primary macrophages were incubated for 24h with the different potential effectors (NS1, HDL, mix NS1-HDL) or with control suspensions (PBS buffer, LPS, mix LPS-HDL). LPS stimulation was used as a positive control in the presence or absence of HDL and provided values consistent between experiments. Phosphate buffer used in the SEC purification step was used as a negative control. Cell culture supernatants were clarified and tested with a Luminex assay to quantify the amount of (A) TNF-α, (B) Il-6, (C) Il-1ß and (D) Il-10 released in the extracellular medium. Data reported on the graphs correspond to biological replicates of macrophages isolated from four blood donors (n=9 for TNF-α, Il-6 and Il-1ß, n=5 for Il-10). Data represent mean +/- SEM. A Mann-Whitney test was used to assess the statistical significance of differences observed between mean cytokine levels in different cell culture supernatants. Not significant: ns, * p < 0.05, *** p < 0.001.


levels of ( B ) NS1 , ( C ) NS1 - ApoA - I , ( D ) NS1 - ApoE and ( E ) NS1 - ApoB NS1 - ApoA - I and NS1 - ApoB complexes are representative of NS1 - HDL and NS1 - LDL complex species , respectively , while the NS1 - ApoE - positive complexes remain to be fully characterized . Errors bars indicate SEM .

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levels of (B) NS1, (C) NS1-ApoA-I, (D) NS1-ApoE and (E) NS1-ApoB NS1-ApoA-I and NS1-ApoB complexes are representative of NS1-HDL and NS1-LDL complex species, respectively, while the NS1-ApoE-positive complexes remain to be fully characterized. Errors bars indicate SEM.


levels of ( F ) total cholesterol , ( G ) HDL - cholesterol and ( H ) triglycerides . Errors bars indicate SEM .

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levels of (F) total cholesterol, (G) HDL-cholesterol and (H) triglycerides. Errors bars indicate SEM.


( I ) Purified NS1 from different flaviviruses ( yellow fever , YF ; ZIKA ; West Nile , WN ; Japanese encephalitis , JE ; The Native Antigen Company ) were spiked in normal human plasma and NS1 - ApoA - I complexes further detected by ELISA . Data represent the mean values of two technical replicates .

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(I) Purified NS1 from different flaviviruses (yellow fever, YF; ZIKA; West Nile, WN; Japanese encephalitis, JE; The Native Antigen Company) were spiked in normal human plasma and NS1-ApoA-I complexes further detected by ELISA. Data represent the mean values of two technical replicates.


Spearman correlations between protein expression and sensitivity to Plk1 inhibitors ( BI2536 , GSK461364 , BRD - K70511574 , and GW - 843682X ) , based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database ( Li et al , 2017 ) . Proteins with expression levels that correlate with area under the curve ( AUC ) values for at least two drugs are included in the graph . The color and size of the data point represent the direction and significance ( p value ) of the correlation . Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity ( i . e . , proteins in red are more highly expressed in resistant cell lines ) .

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Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536, GSK461364, BRD-K70511574, and GW-843682X), based on data from the Cancer Therapeutics Response Portal v2 database and protein expression data derived from the MD Anderson Cell Line Project database (Li et al, 2017). Proteins with expression levels that correlate with area under the curve (AUC) values for at least two drugs are included in the graph. The color and size of the data point represent the direction and significance (p value) of the correlation. Red indicates a positive correlation with drug resistance and green a positive correlation with drug sensitivity (i.e., proteins in red are more highly expressed in resistant cell lines).


Spearman correlations between protein expression and sensitivity to Plk1 inhibitors ( BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins ( cMet , E - cadherin , β - catenin , and Snail ) ; each data point represents an individual non - small cell lung cancer cell line . E - cadherin , β catenin , and cMet protein expression correlated with drug resistance .

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Spearman correlations between protein expression and sensitivity to Plk1 inhibitors (BI2536 GSK461364 AUC values for two Plk1 inhibitors are compared with expression of selected proteins (cMet, E-cadherin, β-catenin, and Snail); each data point represents an individual non-small cell lung cancer cell line. E-cadherin, β catenin, and cMet protein expression correlated with drug resistance.


Three epithelial / resistant non - small cell lung cancer cell lines were treated with 5 ng / ml TGF - β for 14 days to induce a mesenchymal phenotype , which was confirmed with Western blot ( left ) and qPCR ( right ) analysis of epithelial - to - mesenchymal transition ( EMT ) markers ( A ) .

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Three epithelial/resistant non-small cell lung cancer cell lines were treated with 5 ng/ml TGF-β for 14 days to induce a mesenchymal phenotype, which was confirmed with Western blot (left) and qPCR (right) analysis of epithelial-to-mesenchymal transition (EMT) markers (A).


Parental and TGF - β isogenic cell lines were treated with 50nM volasertib for 72 hours . Cells were then harvested , and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β - actin ( B ) .

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Parental and TGF-β isogenic cell lines were treated with 50nM volasertib for 72 hours. Cells were then harvested, and lysates were immunoblotted for cleaved PARP and γH2AX proteins were subsequently quantitated and normalized with β-actin (B).


Apoptosis was measured by the Apo - BrdU assay ( C )

Apoptosis was measured by the Apo-BrdU assay (C)


DNA damage was measured by the Comet assay ( D ) .

DNA damage was measured by the Comet assay (D).


Thirty - three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib , including those involved in the cMet / FAK / Src signaling axis ( blue text ) and those involved in the PI3K / Akt signaling axis ( orange text ) .

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Thirty-three proteins were differentially regulated between epithelial and mesenchymal NSCLC cell lines after treatment with volasertib, including those involved in the cMet/FAK/Src signaling axis (blue text) and those involved in the PI3K/Akt signaling axis (orange text).


The same cell lines treated identically were subjected to immunoblotting for the indicated proteins ( upper ) with densitometric quantification normalized with β - actin ( lower ) .

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The same cell lines treated identically were subjected to immunoblotting for the indicated proteins (upper) with densitometric quantification normalized with β-actin (lower).


Epithelial ( red text ) and mesenchymal ( blue text ) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control and subjected to Western blotting with the indicated antibodies . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - sided Student t test are indicated by * p < 0 . 05 .

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Epithelial (red text) and mesenchymal (blue text) NSCLC cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control and subjected to Western blotting with the indicated antibodies. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-sided Student t test are indicated by *p < 0.05.


H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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H1975 and Calu6 cells were treated with indicated inhibitors for 4 hours or 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Cell viability was measured using CellTiter - Glo in NSCLC cell lines treated with volasertib , tepotinib , or a 1 : 2 ratio of both for 72 hours . Left : representative cell viability graph of cells treated with the indicated drugs . Right : heatmap depicting the calculated combination indices at Fa = 0 . 25 and Fa = 0 . 5 .

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Cell viability was measured using CellTiter-Glo in NSCLC cell lines treated with volasertib, tepotinib, or a 1:2 ratio of both for 72 hours. Left: representative cell viability graph of cells treated with the indicated drugs. Right: heatmap depicting the calculated combination indices at Fa = 0.25 and Fa = 0.5.


Apoptosis was measured using the Apo - BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours .

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Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines treated with the indicated drugs for 24 hours.


Immunoblots from cells treated with indicated drugs for 24 hours ( left ) with densitometric quantification normalized with β - actin ( right ) .

Immunoblots from cells treated with indicated drugs for 24 hours (left) with densitometric quantification normalized with β-actin (right).


All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ .

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All tested cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.


Apoptosis was measured using the Apo - BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting control .

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Apoptosis was measured using the Apo-BrdU assay in the indicated cell lines transfected with 10nM siRNA as indicated for 48 hours. NT, non-targeting control.


Immunoblots from cells transfected with siRNA as indicated for 48 hours ( left ) with densitometric quantification normalized with β - actin ( right ) . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - way analysis of variance with Bonferroni or Benjamini - Hochberg ( BH ) correction for multiple comparison are indicated ( * p < 0 . 01 ) . Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text , respectively .

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Immunoblots from cells transfected with siRNA as indicated for 48 hours (left) with densitometric quantification normalized with β-actin (right). Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg (BH) correction for multiple comparison are indicated (*p < 0.01). Mesenchymal and epithelial NSCLC cell lines are indicated in blue and red text, respectively.


Mice bearing epithelial ( red text ) and mesenchymal ( blue text ) non - small cell lung cancer tumors were treated with vehicle control , volasertib ( 30 mg / kg per week intravenously ) , tepotinib ( 25 mg / kg per day orally ) , or the combination for 5 weeks to generate tumor growth curves of patient - derived xenografts ( PDX ; A ) and cell line xenografts The percent change in tumor volume at the end of treatment ( normalized to day zero ) was calculated with each data point representing an individual mouse , and the bar is the mean value ± standard error of 10 mice for each

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Mice bearing epithelial (red text) and mesenchymal (blue text) non-small cell lung cancer tumors were treated with vehicle control, volasertib (30 mg/kg per week intravenously), tepotinib (25 mg/kg per day orally), or the combination for 5 weeks to generate tumor growth curves of patient-derived xenografts (PDX; A) and cell line xenografts The percent change in tumor volume at the end of treatment (normalized to day zero) was calculated with each data point representing an individual mouse, and the bar is the mean value ± standard error of 10 mice for each group


Tumor growth curve of mesenchymal PDX TC424 ( top ) and cell line xenograft Calu6 ( bottom ) upon treatment for 5 weeks and after cessation of treatment ( recovery phase ) . Data are means ± standard error of the mean from five mice for each group .

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Tumor growth curve of mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) upon treatment for 5 weeks and after cessation of treatment (recovery phase). Data are means ± standard error of the mean from five mice for each group.


Kaplan - Meier survival curves for mesenchymal PDX TC424 ( top ) and cell line xenograft Calu6 ( bottom ) with death due to excessive tumor burden as the endpoint .

Kaplan-Meier survival curves for mesenchymal PDX TC424 (top) and cell line xenograft Calu6 (bottom) with death due to excessive tumor burden as the endpoint.


Quantification of TUNEL - positive cells in paraffin - embedded xenograft tissue sections .

Quantification of TUNEL-positive cells in paraffin-embedded xenograft tissue sections.


Calu6 , H157 , and H1355 TPR - Met - expressing or vector control ( pBABE ) - expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo - BrdU assay .

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Calu6, H157, and H1355 TPR-Met-expressing or vector control (pBABE)-expressing cells were treated as indicated for 24 hours and apoptosis was measured using the Apo-BrdU assay.


Parental and TPR - Met - expressing cell lines were treated with the indicated drugs for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins . β - actin was used as a loading control .

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Parental and TPR-Met-expressing cell lines were treated with the indicated drugs for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins. β-actin was used as a loading control.


Parental and TPR - Met - expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ . Data are means ± standard error of the mean from three independent experiments . Significant differences using two - way analysis of variance with Bonferroni or Benjamini - Hochberg correction for multiple comparisons are indicated .

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Parental and TPR-Met-expressing cell lines were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ. Data are means ± standard error of the mean from three independent experiments. Significant differences using two-way analysis of variance with Bonferroni or Benjamini-Hochberg correction for multiple comparisons are indicated.


Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies . * p < 0 . 05 . * * p < 0 . 01 .

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Mesenchymal NSCLC cell lines with the noted MET alterations were incubated with 50nM volasertib for four hours and subjected to immunoblotting with the indicated antibodies. *p < 0.05. **p < 0.01.


Cell viability measured by CellTiter - Glo in Calu6 parental and Calu6 - VAR cells treated with different concentrations of volasertib for 72 hours .

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Cell viability measured by CellTiter-Glo in Calu6 parental and Calu6-VAR cells treated with different concentrations of volasertib for 72 hours.


Micrograph shows acquisition of a mesenchymal - to - epithelial transition phenotype in VAR cells .

Micrograph shows acquisition of a mesenchymal-to-epithelial transition phenotype in VAR cells.


Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting ( upper ) and reverse - transcriptase PCR ( lower ) , with GAPDH expression used for normalization .

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Basal protein and mRNA expression in parental and VAR cells for the indicated proteins was measured using immunoblotting (upper) and reverse-transcriptase PCR (lower), with GAPDH expression used for normalization.


Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo - BrdU assay .

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Parental and VAR cells were treated as indicated for 48 hours and apoptosis was measured using the Apo-BrdU assay.


Parental and VAR cells were treated with the indicated drug concentrations for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Parental and VAR cells were treated with the indicated drug concentrations for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug - free medium for 15 - 20 days to form colonies , which were counted using ImageJ .

Parental and VAR cells were treated as indicated for 24 hours and allowed to grow in drug-free medium for 15-20 days to form colonies, which were counted using ImageJ.


Epithelial and mesenchymal non - small cell lung cancer ( NSCLC ) cell lines after treatment with 50nM volasertib for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

Epithelial and mesenchymal non-small cell lung cancer (NSCLC) cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Epithelial and mesenchymal NSCLC cell lines after Plk1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Calu6 and H1792 mesenchymal NSCLC cell lines after VIM silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 ( mesenchymal ) and H1975 ( epithelial ) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after Plk1 and INTB1 silencing with siRNA for 48 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 ( mesenchymal ) and H1975 ( epithelial ) NSCLC cell lines after treatment with fibronectin for 24 hours . Cells were then harvested , and lysates were immunoblotted for the indicated proteins .

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Calu6 (mesenchymal) and H1975 (epithelial) NSCLC cell lines after treatment with fibronectin for 24 hours. Cells were then harvested, and lysates were immunoblotted for the indicated proteins.


Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours . Cells were then harvested , and lysates were immunoprecipitated ( IP ) using β1 - integrin , vimentin , and Plk1 antibodies , then immunoblotted for the indicated proteins .

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Calu6 and H1975 NSCLC cell lines after treatment with 50nM volasertib for 24 hours. Cells were then harvested, and lysates were immunoprecipitated (IP) using β1-integrin, vimentin, and Plk1 antibodies, then immunoblotted for the indicated proteins.


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 .

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(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 .

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(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 .

O O O O O O B-MEASURED_VAR O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 ) .

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

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 ) .

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

(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 .

O O O O O O O O O O O O O O B-MEASURED_VAR I-MEASURED_VAR I-MEASURED_VAR O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR 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-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 .

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

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 .

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

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 .

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

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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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 .

O O O O O O O O O O O O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

(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.


Multiple tissue ( eye , brain , adrenal gland , submandibular gland , oesophagus , lung , heart , liver , kidney , spleen , pancreas , stomach , duodenum , jejunum , ileum , caecum , colon , spinal cord , dorsal root ganglia , skeletal muscle , testis , epididymis , female gonad , skull ) northern blot for CAVII . In the P56 mouse , CAVII is prominently expressed in the central nervous system including brain and spinal cord . Gapdh served as the loading control .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-MEASURED_VAR O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O O O O O O O

Multiple tissue (eye, brain, adrenal gland, submandibular gland, oesophagus, lung, heart, liver, kidney, spleen, pancreas, stomach, duodenum, jejunum, ileum, caecum, colon, spinal cord, dorsal root ganglia, skeletal muscle, testis, epididymis, female gonad, skull) northern blot for CAVII. In the P56 mouse, CAVII is prominently expressed in the central nervous system including brain and spinal cord. Gapdh served as the loading control.


( C ) Developmental profile of CAVII protein expression . Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30 . The band of the appropriate size ( ∼ 30 kDa ) was absent in protein lysates from Car7 KO tissue . beta Actin served as the loading control in C and D .

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(C) Developmental profile of CAVII protein expression. Western blot analysis of protein lysates of whole hippocampus shows increasing CAVII levels from P3 to P30. The band of the appropriate size (∼30 kDa) was absent in protein lysates from Car7 KO tissue.beta Actin served as the loading control in C and D.


( D ) In western blots from protein lysates of cultured cells , CAVII was detected in mixed glial / neuron cultures but not in glia cell cultures . The presence of astrocytes in all cultures was confirmed by detection of GFAP . Actin served as the loading control in C and D .

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(D) In western blots from protein lysates of cultured cells, CAVII was detected in mixed glial/neuron cultures but not in glia cell cultures. The presence of astrocytes in all cultures was confirmed by detection of GFAP. Actin served as the loading control in C and D.


Original single - cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons ( baseline pHi 7 . 21 , 7 . 13 and 7 . 11 , respectively ) . Superfusion with CO2 / HCO3 − - free HEPES solution ( upper horizontal bars ) evoked an intracellular alkalinization , which in P14 WT was large and suppressed by 100 μM acetazolamide ( AZ , lower horizontal bar ) , indicating the presence of CA activity ( see Supplementary Materials and methods ) . The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide ( a poorly permeant CA inhibitor ) . Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT ( scale bar 10 μm ) .

O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Original single-cell pHi traces and their time derivatives from P5 and P14 WT and P14 CA VII KO neurons (baseline pHi 7.21, 7.13 and 7.11, respectively). Superfusion with CO2/HCO3−-free HEPES solution (upper horizontal bars) evoked an intracellular alkalinization, which in P14 WT was large and suppressed by 100 μM acetazolamide (AZ, lower horizontal bar), indicating the presence of CA activity (see Supplementary Materials and methods). The possible effect of AZ on extracellular CAs was excluded by adding 10 μM benzolamide (a poorly permeant CA inhibitor). Inset shows an overlay of the BCECF fluorescence signal and Dodt gradient contrast image of CA1 pyramidal neurons in P14 WT (scale bar 10 μm).


Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity . Data from CA II KO and CA II / VII KO neurons were obtained only at > P35 . The number of cells tested is indicated for each bar . The animal numbers for WT mice at the different age points was n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 3 ( P14 - 16 ) , n = 2 ( P17 - 18 ) , n = 2 ( P20 - 30 ) and n = 2 ( > P35 ) ; for CAVII KO mice the numbers were n = 3 ( P5 - 8 ) , n = 2 ( P10 - 11 ) , n = 5 ( P14 - 16 ) , n = 3 ( P17 - 18 ) , n = 4 ( P20 - 30 ) and n = 2 ( > P35 ) ; and for CA II KO and CA II / CAVII KO mice the number was n = 2 ( > P35 ) .

O O O O O O O O B-MEASURED_VAR O O O O O O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR O O O O O O O O O O O O O

Summary of the results obtained using the cytosolic CA activity detection method shown in A and quantified as the percentage of cells showing cytosolic CA activity. Data from CA II KO and CA II/VII KO neurons were obtained only at >P35. The number of cells tested is indicated for each bar. The animal numbers for WT mice at the different age points was n=3 (P5-8), n=2 (P10-11), n=3 (P14-16), n=2 (P17-18), n=2 (P20-30) and n=2 (>P35); for CAVII KO mice the numbers were n=3 (P5-8), n=2 (P10-11), n=5 (P14-16), n=3 (P17-18), n=4 (P20-30) and n=2 (>P35); and for CA II KO and CA II/CAVII KO mice the number was n=2 (>P35).


Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2 / HCO3 − ( horizontal bar , HEPES ) in slices from > P35 WT , CAVII KO and CA II / VII KO mice . The rate of rise and the amplitude of the alkaline shift are identical in WT ( P39 , baseline pHi 7 . 01 ) and CAVII KO neurons ( P40 , 7 . 06 ) , whereas alkalinization develops much more slowly in the CA II / VII KO ( P46 , 7 . 04 )

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Typical single pyramidal neuron pHi responses evoked by withdrawal of CO2/HCO3− (horizontal bar, HEPES) in slices from >P35 WT, CAVII KO and CA II/VII KO mice. The rate of rise and the amplitude of the alkaline shift are identical in WT (P39, baseline pHi 7.01) and CAVII KO neurons (P40, 7.06), whereas alkalinization develops much more slowly in the CA II/VII KO (P46, 7.04)


Excitatory GABAergic response in WT , but not in CAVII KO neurons . Picrotoxin blocked the biphasic response ( PiTX 90 μM ; n = 3 / genotype ) . The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO ( 13 . 53 ± 1 . 27 versus 6 . 39 ± 0 . 96 mV / s , P < 0 . 001 , Student ' s t - test ) . The resting membrane potential of WT and CAVII KO neurons did not differ ( − 71 . 0 ± 1 . 1 versus − 72 . 3 ± 1 . 4 mV , respectively ; n = 15 neurons for both genotypes , P = 0 . 47 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D )

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Excitatory GABAergic response in WT, but not in CAVII KO neurons. Picrotoxin blocked the biphasic response (PiTX 90 μM; n=3/genotype). The mean initial slope of the depolarizing shift was faster in WT than in CAVII KO (13.53±1.27 versus 6.39±0.96 mV/s, P<0.001, Student's t-test). The resting membrane potential of WT and CAVII KO neurons did not differ (−71.0±1.1 versus −72.3±1.4 mV, respectively; n=15 neurons for both genotypes, P=0.47, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D)


Microinjection of GABA ( 5 mM for 100 ms , sr / slm border in the presence of CGP55845 / TTX , arrowheads ) induced a pronounced depolarization in WT neurons ( black ) that was abolished in the absence of CO2 / HCO3 − ( grey ) . The depolarization was smaller in CA VII KO ( red ) . Bar diagram illustrates GABAergic mean peak depolarization in P12 - 16 WT and CAVII KO ( 13 . 3 ± 0 . 5 versus 8 . 3 ± 0 . 6 mV , P < 0 . 001 , Student ' s t - test ) . * P < 0 . 05 , * * P < 0 . 01 and * * * P < 0 . 001 . Error bars denote s . e . m . and n is the number of slices ( C ) or cells tested ( one cell per slice ) ( A , B and D ) .

O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O 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-CONTROLLED_VAR I-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O B-CONTROLLED_VAR O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O O

Microinjection of GABA (5 mM for 100 ms, sr/slm border in the presence of CGP55845/TTX, arrowheads) induced a pronounced depolarization in WT neurons (black) that was abolished in the absence of CO2/HCO3− (grey). The depolarization was smaller in CA VII KO (red). Bar diagram illustrates GABAergic mean peak depolarization in P12-16 WT and CAVII KO (13.3±0.5 versus 8.3±0.6 mV, P<0.001, Student's t-test). *P<0.05, **P<0.01 and ***P<0.001. Error bars denote s.e.m. and n is the number of slices (C) or cells tested (one cell per slice) (A, B and D).