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

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-CONTROLLED_VAR 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

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 pmiR397b : : H2B : GFP transcriptional fusion ( left ) browser AGO1 - loaded signal for miR397b ( right ) .

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

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 - types isolated via Meselect .

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


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

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

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


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

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

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


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

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

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


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

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

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

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

(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 HDL at a 1 : 1 or 5 : 1 mass ) monitored using an interferometric detector . Peaks were integrated for detectors ( interference absorbance at 280nm ) . calculated stoichiometries are indicated for each peak . Solutions were equilibrated at 20 ° C for 2h before sedimentation velocity .

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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 three most representative classes of ( A ) purified HDL particles ( B ) NS1 - HDL complexes . 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 ) or of an NS1 - HDL mixture at a 2 : 1 molar ( orange ) . Of note , HDL particles alone did not generate any signal in 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 .

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

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


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

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

(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.


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

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

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.


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

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

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 ; Native Antigen Company ) were spiked in normal human plasma NS1 - ApoA - I complexes further detected by ELISA . Data represent mean values of two technical replicates .

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

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

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

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

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

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 lung cancer lines were treated with 5 ng / ml TGF - β for 14 days to induce a mesenchymal phenotype , which was confirmed with Western blot ( left ) qPCR ( right ) 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 TGF - β isogenic lines were treated with 50nM volasertib for 72 hours . were then harvested , lysates were immunoblotted for PARP γH2AX were subsequently quantitated normalized with β - actin ( B ) .

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

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 Apo - BrdU ( C )

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


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


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


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

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

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

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

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 Calu6 were treated with indicated inhibitors for 4 hours or 24 hours . were then harvested , lysates were immunoblotted for indicated .

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.


viability was measured using CellTiter - Glo in NSCLC lines treated with volasertib , tepotinib , or a 1 : 2 of both for 72 hours . Left : representative viability graph of treated with indicated drugs . Right : depicting calculated combination indices at Fa = 0 . 25 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 Apo - BrdU in indicated lines treated with indicated drugs for 24 hours .

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


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

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

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


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

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

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 Apo - BrdU in indicated lines transfected with 10nM siRNA as indicated for 48 hours . NT , non - targeting 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

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

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

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

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 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

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


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

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 ) xenograft Calu6 ( bottom ) with due to excessive burden as endpoint .

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

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.


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

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


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

O O O O O B-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

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

O B-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

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 TPR - Met - expressing lines were treated as indicated for 24 hours 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 mean from three independent experiments . Significant differences using two - way of variance with Bonferroni or Benjamini - Hochberg correction for multiple comparisons are indicated .

O B-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

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

O O O O O B-CONTROLLED_VAR 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

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.


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

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

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 .

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


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

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

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 VAR were treated as indicated for 48 hours apoptosis was measured using Apo - BrdU .

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


Parental VAR were treated with indicated drug concentrations for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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

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 VAR were treated as indicated for 24 hours 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 mesenchymal non - small lung cancer ( NSCLC ) lines after treatment with 50nM volasertib for 24 hours . were then harvested , lysates were immunoblotted for indicated .

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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 mesenchymal NSCLC lines after Plk1 silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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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 H1792 mesenchymal NSCLC lines after VIM silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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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 ) H1975 ( epithelial ) NSCLC lines after Plk1 INTB1 silencing with siRNA for 48 hours . were then harvested , lysates were immunoblotted for indicated .

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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 ) H1975 ( epithelial ) NSCLC lines after treatment with fibronectin for 24 hours . were then harvested , lysates were immunoblotted for indicated .

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

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

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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 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 - , 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 ) showing Siglec - F 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 . are 60x super - resolution max z - stack projections taken from coronal slices . ( B - C ) coverage of ( B ) Iba1 ( C ) Iba1 - proximal Siglec - F between CK CK - p25 mice across hippocampal regions . Bars indicate mean ± 95 % CI ; n = 3 - 4 ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , 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 of Siglec - F Iba1 in ( D ) 5XFAD 2 month hypothalamus ( HY ) ( 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 ) coverage of Iba1 - proximal Siglec - F relative to total Iba1 across analyzed thalamic , cortical , hippocampal regions of 6 month WT ( non - Tg ) 5XFAD mice . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 15 - 40 ( 3 - 5 ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . ( G ) coverage of Siglec - F localized to Iba1 compared to total Aβ plaque volume in each field of view across analyzed from 5XFAD non - Tg control mice . Linear regression R2 value is shown for 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 ) coverage of Iba1 - proximal Siglec - F relative to total Iba1 across analyzed thalamic , cortical , hippocampal regions of 6 month WT ( non - Tg ) 5XFAD mice . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 15 - 40 ( 3 - 5 ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , 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 between Siglec - F + Iba1 + mask voxels nearest Aβ plaques in 6 month 5XFAD mice . Each value in CDF represents mean calculated from a single z - stack of a ROI containing ≥ 1 plaque of ≥ 10 μm2 . n = 55 ( 4 ) .

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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 abundances for Siglec - 8 in ( H ) hippocampus ( I ) posterior cingulate cortex post - mortem 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 showing Siglec - 8 Iba1 , MHC - II in cortical matter tissue from a with late - onset AD ( 20x wide - field ; Case ID : 01 - 43 ) . Scale bars = 50 μm . 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 ) 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 . In ( L ) values statistics are shown for each image analyzed . Boolean mask operators indicate : ° = mask dilation , ⋂ = intersection , ⋃ = union , NOT = inverse mask . Box indicates quartiles whiskers indicate last datum within 1 . 5 inter - quartile range ; n = 8 - 43 ( 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 - , 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 of BV - 2 Siglec - F expression . were cotreated with 0 - 10 ng / mL IFNγ 0 - 10 μM tofacitinib . Values are geometric mean 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 - , two - sided . Dotted 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 of iMGLs treated with IFNγ . Siglec - 8 is only shown on non - ( Hoechst 33342 - ) regions . 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 ) of Siglec - 8 on iMGLs treated with PBS or 25 ng / mL IFNα , IFNβ , or IFNγ . Values are mean Siglec - 8 values from Iba1 + ; 33342 - regions . Bars indicate mean ± 95 % CI ; n = 8 ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted 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 ) of Siglec - 8 on iMGLs treated ± 25 ng / mL IFNγ 5 μM tofacitinib . Legend is same as ( C ) . Bars indicate mean ± 95 % CI ; n = 5 - 20 ( 2 replicates ) ; * : p < 5e - 2 , * * : p < 1e - 2 , * * * : p < 1e - 3 , * * * * : p < 1e - 4 ; ns : not significant , using unpaired Student ' s t - , two - sided . Dotted 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 with stable retroviral expression of Siglec - F related human Siglec constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field . 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 - , two - sided . Dotted 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 of live / dead markers Annexin - V propidium iodide on BV - 2 with stable retrovirus expression of Siglec - F constructs . % parent values are shown for early apoptotic ( Annexin - V + ; PI ‑ ) , late apoptotic ( Annexin - V + ; PI ‑ ) , 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 with dox - inducible expression of Siglec - F constructs . Plots indicating doubling time ( normalized to 2xY - > F constructs ) estimated from Incucyte bright - field . 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 - , two - sided . Dotted 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 ) enriched gene sets ( bottom ) taken from - seq of BV - 2 with stable retroviral expression of Siglec constructs , 2xY - > F , empty vector controls . Heatmaps share a colormap range of 0 . 25 to 4 for z - score values ( top ) , normalized enrichment scores ( NES , bottom ) .

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

(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 of C1qa , IL - 1β , β - actin by qPCR in BV - 2 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 . 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 - , two - sided . Dotted indicates mean of 2xY - > F group .

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

(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 β - tubulin in BV - 2 induced to express Siglec - F at 0 48 hours .

O O O O O O 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

(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 three runs . names are shown for altered peptides . Labels are only shown for peptides with maximum directional change from each . Labels colored according to GO terms : magenta = signal transduction , red = endosome , cyan = adhesion , yellow = cytoskeleton , green = metabolism .

O O O O O O O O B-MEASURED_VAR 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 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

(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 ) 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 .

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

(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.


showing phosphosites from CD33 , Siglec - 5 , Siglec - 8 overexpression that were increased with Siglec expression shared their directional change with Siglec - F . Siglec - F column indicates average of 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

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).


showing phosphosites from CD33 , Siglec - 5 , Siglec - 8 overexpression that were decreased with Siglec expression shared their directional change with Siglec - F . Siglec - F column indicates average of 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 of myc , SHP - 2 , SHP - 1 from myc co - IP eluates . Co - IP lysates were prepared from BV - 2 stably expressing Siglec - F optionally treated with 1 μM SHP099 .

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

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

O O O O O O O O O B-CONTROLLED_VAR 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 of monomeric Aβ uptake Siglec - F expression for ( B ) Siglec - F ( C ) Siglec - F 2xY - > F . Siglec - expressing were plated with empty vector control in same well alongside substrates .

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

(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.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse 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 - , two - sided . Dotted 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 B-MEASURED_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

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.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse 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 - , two - sided . Dotted 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 B-MEASURED_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

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.


of relative mean intensities ( MFI ) between empty vector Siglec - expressing populations . Relative values are shown for each mouse 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 - , two - sided . Dotted 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 B-MEASURED_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

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 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 - , two - sided .

O O O O B-MEASURED_VAR 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

(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 stably expressing Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from same experiment as ( G ) .

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

(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 with stable expression of Siglec - F with 0 - 2 . 5 μM SHP099 treatment . Data is from 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 - , two - sided . Dotted indicates mean of untreated 2xY - > F group .

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 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

(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 , submandibular , oesophagus , lung , heart , liver , kidney , spleen , pancreas , stomach , duodenum , jejunum , ileum , caecum , colon , spinal cord , dorsal root ganglia , skeletal muscle , testis , epididymis , gonad , skull ) northern blot for CAVII . In P56 mouse , CAVII is prominently expressed in central nervous system including brain spinal cord . Gapdh served as 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 B-MEASURED_VAR 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

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

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

(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 lysates of cultured , CAVII was detected in mixed glial / neuron cultures but not in glia cultures . presence of astrocytes in cultures was confirmed by detection of GFAP . Actin served as loading control in C D .

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

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

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

O O O O O B-MEASURED_VAR O O O O O O O O O O B-MEASURED_VAR O O O O B-CONTROLLED_VAR I-CONTROLLED_VAR 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 B-CONTROLLED_VAR I-CONTROLLED_VAR O B-CONTROLLED_VAR I-CONTROLLED_VAR O B-MEASURED_VAR 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 CA II / VII KO mice . rate of rise amplitude of alkaline shift are identical in WT ( P39 , baseline pHi 7 . 01 ) CAVII KO neurons ( P40 , 7 . 06 ) , whereas alkalinization develops much more slowly in CA II / VII KO ( P46 , 7 . 04 )

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

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 biphasic response ( PiTX 90 μM ; n = 3 / genotype ) . mean initial slope of 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 - ) . resting potential of WT 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 - ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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

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

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).


In presence of AP5 / CNQX / CGP55845 , microinjection of GABA ( 5 mM , sr / slm border ) triggered SR 95531 - sensitive field potential spikes in WT slices already upon 8 - ms injection , while even 16 - ms injection evoked hardly any spikes in CAVII KO ( left ) . Mean number of spikes was plotted against GABA puff duration in P12 - 16 WT CAVII KO ( P < 0 . 01 P < 0 . 001 , Student ' s t - ; right ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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In the presence of AP5/CNQX/CGP55845, microinjection of GABA (5 mM, sr/slm border) triggered SR 95531-sensitive field potential spikes in WT slices already upon 8-ms injection, while even 16-ms injection evoked hardly any spikes in CAVII KO (left). Mean number of spikes was plotted against GABA puff duration in P12-16 WT and CAVII KO (P<0.01 and P<0.001, Student's t-test; right). *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)


After P35 , depolarizations upon GABA microinjection ( arrowheads ) in presence of CGP55845 / TTX were indistinguishable in WT ( black ) , CA VII KO ( red ) CA II KO ( blue ) but smaller in CA II / VII KO ( green ) . depolarization in WT was abolished in absence of CO2 / HCO3 − ( grey , n = 5 ) . Bar diagram summarizes mean peak depolarization for each genotype ( right ; ANOVA , Bonferroni ) . * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 . Error bars denote s . e . m . n is number of slices ( C ) or tested ( one per slice ) ( A , B D )

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After P35, depolarizations upon GABA microinjection (arrowheads) in the presence of CGP55845/TTX were indistinguishable in WT (black), CA VII KO (red) and CA II KO (blue) but smaller in CA II/VII KO (green). The depolarization in WT was abolished in the absence of CO2/HCO3− (grey, n=5). Bar diagram summarizes mean peak depolarization for each genotype (right; ANOVA, Bonferroni). *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)


Representative raw traces of epidural EEG recordings during hyperthermia ( HT ) from a WT a CA VII KO mouse . Seizures were reliably induced in WT ( n = 9 ) but not in CA VII KO ( n = 7 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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Representative raw traces of epidural EEG recordings during hyperthermia (HT) from a WT and a CA VII KO mouse. Seizures were reliably induced in WT (n=9) but not in CA VII KO (n=7). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Specimen recordings of breath rate from a WT a CA VII KO mouse . baseline breath rate ( WT 153 ± 11 breaths / min CA VII KO 157 ± 8 breaths / min ) was increased during HT in both genotypes ( WT 272 ± 28 breaths / min CA VII KO 279 ± 12 breaths / min ) . bar diagram summarizes mean increase in breath rate in WT ( 177 ± 12 % ) CA VII KO mice ( 178 ± 6 % , P = 0 . 98 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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Specimen recordings of breath rate from a WT and a CA VII KO mouse. The baseline breath rate (WT 153±11 breaths/min and CA VII KO 157±8 breaths/min) was increased during HT in both genotypes (WT 272±28 breaths/min and CA VII KO 279±12 breaths/min). The bar diagram summarizes the mean increase in breath rate in WT (177±12%) and CA VII KO mice (178±6%, P=0.98).Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Hyperthermia caused a comparable alkalosis in blood pH in both WT CA VII KO mice . blood pH did not differ between two genotypes under control conditions ( P = 0 . 37 ) or during HT ( P = 0 . 44 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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Hyperthermia caused a comparable alkalosis in blood pH in both WT and CA VII KO mice. The blood pH did not differ between the two genotypes under control conditions (P=0.37) or during HT (P=0.44). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Electrographic seizures were not detected in WT mice when hyperthermia was induced in continuous presence of 5 % CO2 ( n = 5 ) . Error bars denote s . e . m . , P - values are on Student ' s t - ( * * * P < 0 . 001 ) , number is given in bar diagrams . Rectal temperatures are given above traces .

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Electrographic seizures were not detected in WT mice when hyperthermia was induced in the continuous presence of 5% CO2 (n=5). Error bars denote s.e.m., all P-values are based on Student's t-test (***P<0.001), and animal number is given in bar diagrams. Rectal temperatures are given above the traces.


Diazepam ( DZP , 150 μg / kg ) reduced latency to seizure onset in P14 rat pups ( P < 0 . 05 ) . There was no statistical difference between 50 μg / kg saline - injected . DZP 2 . 5 mg / kg completely blocked seizures . DZP was given intraperitoneally 15 min before hyperthermia ( HT ) onset .

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Diazepam (DZP, 150 μg/kg) reduced the latency to seizure onset in P14 rat pups (P<0.05). There was no statistical difference between 50 μg/kg and saline-injected animals. DZP 2.5 mg/kg completely blocked the seizures. DZP was given intraperitoneally 15 min before hyperthermia (HT) onset.


Diazepam given at 150 μg / kg did not affect baseline breath rate , HT induced an analogous increase in breath rate in saline - diazepam - injected rats ( P > 0 . 05 ) . At a higher ( 2 . 5 mg / kg ) , DZP suppressed breathing significantly ( P < 0 . 01 ) HT caused only a small increase in breath rate .

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Diazepam given at 150 μg/kg did not affect the baseline breath rate, and HT induced an analogous increase in the breath rate in saline- and diazepam-injected rats (P>0.05). At a higher concentration (2.5 mg/kg), DZP suppressed breathing significantly (P<0.01) and HT caused only a small increase in the breath rate.


In slice preparation , DZP ( 1 μM ) potentiated high - frequency stimulation ( 40 pulses at 100 Hz , horizontal bar ) - evoked GABAAR - mediated depolarization . Sample trace from a slice from a P14 rat pup where stimulation to border of stratum radiatum stratum lacunosum - moleculare was given in presence of AP5 , CNQX CGP 55845 . bar diagrams summarize effects of DZP on duration of depolarization ( time to half - maximum ) number of action potentials associated with depolarizing phase . P - values ( * P < 0 . 05 , * * P < 0 . 01 * * * P < 0 . 001 ) are on ANOVA , Bonferroni ( A , B ) or Student ' s t - ( C ) , error bars denote s . e . m . number of ( A , B ) or ( C ) is indicated in bar diagrams .

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In slice preparation, DZP (1 μM) potentiated the high-frequency stimulation (40 pulses at 100 Hz, horizontal bar) -evoked GABAAR-mediated depolarization. Sample trace from a slice from a P14 rat pup where stimulation to the border of stratum radiatum and stratum lacunosum-moleculare was given in the presence of AP5, CNQX and CGP 55845. The bar diagrams summarize the effects of DZP on the duration of the depolarization (time to half-maximum) and the number of action potentials associated with the depolarizing phase. P-values (*P<0.05, **P<0.01 and ***P<0.001) are based on ANOVA, Bonferroni (A, B) or Student's t-test (C), and error bars denote s.e.m. The number of animals (A, B) or cells (C) is indicated in bar diagrams.


CA VII expression during human brain development . plots show log2 - transformed exon array signal in neocortex ( NCX ) hippocampal formation ( HIP ) from early period to late adulthood . solid between periods 7 8 separates prenatal from postnatal period .

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CA VII expression during human brain development. Line plots show the log2-transformed exon array signal intensity in the neocortex (NCX) and hippocampal formation (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


CA VII expression during human brain development . plots show log . plots show normalized of absolute copy number of CA VII to Gapdh by droplet digital PCR ( ddPCR , right ) in neocortex ( NCX ) hippocampus ( HIP ) from early period to late adulthood . solid between periods 7 8 separates prenatal from postnatal period .

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CA VII expression during human brain development. Line plots show the log. Line plots show the normalized ratio of absolute copy number of CA VII to Gapdh by droplet digital PCR (ddPCR, right) in the neocortex (NCX) and hippocampus (HIP) from the early fetal period to late adulthood. The solid line between periods 7 and 8 separates the prenatal from the postnatal period.


Normalised Wnt / STOP signalling activity by of expression of two Wnt / STOP target , β - catenin c - Myc , in mitotic ( M ) asynchronised ( A ) after silencing 47 Wnt signalling regulators .

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Normalised Wnt/STOP signalling activity by quantification of the protein expression of two Wnt/STOP target proteins, β-catenin and c-Myc, in mitotic (M) and asynchronised cells (A) after silencing 47 Wnt signalling regulators.


ELISA validation of selected Wnt / STOP signalling regulators ( n = 3 ) .

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ELISA validation of selected Wnt/STOP signalling regulators (n=3).


Immunoblotting ( D ) of stability of four STOP target in both G1S natural mitotic treated with BCL9 siGenome pools CHX ( n = 3 ) .

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Immunoblotting and (D) quantification of the stability of four STOP target proteins in both G1S and natural mitotic cells treated with BCL9 siGenome pools and CHX (n=3).


Representative pictures ( left panel ) normalised biosensor on live imaging of with a GSK3 - GFP biosensor following different treatments before after metaphase ( n = 10 ) . Scale bars represent 10 μm .

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Representative pictures (left panel) and the normalised biosensor intensity based on live cell imaging of the cells with a GSK3-GFP biosensor following different treatments before and after metaphase (n=10). Scale bars represent 10 μm.


Myc β - catenin target gene enrichment on transcriptome targets regulated by BCL9 .

Myc and β-catenin target gene enrichment analysis based on the transcriptome targets regulated by BCL9.


representative xenograft tumours ( top panel ) tumour growth ( bottom panel ) of scramble BCL9 stable knockdown . LF : left flank of mouse , RF : right flank . n = 6 for shS1 vs . shB1 , n = 5 for shS1 vs . shB2 .

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The representative xenograft tumours (top panel) and tumour growth analysis (bottom panel) of scramble and BCL9 stable knockdown cells. LF: left flank of mouse, RF: right flank. n=6 for shS1 vs. shB1, n=5 for shS1 vs. shB2.


Immunohistochemical of xenograft ; red arrows indicate representative mitotic . Scale bars represent 200 μm .

Immunohistochemical staining of the xenograft; the red arrows indicate the representative mitotic cells. Scale bars represent 200 μm.


Immunoblotting of BCL9 cycle expression in double thymidine synchronised ; black arrow indicates delayed band of mitotic BCL9 .

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Immunoblotting analysis of BCL9 and cell cycle protein expression in double thymidine synchronised cells; the black arrow indicates the delayed band of mitotic BCL9.


Immunofluorescence confocal of BCL9 localization on spindle by antibody . Yellow arrows indicate spindle pole . Scale bars represent : 2 μm for interphase anaphase ; 5 μm for prophase , metaphase telophase .

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Immunofluorescence confocal analysis of BCL9 localization on the spindle by antibody staining. Yellow arrows indicate the spindle pole. Scale bars represent: 2 μm for interphase and anaphase; 5 μm for prophase, metaphase and telophase.


Immunofluorescence confocal of BCL9 localization on centrosome . Yellow arrows indicate centrosome . Scale bars represent : 5 μm for control treatment , 10 μm for BCL9 peptide treatment .

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Immunofluorescence confocal analysis of BCL9 localization on the centrosome. Yellow arrows indicate the centrosome. Scale bars represent: 5 μm for control treatment, 10 μm for BCL9 peptide treatment.


Confocal microscope 3D - radial histograms of spindle angle in with siRNA treatment ; yellow arrows indicate centrosome . Scale bars represent 5 μm .

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Confocal microscope 3D-analysis and radial histograms of the spindle angle in the cells with siRNA treatment; yellow arrows indicate the centrosome. Scale bars represent 5 μm.


Confocal ( left panel ) live imaging ( right panel ) of chromosome segregation in H2BGFP - following siRNA treatment ; yellow arrows indicate typical abnormal chromosomes by BCL9 knockdown . 23 H2BGFP - mitotic were monitored for each time experiment ; data are shown from three independent experiments . Scale bars represent 5 μm . of aberrant chromosome segregation rate by live imaging of H2BGFP upon Wnt3a or DKK1 treatment . 23 H2BGFP - mitotic were monitored for each time experiment ; data are shown from three independent experiments .

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Confocal (left panel) and live cell imaging (right panel) analysis of chromosome segregation in H2BGFP-cells following siRNA treatment; yellow arrows indicate the typical abnormal chromosomes by BCL9 knockdown. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments. Scale bars represent 5 μm. Quantification of aberrant chromosome segregation rate by live cell imaging of H2BGFP cells upon Wnt3a or DKK1 treatment. 23 H2BGFP-mitotic cells were monitored for each time experiment; data are shown from three independent experiments.


of duration of NEB to ANA ( NEB : envelope breakdown , ANA : anaphase ) , NEB to in interphase after division ( blue colour bar ) or in mitosis ( red bar ) , or growth well in 30 hours ( pink bar ) by live imaging in monitored H2BGFP with different treatments .

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Quantification of the duration of NEB to ANA (NEB: nuclear envelope breakdown, ANA: anaphase), NEB to cell death in interphase after cell division (blue colour bar) or in mitosis (dark red bar), or growth well in 30 hours (pink bar) by live cell imaging analysis in monitored H2BGFP cells with different treatments.


Volcano plot of BCL9 mitotic interactome by SILAC proteomics approach . Red dashed indicates a strict cut off by a normalised SILAC H / L > 2 .

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Volcano plot of the BCL9 mitotic interactome by the SILAC proteomics approach. Red dashed line indicates a strict cut off by a normalised SILAC H/L ratio > 2.


An independent immunoprecipitation for validation of potential BCL9 interactors in both asynchronised mitotic . relative binding of CLTC with BCL9 is quantified labelled on top of CLTC blot .

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An independent immunoprecipitation assay for validation of potential BCL9 interactors in both asynchronised and mitotic cells. The relative binding of CLTC with BCL9 is quantified and labelled on top of the CLTC blot.


Immunoprecipitation of endogenous BCL9 Axin1 interaction in synchronised HeLa , from G1S to G2M . P - H3S10 , a mitotic marker indicating mitosis .

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Immunoprecipitation assay of the endogenous BCL9 and Axin1 interaction in synchronised HeLa cells, from G1S to G2M. P-H3S10, a mitotic marker indicating mitosis.


Duolink ( A ) its ( B ) of interaction by assessing PLA in mitotic after BCL9 or Axin1 knockdown ( n = 20 ) . Scale bars represent 5 μm .

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Duolink analysis (A) and its quantification (B) of the interaction by assessing the PLA complex in mitotic cells after BCL9 or Axin1 knockdown (n=20). Scale bars represent 5 μm.


Duolink of interaction of BCL9 CLTC in mitotic . Scale bars represent 5 μm .

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Duolink analysis of the interaction of BCL9 and CLTC in mitotic cells. Scale bars represent 5 μm.


Immunoprecipitation of interaction of truncated BCL9 forms in mitosis ; purple stars indicate domain required for clathrin binding , green stars indicate domains for β - catenin binding . domain required for both clathrin β - catenin complexes binding is indicated by a red .

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Immunoprecipitation analysis of the interaction of truncated BCL9 forms in mitosis; the purple stars indicate the domain required for clathrin complex binding, and the green stars indicate the domains for β-catenin complex binding. The domain required for both clathrin and β-catenin complexes binding is indicated by a red line.


of relative binding of truncated forms with clathrin or β - catenin ; red frame indicates common domains of BCL9 for binding of both complexes .

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Quantification of the relative binding of the truncated forms with the clathrin complex or β-catenin complex; the red frame indicates the common domains of BCL9 for binding of both complexes.


Immunoprecipitation of BCL9 - MF interaction in CLTC knockdown mitotic .

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Immunoprecipitation analysis of the BCL9-MF interaction in CLTC knockdown mitotic cells.


Immunoprecipitation of endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF - overexpressing mitotic .

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Immunoprecipitation analysis of the endogenous clathrin interaction with LRP6 signalosome components in BCL9 MF-overexpressing mitotic cells.


Immunoblotting of signalosome components by immunoblotting in BCL9 , CLTC or in both BCL9 CLTC knockdown mitotic with CHX . of stability of several LRP6 signalosome components by immunoblotting of mitotic with CHX .

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Immunoblotting analysis of signalosome components by immunoblotting in BCL9, CLTC or in both BCL9 and CLTC knockdown mitotic cells with CHX. Quantification of the stability of several LRP6 signalosome components by immunoblotting of mitotic cells with CHX.


Immunofluorescence confocal of Ser1490 - phosphorylated LRP6 CLTC co - localization in mitotic HeLa ; arrows indicate colocalization . Scale bars represent 5 μm .

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Immunofluorescence confocal analysis of Ser1490-phosphorylated LRP6 and CLTC co-localization in mitotic HeLa cells; white arrows indicate the colocalization. Scale bars represent 5 μm.


Immunofluorescence of α - tubulin , Axin1 CLTC triple - in mitotic . arrows indicate colocalization . Scale bars represent 5 μm .

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Immunofluorescence analysis of α-tubulin, Axin1 and CLTC triple-staining in mitotic cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.


Immunofluorescence of colocalization of LRP6CAGFP CLTC or CD63 in interphase . arrows indicate colocalization . Scale bars represent 5 μm .

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Immunofluorescence analysis of colocalization of LRP6CAGFP and CLTC or CD63 in interphase cells. The white arrows indicate the colocalization. Scale bars represent 5 μm.


of signalosome components mitotic Wnt target expression level by immunoblotting in natural mitotic treated for 1 hour with DMSO or MDC , an inhibitor of clathrin - mediated - endocytosis .

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Quantification of signalosome components and mitotic Wnt target expression level by immunoblotting in natural mitotic cells treated for 1 hour with DMSO or MDC, an inhibitor of clathrin-mediated-endocytosis.


A representative image of soft agar oncogenic colony formation assays for stable knockdown .

A representative image of soft agar oncogenic colony formation assays for stable knockdown cells.


A representative image of mouse xenograft tumours for stable knockdown at end point of experiment .

A representative image of the mouse xenograft tumours for stable knockdown cells at the end point of the experiment.


In vitro CDK1 / cyclin B1 kinase for immunoprecipitation - purified BCL9 - Flag or T172A - Flag .

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In vitro CDK1/cyclin B1 kinase assay for immunoprecipitation-purified BCL9-Flag or T172A-Flag proteins.


Immunoprecipitation of transiently - expressed BCL9 - MF or T172A - MF interactors in synchronised , from G1S to G2 M phase . relative level of BCL9 - MF or T172A - MF immunoprecipitation is determined ; red numbers indicate up - regulation , green numbers indicate down - regulation compared to starting time point .

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Immunoprecipitation analysis of transiently-expressed BCL9-MF or T172A-MF interactors in synchronised cells, from G1S to G2 M phase. The relative level of BCL9-MF or T172A-MF immunoprecipitation is determined; red numbers indicate up-regulation, and green numbers indicate down-regulation compared to the starting time point.


Immunoprecipitation of transiently - expressed BCL9 - MF interaction in mitotic treated with RO3306 .

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Immunoprecipitation analysis of the transiently-expressed BCL9-MF interaction in mitotic cells treated with RO3306.