Male and Female Cynomolgus monkeys (Macaca fascicularis) aged from 4.8 to 5.9 years were purchased from Le Tamarinier and Noveprim Ltd. (Mahebourg, Mauritius). Six animals were group-housed in aviaries or interconnected mobile cages and two animals were individually housed in interconnected mobile cages. Animals were housed under controlled conditions (20–2 4°C, 40–70% humidity, 10–15 renewals per hour of filtered, non-recycled air, 12-h light cycle) with free access to filtered tap water and daily distribution of expanded diet (sodium dodecyl sulfate SDS, France) and fruits or vegetables. Animals used for the isolation of brain microvessels were at disposal following pre-clinical studies. Prior to the isolation of brain cortical microvessels, animals were submitted to a washout period of a minimum of 1 month.
Male mice C57BL6/J aged from 6 to 8 weeks were purchased from Charles River Laboratories (France)
Pregnant mice C57BL/6JRj were purchased from Janvier Lab’s (France) between E10 and E12.
After arrival, mice were housed individually (for pregnant mice) or grouped (for male, 6 animals per cage) in an enriched environment in a pathogen-free facility at a constant temperature of 22 ± 2°C and humidity (50 ± 10%) on a 12-h light/dark cycle with ad libitum access to food and water.
Male rats Wistar: crl WI were purchased from Charles River Laboratories (Germany) After arrival, rats were housed individually in an enriched environment in a pathogen-free facility at a constant temperature of 22 ± 2°C and humidity (50 ± 10%) on a 12-h light/dark cycle with ad libitum access to food and water.
Isolation of Brain Microvessels from Cynomolgus Monkey and rodents Cortex
Brains from Cynomolgus monkeys or rodents were collected shortly after the euthanasia of the animal into ice-cold Hibernate A medium (ThermoFisher). All subsequent steps were performed at 4 °C and under a biological safety cabinet. Brain cortex was isolated and placed in petri dishes containing ice-cold Hanks’ Balanced Salt solution HBSS. The meninges and the cortical white matter were removed. The collected tissues were transferred into a new sterile container with HBSS, finely minced with a scalpel, and then pelleted by centrifugation (5 min at 600 g, 4°C). The pellet was resuspended in a collagenase/dispase solution (Roche, Meylan, France, Collagenase 0.1 U/mL; Dispase 0.8 U/mL prepared in Ca2+/Mg2+ free HBSS) containing type I DNAse at 20 U/mL and Tosyl-L-lysyl-chloromethane hydrochloride (TLCK) at 0.147 g/mL, and incubated at 37°C for 60 min, under vigorous agitation. The digested tissue was carefully homogenized, and centrifuged for 5 min at 600 g, 4°C. The resultant pellet was resuspended in HBSS containing 20% Bovine serum albumin (BSA) and centrifuged at for 30 min at 2000 g, 4°C. The myelin ring-containing supernatants were discarded, and the vessel-containing pellet was resuspended and re-incubated in the collagenase/dispase solution in presence of DNAse and TLCK for 30 min at 37°C (except microvessels from mice). This suspension was re-pelleted by centrifugation 5 min at 600 g, 4°C, and the final pellet (named passage 0 Day in vitro 0 (P0D0) fraction from this point onwards) is resuspended in endothelial cell medium endothelial basal medium (EBM-2) supplemented with Kit endothelial cell growth medium micro vascular (EGM-2 MV) Single Quots, Lonza, Basel, Switzerland) containing 3 g/mL puromycin, before set up in pre-coated (collagen IV 100 g/mL, fibronectin 10 g/mL, Sigma, Saint Quentin Fallavier, France) cell culture flasks, and incubated at 37°C, 5% CO2 for 7 days. Every 2 days the cell medium was changed, the supplemented puromycin concentration lowered to 2 g/mL, and subsequently removed. Following 7 days of expansion at P0D7, Brain Endothelial Cell (BEC)s from cortex, were further singularized and re-plated de novo for further 7-day cell expansion (P1D7).
Stable cell lines generation
Various human influenza hemagglutinin (HA) human ITM2A cDNA with coding sequence: NM_004867 and its variants were synthetically made by GeneART and introduced in a PiggyBac® transposon mammalian expression vector pBH 6450 with a Cytomegalo virus (CMV) promoter. HA tag is inserted at different position in ITM2A sequence: at NH2 or COOH side or in the Brichos domain: at NH2 or COOH side of the Brichos domain or in the middle in order to disrupt the eventual function of this domain. Plasmids were named: pBH-hITM2A HA NH2, pBH-hITM2A HA COOH, pBH-hITM2A wild type (wt), pBH-hITM2A Brichos HA COOH, pBH-hITM2A Brichos HA NH2, pBH-hITM2A4 Brichos HA mid.
Various Green Fluorescent Protein (GFP) ITM2A constructs with coding sequence: NM_004867 for human and NM_008409 for mouse were introduced in pcDNA6.2™C-Emerald (Em)GFP or pcDNA6.2™N-EmGFP expression vectors with CMV promoter and blasticidin resistance. GFP tag is inserted at different positions in mouse or human ITM2A sequences: at NH2 or COOH sides. Plasmids were named: pcDNA6.2™N-EmGFP -hITM2A, pcDNA6.2™N-EmGFP -mITM2A, pcDNA6.2™C-EmGFP -hITM2A, pcDNA6.2™C-EmGFP -mITM2A.
Cells and cloning
HEK293/CVCL_0045 cells were obtained from Deutsche Sammlung von Mikroorganismen und Zellkulturen (DSMZ). For thawing: cells are thawed rapidly in water bath at 37°C, centrifuge at 900 g for 4mn, pellet is re-suspended in culture medium. Cells are cultured in the following medium: Dulbecco’s Modified Eagle Medium Gibco 21969 (glutamine-free: selection pressure for HA tag or Blasticidin 15µg/mL: selection pressure for GFP tag); 10% Foetal Calf Serum Eurobio CVFSVF06 heat inactivated Australian; Penicillin / Streptomycin Gibco 15140 100 U / mL final. At confluence, the cells are rinsed with Phosphate-buffered saline (PBS) (-Ca2+, -Mg2+), detached by enzymatic treatment (Accutase Sigma A6964 or trypsin) at 37°C for 3mn, centrifuge at 900g for 4mn. The pellet is re-suspended in culture medium and diluted to 1/10 in fresh medium for seeding in new flasks.
Human Cerebral Microvascular Endothelial Cell (hCMEC)/D3 cells were obtained from Cedarlane. Cells are cultured in the medium cell biologics add with supplements for endothelial cells H1168 as described above.
HEK293/CVCL_0045 cells were transfected with pcDNA6.2™-EmGFP or cotransfected with PiggyBac® transposon expression vectors bearing wt ITM2A and variants cDNAs and transposase plasmids 6209 (10:1) by using Lipofectamine 2000®, following manufacturer instructions. Transfections were performed at 50% confluence in 24 wells plate with 500 ng of plasmids. For stable transfections, clones are obtained by limit dilution in 96 well plates with blasticidin selection for pcDNA 6.2 or in glutamine free media corresponding to the glutamine synthase selection marker of pBH 6450. Each individual cell is amplified in a 6 wells plate, then ITM2A expression is checked first with fluorescent microscope for GFP tag, second by Western Blot for all ITM2A constructions.
The human and murine ITM2A extra-cellular domain (ECD) gene sequence was synthesized and fused to a human Fc in a mammalian expression vector. After preparation of transfection-grade DNA, a transient transfection of HEK293 cells was performed. After 7 days of cultivation, the ITM2A-Fc containing culture supernatant was isolated and purified by Protein A affinity chromatography according to standard protocols. After buffer exchange to PBS, the purified antigen was analyzed by UV/VIS spectrometry and SDS-PAGE.
The target protein (ITM2A-human Fc) and negative antigen (Protein N Standard, Siemens, QQIM13) were immobilized onto the wells of an Enzyme-Linked Immunosorbent Assay (ELISA) plate (Corning, 9018) for 1 h at room temperature (RT) (1 µg each). After removal of non-bound antigen, ELISA wells were blocked with a 2% BSA solution for 16 h at 4 °C. After washing of the plates with PBS-T (PBS containing 0.05% Tween 20), the antibody-phage library was added to the immobilized negative antigen and incubated for 1 h at RT to remove Fc specific or polyreactive antibody-phage. Additionally, 5 µg Protein N Standard was added as soluble competitor. Non-bound antibody-phage were recovered and incubated on the immobilized target antigen for 2 h at RT. Non-bound or weakly bound antibody-phage were removed by washing with PBS-T (10x) before antigen-specific phage were recovered by Trypsin (10 µg/ml) elution for 30 min at 37 °C. The antibody-phage were rescued by infection of TG1 cells (OD600=0.5) for 30 min at 37 °C. After propagation of the cells for additional 30 min at 37 °C and 500 rpm, ampicillin (100 µg/ml) and glucose (100 mM) were added to the 2YT culture medium. Bacterial propagation was continued for 1 h at 37 °C and 500 rpm. Then, bacteria were co-infected with M13K07 helper phage, incubated at 30 min at 37 °C followed by another incubation for 30 min, 37 °C and 500 rpm. Double-infected bacteria were centrifuged (4000 g, 10 min) and the cell pellet was resuspended in fresh 2YT, containing Ampicillin (100 µg/ml) and Kanamycin (50 µg/ml). For the amplification of antibody-phage particles, the incubation was continued for 16 h at 30 °C. Then, the culture was centrifuged (4000 g, 10 min) and antibody-phage containing supernatant was recovered and used for the next panning cycle. Three panning cycles were performed in total. In each cycle, the number of washing steps was increased (cycle 2: 20x, Cycle 3: 30x) to increase the stringency of the selection.
Antibody screening and sequence analysis
After the third panning cycle, the eluted phage were used to infect XL1 (OD600=0.5) for 30 min at 37 °C and streaked out on 2YT agar plates, containing ampicillin (100 µg/ml), glucose (100 mM) and tetracycline (20 µg/ml). Incubation was continued at 37 °C until single colonies were observed. Single clones were isolated and transferred into 96-well plates, containing 2YT, ampicillin (100 µg/ml), glucose (100 mM) and tetracycline (20 µg/ml). The bacteria were cultivated for 16 h at 37 °C and 300 rpm. Then, 15 µl of the overnight cultures were used to inoculate new 96-well plates, containing 2YT medium, ampicillin (100 µg/ml), tetracycline (20 µg/ml) and IPTG (50 µM). The bacteria were cultivated for 16 h at 30 °C and 300 rpm. The cultures were centrifuged (4000 g, 10 min) and Single-Chain Variable Fragment (scFv) containing supernatants recovered. The scFv containing supernatants were used for antibody screening. In brief, supernatants were diluted with a 2% BSA solution (in PBS, containing 0.05% Tween20), added to the immobilized antigens in a 384 well ELISA plate (20 ng/well) and incubated for 1 h at RT. After washing, binding of the scFv antibodies to human ITM2A-human Fc, murine ITM2A-human Fc, Protein N Standard or BSA was detected via a Myc-tag using a secondary horseradish Peroxidase (HRP) conjugated antibody. The binding was quantified by TMB reaction and absorbance reading at 450 nm. Target specific antibody clones were isolated, and the DNA sequence of the respective scFv antibody analyzed by sanger sequencing.
Immunoglobulin G (IgG) expression
The VH and VL sequence of selected antibody clones was amplified by PCR and cloned into mammalian IgG expression vectors. After preparation of transfection-grade DNA, a transient transfection of HEK293 cells was performed. After 7 days of cultivation, the IgG containing culture supernatant was isolated and purified by Protein A affinity chromatography according to standard protocols. After buffer exchange to PBS, purified antibodies were analyzed by UV/VIS spectrometry, SDS-PAGE and flow cytometry analysis for cell binding.
Transcriptome Sequencing (RNAseq) and mRNA Expression Analysis COMPACT IMI consortium
As described previously by Li et al (11).
RNA Samples for Library Preparation
As described previously by Chaves et al (34) Frozen cell pellets from the cellular P0D0, P0D7 and P1D7 fractions were lysed using QIAzol Lysis Reagent (Qiagen, #79306, Courtaboeuf, France). Total RNA was then isolated from the lysates on a QIAcube instrument (Qiagen) using the Rneasy Mini QIAcube kit (Qiagen, #74116) and following the manufacturer’s instructions. The RNA concentration was determined using the Qubit RNA HS Assay Kit (Invitrogen, #Q32852, Illkirch-Graenstaden, France) and the quality and integrity was assessed on a Bioanalyzer 2100 (Agilent Technology, Les Ulis, France) with an Agilent RNA 6000 nano kit (Agilent Technology, #5067-1511).
RNA Sequencing (RNAseq)
As described previously by Chaves et al (34), the RNAseq libraries were prepared with 30 ng of input total RNA using the NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (New England Biolabs, #E-7765S, Évry-Courcouronnes, France) with the NEBNext rRNA Depletion Kit (New England Biolabs, #E6310L) and following the manufacturer’s instructions. The libraries were then paired end sequenced (75 cyclesx2) on the NovaSeq 6000 instrument (Illumina, Paris, France) using the NovaSeq 6000 SP Reagent Kit (300 cycles; #20027465, Illumina).
Reverse transcription quantitative Polymerase Chain Reaction (RTqPCR)
Cells or tissus were lysed with RLT buffer from Qiagen added with 1% β-mercapto-ethanol according to manufacturer instructions. mRNA was extracted with Qiagen MiniKit followed by DNase step using the Qiacube robot. Reverse Transcription is achieved from 250 ng of mRNA with High Capacity cDNA Reverse Transcription Kit from AppliedBiosystem Ref 4368813. qPCR is performed from cDNA diluted 1/20 using QuantStudio™ 7 in 384 wells with the TaqMan system standard mode and following manufacturer instructions. Primers and Probes Taqman were bought at thermoficher scientific inventoried assay ref 4331182, ITM2A: Mm00515208_m1; Actin: Mm01205647_g1; glyceraldehyde-3-phosphate deshydrogenase (GAPDH) Mm99999915_g1; Platelet endothelial cell adhesion molecule (PECAM) 1 Mm01242576_m1. Raw data were analyzed with QuantStudio™ Real-Time PCR software.
Cultured cells were fixed in 4% paraformaldehyde for 15 min, at RT, and subsequently permeabilized and blocked in Blocking Buffer Odyssey LiCor containing 0.2% Triton X-100. Primary antibodies were incubated overnight at 4°C (anti-ITM2A polyclonal AF4876 and 14407-1-AP, monoclonal non-commercial provider Yumab: YU93-G04, YU147-A01 YU147-E02 YU147-H07, anti-EmGFP A31852 or A11122, anti-HA 901509), and appropriate secondary antibodies conjugated with Alexa fluorophores (Invitrogen) and Hoechst 33432 (Invitrogen) for nuclei staining were subsequently used for 2h at RT. Images were acquired on a Perkin Elmer Operetta CLS system.
Colocalization confocal imaging
Confocal microscope images were acquired with SP8 LEICA microscope, 40X objectives. For the acquisition ITM2A was directly visualized by EmGFP 488. Organelles were stained with following primary antibodies anti Lysosomal-associated membrane protein 1 (LAMP1) ab24170 for lysosome detection, anti-giantin ab37266 for Golgi. Antibody anti-organelle were detected by staining with appropriate secondary antibody Alexa 633 goat anti mouse A21050 1/1000 or goat anti rabbit A21070 1/1000
NHP-derived BEC were lysed using ice-cold radio-immunoprecipitation assay (RIPA) or cell lysis buffer containing protease inhibitor cocktail (ThermoFisher) centrifuged at 15,000 g for 15 min, and supernatant fractions were collected. Samples were added with SDS and loading buffer then denaturized by heat 95°C for 5 min. These were loaded into 4–12% Tris-Glycine SDS-page gels (Invitrogen), and let to migrate for 1h at 180V. Samples were then transferred onto polyvinylidene fluoride (PVDF) or nitrocellulose membranes using an iBlot 2 Dry Blotting System (Invitrogen) on the P0 program (20 V for 1 min, 23 V for 4 min, 25 V for 2 min). PVDF membranes were then rinsed with Tris-buffered saline with 0.1% Tween 20 (TBST) and blocked for 1 h in 5% non-fat dry milk in TBST (blocking buffer). Membranes were first probed overnight at 4°C with primary antibodies in blocking buffer (anti-ITM2A polyclonal AF4876 and 18306-1-AP, EmGFP A11122, anti-HA 901509, anti α-tubulin T9026) and then probed with secondary antibodies diluted in TBST for 1 h at RT (1:10,000 diluted HRP-coupled goat anti-mouse IgG or goat anti-rabbit IgG, GEHealthcare). Following secondary antibody incubation, membranes were rinsed thoroughly with TBST, imaged using a LICOR Odyssey Imager and bands quantified using Multi-Gauge v3.0.
Post-mortem human brain samples (occipital cortex, devoid of pathological findings) from three 69 to 79-year-old, male, non-demented, control donors were obtained from external biological resource centers in full accordance with legislation and ethical standards. Microvessels were isolated as described above for monkeys. Tissus or cells were lysed in Preomics 2X buffer then crushed gently with gentle MACS Dissociator Miltenyi Biotec, program Protein for 1min. Samples were centrifugated 10min at 4000g, 4°C, supernatants were collected and dilute to 1X Preomics buffer. Benzonase 1/100 was added and incubated 10 min à 95°C, 1000 rpm. Samples were centrifugated 20mn at 13000g and supernatants were collected. Proteins are quantified by spectrophotometry using bicinchoninic acid method and spectraMax i3x. 25 µg of protein were digested on Preomics filter in 50µL following manufacturer instructions during 3h at 37°C, after evaporation, samples were diluted at 0,5µg/µL in 50µL LC-Load buffer, vortexed and sonicated. Heavy peptides were added to the sample solution to inject 5 fmol of heavy peptides and 2µg of proteins. 7 heavy stable isotope labelling (SIL) peptides spiked in final digest prior to Parallel Reaction Monitoring-Mass Spectrometry analysis on Q-Exactive HF /NanoRSLC 3500. From ITM2A_MOUSE Integral membrane protein 2A Q61500, spiked SIL peptides for detection were: IAFNTPTAVQK, NLVELFGK, EDLVAVEEIR, DLLLGFNK. Spiked SIL peptides used for hTFRC detection were: DSAQNSVIIVDK, LTVSNVLK, SGVGTALLLK, AAAEVAGQFVIK, LTTDFGNAEK.
HEK293 ITM2A cells are cultured on 96 wells plate coated with poly-L-lysine at 50000 cells/well. After 24h cells were incubated 1h at 4°C in cell medium with 5µg/mL of studied antibody (Anti ITM2A RandDsystem AF4876, Yumab antibodies anti ITM2A YU147-A01, E02 and H07, YU93-G04, GFP antibodies A31852 or A11122, anti-HA 901509). Cells were washed with ice cold PBS then incubated 1h at 37°C or at 4°C for control. Cells were washed 2 times with ice cold PBS, then 2mn with acid buffer (glacial acid acetic 1/167 in PBS, pH = 2,5) and finally 2 times with ice cold PBS. Cells were fixed and stained as described above. Images were acquired with Operetta High Content Screening (PerkinElmer) objective 40X water confocal mode.
In Vitro Transcytosis and Permeability Measurements Brainplotting models
All human samples were provided by Brainplotting (35)(iPEPS, Institut du Cerveau et de la Moelle épinière, Hôpital Universitaire de la Pitié-Salpêtrière, Paris, France) in partnership with Sainte-Anne Hospital, Paris (neurosurgeon Dr. Johan Pallud) and harvested during tumor scheduled resection surgery with written informed consent from the patients (authorization number CODECOH DC-2014-2229). Human brain microvessels were obtained from surgical resections of one patient: a 35-years-old female suffering from of a diffuse oligoastrocytic grade III glioma. Microvessels were isolated from peritumoral or healthy brain tissue using an enzymatic procedure(34) adapting methods previously published for rats(36, 37). Briefly, tissue samples were carefully cleaned from meninges and excess of blood; then, an enzymatic mix was used to dissociate the tissues and microvessels were isolated by retention on a 10 mM mesh. Cells were cultured in EBM-2 medium (Lonza, Basel, Switzerland) supplemented with 20% serum and growth factors (Sigma)(36, 37). After seeding brain capillaries in petri dishes, brain primary microvascular endothelial cells were shortly amplified and seeded (P1D0) on Transwell (Corning) with microporous membranes (pore size: 0.4 mm) in monoculture.
Test (1 µg/mL of internal anti-human/cynomolgus Transferrin Receptor C (TFRC) antibody or anti-ITM2A YU93-G04) and control antibodies (1µg/mL mouse IgG, clone MG1-45, BioLegend) were added onto the upper chamber on day adapted to the transport assay defined by Brainplotting. Fresh endothelial cell medium with none of these compounds was added onto the bottom chamber. Final aliquots from both chambers were taken 240 min following incubation at 37°C, 5% CO2. Compound levels in mother solutions (T = 0 min), upper and lower compartments (T = 240 min) were determined by ELISA assay (MESOQuickPlex SQ120, MesoScale Discovery, Rockville, MD, USA). Apparent permeability (Papp) coefficients were calculated using the following formula:
Papp (cm/min) = (V /(A x Cluminal)) x (Cabluminal/ t)
where V = volume of cell medium in the bottom chamber (mL), A = surface area of the insert (cm2), Cluminal = compound concentration loaded in the upper chamber (µM), Cabluminal = compound concentration measured in the bottom chamber (µM); t = time of the assay (min).
Immunoassay Method ELISA (in vitro and in vivo studies)
Standard 96-well sector plates (Meso Scale Discovery) were coated with 0.5 μg/mL of Fab’2 anti-Human (709-006-098 Jacksonimmuno) or anti-mouse (M0284 Sigma-aldrich) IgG in PBS and then incubated for 1 h under agitation at RT. After incubation, plates were washed three times with PBS-Tween 0.05% (Calbiochem, 524653) and blocked for 1 h at RT with 0.1% BSA solution (A7030, Sigma). After blocking the plates, collected samples in transport assay and standards were incubated on plates for 2 h at RT. After incubation, plates were washed three times with PBS-Tween 0.05%, and bound antibody was detected with SULFO-TAG conjugated goat anti-mouse antibody (R32AC-1, Meso Scale Discovery) or goat anti-human antibody (R32AJ-1, Meso Scale Discovery) using Tripropylamine containing read buffer (R92TC-2, Meso Scale Discovery). Concentrations were determined from the standard curve using a four-parameter non-linear regression program (Discovery Workbench version 4.0 software).
In vivo experimentations
Mouse brain collection
Each mouse was anaesthetized in isoflurane gas chamber then transcardiacally perfused with Li-heparinate solution at final concentration 20 U/mL in sterile PBS. Perfusion was realized with 48mL delivered at the speed of 8 mL/mn. Brain samples were collected. Each mouse was decapitated immediately after perfusion. Perfused brain was removed, cerebellum and brainstem were separated and eliminated. Brain cortex were washed in ice-cold PBS, collected in preweighted Precellys tube and stored immediately at -80°C freezer (or dry ice) until use. Then, the preweighted hemispheres were thawed and homogenized in 5 vol. (v/w) of brain lysis buffer (1% NP-40 in PBS containing complete mini ethylene diamine tetra-acetic acid - free protease inhibitor cocktail tablets, Pierce) using bead homogenizer. Homogenized brain samples were then rotated at 4°C for 1 hour before centrifuge at 20000 g for 20 min.
Pharmacokinetic study in vivo in mouse
5-10 mg/kg (35-70 nmol/kg) in a single dose of anti-ITM2A (clone YU93-G04) or control (anti- trinitrophenyl (TNP), batch VA2-17-419-1, internal production) antibodies were administrated by caudal intravenous injection into mice, C57Bl/6, male, 20-25g (n = 3/condition). 5h post-injection, plasma and saline-perfused brains were collected, then concentration was determined by ELISA immunoassay as described above
In vivo panning Yumab in mice
Yumab provided amplified phage library to Sanofi. First, a naïve human antibody phage library was enriched for antigen specific antibodies against ITM2A proteins. The antibody phage output was amplified and purified by poly-ethylene glycol (PEG)/ NaCl purification. Purified antibody phages were directly used for the in vivo panning: 1011 antibody phage particles (~10 µl) were mixed from each panning output. The antibody phage mix was injected into the mouse. Brains were isolated after 1 h and 24 h (2x each). Brains were collected as described above.
Antibody phages in the homogenate were used for infection of E. coli. Bacteria were selected and used for production of monoclonal scFv antibodies. About 800 clones were picked and screened by scFv in ELISA assay.
The statistical significance of differences between groups was analyzed using GraphPad Prism v9.0.0 software (GraphPad Software, San Diego, CA, USA) Ordinary one-way Analysis of Variance (ANOVA) (non-parametric or mixed) with Dunnett’s multiple comparisons test, Ordinary two-way ANOVA (mixed) with Sidak’s, Kruskal Wallis or Mann-Whitney multiple comparison test or unpaired t test (non-parametric). The application of these statistical methods to specific experiments is noted in the Fig. legends.