EMBO/SourceData huggingface dataset:
Figure 2A iPSCs obtained from a control (WT#1; (Lenzi et al, 2015), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) were differentiated into myocytes by MYOD/BAF60c overexpression. Cells were collected before the induction (iPSC) and after 9 days in DM (Myo) and RNA was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD expression. GAPDH was used as loading control. Representative results are shown (n=3).B Representative immunofluorescence for Myosin Heavy Chain (MHC in red) in combination with DAPI staining (in blue) of iPSCs obtained from WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2) differentiated for 9 days into myocytes by MYOD/BAF60c overexpression. Scale bar 100 μm. C Histogram represents fusion index quantification. At least 7 randomly chosen microscope fields of two independent biological samples were analysed (n=2). Data are presented as mean ± SD of the biological replicates.D RNA extracted from myocytes obtained by differentiation of WT#1, GS∆44 (GS∆44#8) and GS∆44 edited clones (GS∆44#8.2 ∆CELF2a) was analysed by RT-PCR for CELF2a, CELF2b, CELF2c and DMD exon 45 skipping. GAPDH was used as loading control. The arrow indicates the DMD exon 45 skipped band. Representative results are shown (n=3).E Western blot on proteins (40 μg) extracted from myocytes obtained by differentiation of WT (WT#1), GS∆44 (GS∆44#8) and edited GS∆44 (GS∆44#8.2 ∆CELF2a) iPSC clones probed with antibodies against dystrophin (DMD). Actinin (ACTN) was used as a loading control. For the WT 5% sample, 2 μg of proteins were diluted in 38 μg of proteins belonging from GS∆44#8 to reach the total amount of 40 μg. Representative results are shown (n=3).
Figure 3A DNA accessibility (ATAC-sequencing) and chromatin marks (ChIP-seq) signals in the Celf2 region (+/- 50kb) using the UCSC genome browser. ATAC-seq peaks called in the two conditions are reported below each track. The differential peaks specific for one of the two conditions are reported in the tracks ATACseq_specific peaks_WT and ATACseq_specific peaks_GS∆44. ChIP-seq signals for H3K4me3, H3K27ac and H3K27me3 in WT and GSΔ44 myoblasts (WT myoblasts in light orange; GSΔ44 myoblasts in light blue). ChIP-seq for MyoD in control cells was obtained from (MacQuarrie et al, 2013) (GSM1218849). Orange boxes highlight the regions containing the first exon of each isoform (a, b and c).D Chromatins extracted from Control (WT) and GS∆44 myoblasts were immunoprecipitated with anti-MyoD and control IgG antibodies. Regions corresponding to the MyoD binding sites present in Peak A (see Figure EV3D) and in the MAFA and LSTZ2 genes were analysed by qPCR. Fold enrichments of PeakA over MAFA or LSTZ2 immunoprecipitation (IP) are plotted for WT and GS∆44 samples. The mean ± SEM of triplicates from one representative among three experiment is shown. Statistical significance of differences between means was assessed by a two-tailed t-test and P<0.05 was considered significant. *P=0.0339, **P=0.0067.
Figure 4B RT-PCR analyses of indicated RNAs in control (WT), ∆44 and in GS∆44 myocytes collected after 9 days upon the induction of differentiation. GAPDH was used as control. Representative results are shown (n=3).C RT-PCR analyses for CELF2a and DUXAP8 expression in WT and GS∆44 myoblasts (GM) and myocytes (DM). GAPDH was used as control. Representative results are shown (n=3).D RT-PCR analysis of DUXAP8 expression in RNA extracted from GS∆44, GS∆44's mother (GSM) and GS∆44's father (F) fibroblasts. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).E RT-PCR analysis for DUXAP8 expression in RNA extracted from WT and GS∆44 myocytes and from WT (WT#1), GS∆44 (GS∆44#2, GS∆44#8) and GSM (GSM#1) iPSCs induced to differentiation for 9 days. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).F RT-PCR for the indicated RNAs on extracts from GS∆44 myoblasts treated with either control siRNAs (siSCR) or siRNAs against DUXAP8 (siDUXAP8) and collected two days after transfection. GAPDH was used as control. Lane (-) indicates the negative control. Representative results are shown (n=3).G qPCR analysis of DUXAP8 and CELF2a expression in control myoblasts transfected with a plasmid for the overexpression of DUXAP8 (OE DUXAP8) or an empty vector (Empty), and collected after 96h. Relative mRNA levels were calculated with the delta delta Ct method. qPCR reactions were normalized against an internal control (GAPDH) and plotted relative to the expression level in the sample treated with the empty vector which was set to a value of 1. The mean ± SEM of triplicates from one representative experiment is shown (n=3).
Figure 2Microscope images of the tissue and microinjection pipette before (top) and after (bottom) annotation of the tissue surface and microinjection micropipette tip by the experimenter. Scalebars: top 100µm, bottom 10µm. Ap indicates the apical surface and BL indicates the basal lamina.
Figure 3(Left) Confocal image of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different pressures. Microinjections were attempted at pressure of 75 mbar (left area), 100 mbar (center area), and 125 mbar (right area). (Right) Percentage of successful injections achieved by the Autoinjector at 75 mbar (35%, n = 314 total, 110 successful), 100 mbar (35%, n = 185 total, 65 successful), and 125 mbar (8%, n = 287 total, 23 successful).(Left) Confocal images of a section of mouse telencephalon fixed and stained immediately after microinjection. Dashed lines indicate locations in the slice where microinjections were attempted at different depths keeping the pressure constant at 75 mbar. Microinjections were attempted at depths of 10 μm (left area), 15 μm (center area), and 25 μm (right area) into the apical surface. (Right) Percentage of successful injections achieved by the Autoinjector at depths of 10 μm (68%, n = 163 total, 111 successful), 15 μm (22%, n = 170 total, 37 successful), and 25 μm (11%, n = 169 total, 19 successful) into the apical surface.Cells targeted at a constant spacing of 30 μm, scale bar is 10 μm.
Figure 4Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Fluorescence images of tissue after microinjection with dextran-A555 (magenta). The tissue was stained for Tbr2 (green) and TuJI (white). Microinjected cell at 0 h; injected cells show the typical bipolar morphology of an AP and are mainly negative for the BP marker Tbr2. Two-daughter cell progeny 24 h after microinjection; the top cell shows the bipolar morphology characteristic of an AP and is negative for Tbr2; the bottom cell is a newborn BP positive for Tbr2. Both cells are negative for the neuronal marker TuJI (white). Four-daughter cell progeny 48 h after microinjection; note that daughter 1 and 2 reside in the VZ, while daughter 3 and 4 are positive for Tbr2 and TuJI and reside in the SVZ. Distribution of microinjected cells and their progeny in the VZ, SVZ and CP (cortical plate). n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Distribution of microinjected cells into 9 bins based on the distance from the ventricular surface (0 µm) and expressed as % of total. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dextran-A555 with slice culture for 24 or 48 h After fixation slices were stained for Tbr2 and TuJI. Expression of Tbr2 in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h. Expression of TuJI in microinjected cells and their progeny at 0, 24 an 48 h. n = 147 cells total for 0 h, 151 cells total for 24 h, and 26 cells total for 48 h.Automated microinjection was performed on organotypic slices of mouse E14.5 dorsal telencephalon using Dx-A488 along with mRNA for RFP Organotypic slices of mouse E14.5 dorsal telencephalon injected with Dextran-A488 (green) and RFP poly-A+-mRNA. After 24h in culture the progeny expresses RFP (magenta; blue: DAPI, n = 17 cells injected, 8 RFP positive). Percentage of RFP-positive and RFP-negative cells (n = 17 cells injected).
Figure 5Automated microinjection was performed on organotypic slices of mouse E14.5 - E15.5 dorsal telencephalon using a solution containing Dextran-A555 (not shown) and Alexa488 (green). Slices were fixed after microinjection (n = 71 cells total) and were stained for Tbr2 (white, H-J). (A-C) Representative examples of a non-coupled cell (A, cartoon on the right), a 2-cell cluster (B, cartoon on the right) and a 5-cell cluster (C, cartoon on the right). The asterisks indicate the Dx-A555-positive, microinjected cell. Percentage of microinjected cells found in a coupled cluster (coupled, green). Cluster size, expressed as % of total. Distribution of coupled cells expressed as the distance from the ventricular surface (0 µm = ventricle surface. Distribution of coupled cells divided into 11 bins and expressed as % of total. the images on the left are maximum intensity projections (MIP) of 18, 17, 23, 12 and 30-focal planes, respectivelyA representative picture of a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2. Microinjected cells are stained for Tbr2. A representative picture of (H) a 2-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 2) and (I) a 7-cell cluster (cartoon in the middle, nuclei are numbered from 1 to 7), the latter containing one Tbr2 positive cell. Microinjected cells are stained for Tbr2. The asterisks indicate the Dx-A555-positive, microinjected cell. comparison of Tbr2-positive cells (expressed as % of total) among all the cells in the VZ (VZ; n=1003 from 4 different confocal images from 3 independent experiments; error bar represents standard deviation) and in coupled clusters in the VZ (coupled clusters, VZ; n= 104 from two independent experiments). Coupled cells were scored based one presence/absence of apical and/or basal polarity cues, and results are expressed as % of total. the images on the right are single optical sections corresponding to the nucleus (white dotted line); scale bars are 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.
Figure 6Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 dorsal telencephalon using Dx-A555 Phase contrast image of automated microinjection into mouse neurons. Scale bar is 100 µm.Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection and stained for Reelin (white, C) Left: overview image of a mouse microinjected area (arrow indicates a pyramidal neuron, arrowhead indicates a Cajal-Retzius neuron; image is MIP of 20 focal planes). Microinjected neurons show different morphology and positivity for the Cajal-Retzius marker Reelin (white). Right, top row: the pyramidal neuron indicated with an arrow in the overview image is negative for Reelin. Right, bottom row: the Cajal-Retzius neuron indicated with an arrowhead in the overview image is positive for Reelin. Images on the top and bottom row left (LY MIP) are a MIP of 2 and 3 focal planes, respectively. The three images on the top and bottom right (LY, Reelin and DAPI) are single optical sections. Scale bar is 20 µm for MIP and 10 µm for single focal planes. VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.Automated microinjection of mouse neurons. Automated microinjection was performed on organotypic slices of mouse E16.5 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green Slices were fixed after microinjection and stained for SatB2 (white, Overview image of a mouse microinjected area. The arrow indicates a pyramidal neuron; the image is MIP of 30 focal planes, scale bar: 50 µm. Row on the right: the microinjected neuron with pyramidal morphology indicated with an arrow in the overview is positive for the upper-layers marker SatB2 (white). Images are single focal planes. Scale bar is 10 µm. Proportion of neurons positive or negative for Reelin (n = 12 cells total). Proportion of neurons positive or negative for SatB2 (n = 39 cells in total). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.Automated microinjection of human (G,H) neurons. Automated microinjection was performed on organotypic slices of human 12 wpc dorsal telencephalon using Dx-A555 (not shown) and LY (green, Slices were fixed after microinjection Microinjected neurons in the developing human telencephalon. Top, MIP of 20 focal planes (scale bar: 50µm). Bottom: high magnification (scale bar: 10 µm). VZ, ventricular zone; SVZ, subventricular zone; CP, cortical plate.
Figure 1D Distributions of pairwise Ka/Ks (nonsynonymous to synonymous nucleotide substitution rates) ratios between PIWI duplicates in arthropods (n = 17 in Drosophila and n = 37 in others, Mann-Whitney U test, *P<0.05). The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.
Figure 2A PIWI genes are present in the locust brain. Rp49 served as a loading control (n=4 biological replicates). Data information: data are presented as mean ± SEM.C The fluorescence signals of PIWI proteins were determined in locust brains. Hochest33342 was used to label cell nuclei in locust brains. The scale bars represent 20 µm.
Figure 3G Food intake by dsPiwi1-treated locusts and dsGFP-treated control locusts within 1 h (n=5 biological replicates) , ***P<0.001 (Student's t-test). Data information: All assays were performed on fourth-instar locusts.
Figure 4C Representative images of Nile red staining of lipid droplets in the fat bodies of dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). The scale bars represent 50 µm. D Quantification of lipid droplet staining in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). E Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.E, F Measurements of glycogen (n=5 biological replicates) and TAGs (n=10 biological replicates) in fat bodies. Data information: All data are presented as the mean ± SEM (Student's t-test, **P<0.01, ***P<0.001, ns: not significant). All assays were performed on four-day-old fourth-instar locusts.
Figure 5B Food intake within 1 h following treatment with candidate genes (dsNPF1-treated, n=4 biological replicates; dsCG10621-treated, n=5 biological replicates; dsMFS3-treated, n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, **P<0.01).D, E Representative images (D) and quantification (E) of Nile red staining of fat body lipid droplets in dsNPF1-treated locusts and dsGFP-treated controls (n=5 biological replicates). The scale bars represent 50 µm, the data are shown as the mean ± SEM (Student's t-test, **P<0.01).G Food intake within 1 h in the food instake assays (n=5 biological replicates), the data are shown as the mean ± SEM (Student's t-test, *P<0.05, ***P<0.001) . H Food intake within 2 h in the food instake assays (n=5 biological replicates). Five locusts were included in each replicate, the data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01).
Figure 6B mRNA levels of differentially expressed piRNAs located within the flanking region of the NPF1 gene in dsPiwi1-treated locusts and dsGFP-treated control locusts (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).C The mRNA expression of NPF1 was measured by qPCR after a mixture of inhibitors or mimics of piRs-3-I2 and piRs-3-I3 was injected (n=5 biological replicates). Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).E Body weight measurement of locusts administered inhibitors (NC, n=25 locusts; Inhibitors- piRs-3-I2+I3, n=23 locusts) and mimics (NC, n=37 locusts; Inhibitors-piRs-3-I2+I3, n=39 locusts) of piRs-3-I2 and piRs-3-I3. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value.F RNA-binding protein immunoprecipitation assay of piRs-3-I2 and piRs-3-I3 in Piwi1 immunoprecipitates from brain tissue extracts (n=6 biological replicates). Normal mouse IgG was used as a negative control.
Figure 7A U6 RNA was used as a nuclear RNA control (relative value set to 1 in the nuclear fraction), and glycine tRNA was used as the cytoplasmic RNA control (relative value set to 1 in the cytoplasmic fraction) (n=4 biological replicates), U6 and tRNA-Gly was used as an endogenous control.B Relative expressions of piRs-3-I2 and piRs-3-I3 determined by qPCR and high-throughput sequencing in the nucleus (n=3 biological replicates). The brain tissues were homogenized and then divided into two parts of equal volume; one was prepared for high-throughput sequencing and the other was used for piRNA quantification in nuclear and cytoplasmic fractions after subcellular RNA fractionation. The relative piRNA expression in nucleus was determined by multiplying the relative nuclear piRNA fraction estimate by qPCR quantification by the normalized count of piRNA in high-throughput sequencing libraries. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).C Expression level of pre-NPF1 in the nucleus after a mixture of inhibitors (piRs-3-I2 and piRs-3-I3) was injected (n=4 biological replicates). D qPCR expression analyses of pre-NPF1. The precursor mRNA expression level was measured by qPCR after the dsPiwi1 treatments (n=6 biological replicates). E Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).F Analysis was performed to amplify pre-NPF1 and piRs-3-I3 in hnRNP F/H immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates; piRs-3-I3 n=4 biological replicates). G Analysis was performed to amplify pre-NPF1 in U2AF65 immunoprecipitates from extracts of brain tissue treated with piRs-3-I3 mimics compared to the negative controls (pre-NPF1 n=3 biological replicates). HJ Expression level of the mature NPF1 gene and pre-NPF1 in 293T cells transfected with pre-NPF1 and mutated pre-NPF1 (n=5 biological replicates). NC means negative control. The center line of the boxplots represents median value, the bounds of the box represent 75th and 25th percentile, and the whiskers represent maximum and minimum value. Data information: The qPCR data are shown as the mean ± SEM (Student's t-test, *P<0.05, **P<0.01, ***P<0.001).
Figure 1(A) Gating strategy used to identify ILCs and ILC subsets in humans (hILCs).(B) Representative histogram and summary of the percentage and of the gMFI of c-Maf expression in hILC subsets (ANOVA analysis: F(2,18)=305.4, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.(C Gating strategy for cKitlow and cKithigh ILC2s (C)D) expression level (percentage and gMFI) of c-Maf in cKitlow and cKithigh ILC2s (D) (paired T tests, ****p<0.0001). Data information: Each dot represents the expression level in one donor (n=7). Bars represent mean ± SEM.
Figure 3(G) Human ILC2 sorted from peripheral blood and in vitro expanded were transduced with Luc-sh or Maf-sh as in (C). hILC2s were then stimulated for 48h with IL-33 and the concentration of the indicated cytokines in the supernatants was measured (Multiple Mann-Whitney tests, *p<0.05, **p<0.01) Bars represent mean ± SEM. Data information: Each dot represents data from one individual mouse or from one donor.
Figure 5(E) ChIP-qPCR analysis of c-Maf occupancy on IL-4, IL-5 and IL-13 promoters in untreated mouse and human ILC2s (IL-2 stimulation) and in mouse and human ILC2s treated with IL-33 (n=3 biological replicates).
Figure 6(C) Representative histograms of c-Maf and GATA3 expression in ILC2s from adult PBMC or cord blood cells and percentages of c-Maf+ and GATA3+ ILC2s from adult PBMC or cord blood (n=5 different donors; T test, ****p<0.0001).(D) ILC2 expression of the indicated markers. Each dot represent sample from one donor. Bars represent mean ± SEM.(E, F) c-Maf quantification by qPCR (E) and cytokine concentration (F) in the supernatants of adult and cord blood ILC2s unstimulated, stimulated for 3h with PMA-Ionomycin (3h stim) and stimulated for an overnight with IL-33 and then 3h with PMA-Ionomycin (o.n. stim). ILC2s were sorted from 3 different donors. (E) Multiple T tests, unstimulated: p=0.000101; 3h stim: p=0.0036. (F) Multiple T tests, IL-13: p=0.022; IL-5: p=0.015 (*p<0.05). Bars represent mean ± SEM.
Figure 1C - Left: Percentage of total live cells expressing ChAT-eGFP derived from IWAT (including inguinal lymph node; n = 7), IWAT alone (n = 8) and lymph node alone (n = 8). Right: Proportion of ChAT-eGFP+ cells comprised by T cells, B cells and MΦ in each tissue type.F - Left: relative mRNA expression of Chat in ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 9). Chat expression was analyzed by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method. Right: quantification by LC/MS-MS of Ach concentration in SVF isolated from ChATfl/fl and ChATfl/fl;Vav-iCre IWAT (n = 6).
Figure 2Body weight (C) of ChATfl/fl;LysM-Cre (n = 7 for ChATfl/fl, n = 8 for Cre ChATfl/fl;Cd4-Cre (n = 6 for ChATfl/fl and n = 7 for Cre ChATfl/fl;Mb1-Cre (n = 12 in C, n = 9 for ChATfl/fl, n = 8 for Cre and littermate ChATfl/fl mice housed at RT.E-G - mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl;LysM-Cre (n = 24 for ChATfl/fl_RT, n = 25 for Cre_RT, n = 20 for ChATfl/fl_CE, n = 20 for Cre_CE) (E), ChATfl/fl;Cd4-Cre (n = 17 for ChATfl/fl_RT, n = 22-23 for Cre_RT, n = 19 for ChATfl/fl_CE, n = 22 for Cre_CE) (F), ChATfl/fl;Mb1-Cre (n = 10 for ChATfl/fl_RT, n = 11 for Cre_RT, n = 9-12 for ChATfl/fl_CE, n = 13 for Cre_CE) (G) and littermate ChATfl/fl mice housed at RT or 4oC (CE) for 6 h. An insert graph in (E) highlights mRNA expression of Chrna2 and thermogenic genes in IWAT of ChATfl/fl and ChATfl/fl;LysM-Cre mice after 6 h CE.K - Average whole-body oxygen consumption rate (OCR) of ChATfl/fl (n = 10) and ChATfl/fl;LysM-Cre (n = 14) mice housed in metabolic chambers at RT or CE for 6 h (from 9 a.m. to 3 p.m.).
Figure 3A - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). B - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice housed at RT (n = 7) or 10°C for 4 h (n = 3). C - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). D - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from β-less ChAT-eGFP mice housed at RT or 10°C for 4 h (n = 4). E - ChAT-eGFP+ cells as a percentage of total IWAT SVF cells from ChAT-eGFP mice treated with vehicle (veh) or 1 mg/kg NE for 2 h (n = 6). F - Percentages of total T cells, B cells and MΦ that are ChAT-eGFP+ in IWAT from ChAT-eGFP mice treated with veh or 1 mg/kg NE for 2 h (n = 6). J - Relative mRNA expression of Adrb1, Adrb2 and Adrb3 in IWAT MΦ sorted from WT mice (n = 4). Expression was measured by qPCR and normalized to levels of Tbp using the 2-ΔΔCt method.