EMBO/soda-nel huggingface dataset:
Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance (2020)
Figure 2

Figure 2. B-cell compartment is moderately affected in Bbs4-deficient mice

Cells isolated from the bone marrow (BM) (A-C) and spleens (SPL) (D-F) of 18-25 weeks old Bbs4+/+ (WT), Bbs4GT/GT, and Bbs4KO/KO mice were analyzed by flow cytometry. Number of analyzed mice (n) is indicated. Six (Bbs4GT/GT strain and Bbs4WT/WT controls) or eight (Bbs4KO/KO strain and Bbs4WT/WT controls) independent experiments were performed.

(A) Percentage of B220low positive cells in the bone marrow. Up: Bbs4+/+ n=9, Bbs4GT/GT n=10. Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.

(B) Percentage of B-cell precursors (IgM- IgD-) in the bone marrow. Gated on viable B220+ cells. Up: Bbs4+/+ (n=9), Bbs4GT/GT (n=10). Down: Bbs4+/+ (n =11), Bbs4KO/KO (n=10). Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.

(C) Percentage of pre-B cells (CD43- CD24high) in the bone marrow of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10). Gated on viable B220+ IgM- IgD- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.

(D) Percentage of late mature (IgM- IgD+) B cells in the spleen of Bbs4+/+ (n=11) and Bbs4KO/KO mice (n=10) Gated on viable CD19+ cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.

(E) An alternative analysis of the experiment shown in (D). Geometric mean fluorescence intensity (MFI) of BV421 (IgM) on IgD+ B cells was determined. Mean of MFI values for each genotype per experiment was quantified, and obtained values were normalized to Bbs4+/+ (=1) for each experiment. Two-tailed One Sample Wilcoxon Signed Rank Test was used for the statistical analysis.

(F) Percentage of splenic marginal zone (MZ) B cells (CD23- CD1d+) in Bbs4+/+ controls (n=10) and Bbs4KO/KO mice (n=9) was determined. Gated on viable CD19+, IgD- IgM+, CD138- cells. Statistical significance was calculated using two-tailed Mann-Whitney test. Medians are shown.
Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance (2020)
Figure 3

Figure 3. BBS4 is not required for T-cell and B-cell antigenic responses

(A,B) Splenocytes isolated from Bbs4+/+ (n=4 mice) and Bbs4GT/GT (n=3) B-cell transgenic B1-8 littermates were activated with 4-hydroxy-3-nitrophenylacetic acid succinimide ester-loaded T2-Kb cells in three independent experiments. Percentage of activated B cells (gated as CD69+ B220+ IgLλ+ viable cells) in the samples without T2-Kb (negative control), and in the samples with T2-Kb added in ratios 1:10 or 1:3 was determined by flow cytometry. (A) Representative mice are shown. (B) Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.

(C) B cells from 18-20 weeks old Bbs4KO/KO (red circle, n=3 mice), and control mice (Bbs4+/+, dark grey square, n=4, or Bbs4+/KO, light grey square, n=2) were activated with F(ab')2-goat anti-mouse IgM (Mu chain). Percentage of activated B cells (gated as CD69+ CD19+ viable cells) was determined by flow cytometry. Representative experiment out of three biological replicates is shown. Mean±SEM. Statistical significance was calculated using two-tailed Mann-Whitney test.

(D) CFSE-loaded T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were incubated with DDAO-labeled WT splenocytes loaded with OVA peptide or with the indicated altered peptide ligands for 20 min. Percentage of T cells conjugated with the APCs was determined by flow cytometry. Four biological replicates were performed. Mean+SEM. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all peptides.

(E,F) 500 or 1000 T cells isolated from lymph nodes of Bbs4FL/FL OT-I Rag2KO/KO (OT-I) and CD4-Cre Bbs4FL/FL OT-I Rag2KO/KO (Bbs4 cKO, OT-I) littermates were adoptively transferred into RIP.OVA mice followed by infection with Listeria monocytogenes expressing ovalbumin (LM-OVA) on the next day. (E) Glucose level in the urine of mice was monitored on a daily basis. The mouse was considered diabetic when it had urine glucose level ≥ 1000 mg/dL for two consecutive days. Statistical significance was calculated by Log-rank (Mantel-Cox) test. (F) Glucose concentration in blood on day 7 post-infection. 500 OT-I ctrl (n=13 mice), 500 OT-I Bbs4 cKO (n=13), 1000 OT-I ctrl (n=9), 1000 OT-I Bbs4 cKO (n=11), analyzed in four independent experiments, mean is shown. Statistical significance was calculated using two-tailed Mann-Whitney test.
Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance (2020)
Figure 4

Figure 4. The role of Bbs4 in B-cell development is not intrinsic

(A-D) Cells isolated from bone marrow (A, B), lymph nodes and spleen (C, D) of Bbs4FL/FL (control) and Vav-iCre Bbs4FL/FL (cKO) mice were analyzed by flow cytometry. (A) Percentage of B220low in bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). (B) Percentage of B-cell precursors (B220+, IgM- IgD-) in the bone marrow. Representative experiment out of four in total is shown. Bbs4FL/FL (n=6 mice), Vav-iCre Bbs4FL/FL (n=9). (C) Percentage of CD19+ B cells in spleens and lymph nodes of Vav-iCre Bbs4FL/FL (n=12 mice) and Bbs4FL/FL (n=9) mice, six independent experiments. (D) Percentage of late mature B cells (CD19+, IgM-, IgD+) in spleens of Vav-iCre Bbs4FL/FL (n=12 mice), and Bbs4FL/FL mice (n=9). Representative experiment out of six in total is shown. Statistical significance was calculated using two-tailed Mann-Whitney test, p >0.05 for all tests. Median is shown.

(E) Expression of Cxcl12 and Il-7 in the bone marrow from Bbs4+/+ Bbs18+/+ (WT) mice (black circles, n=7 mice), Bbs4+/KO (Het, green circle, n=3), Bbs18+/KO (Het, blue circle, n=3), Bbs4KO/KO (green square, n=5), and Bbs18KO/KO (blue square, n=2) was analyzed by RT-qPCR in five independent experiments. The expression was normalized to Gapdh. The statistical significance of the difference between the BBSome-deficient mice (Bbs4KO/KO and Bbs18KO/KO) and controls were calculated using two-tailed Mann-Whitney test. Median is shown.

(F) MEF cell lines were derived from a single Bbs4+/+ (WT) or a single Bbs4KO/KO embryos. The expression of Cxcl12 in these cell lines was analyzed by RT-qPCR. The expression was normalized to Gapdh and to Bbs4+/+ MEFs (=1) for each experiment. Three biological replicates. One sample t test was used for the statistical analysis.

(G) Expression of Cxcl12 and canonical-WNT responsive gene Alpl in untreated (NT) ST2 cells, or ST2 cells treated with WNT3A or WNT5A was analyzed by RT-qPCR. The expression was normalized to Gapdh and untreated ST2 sample (=1) for each experiment. Four biological replicates were performed. One sample t test was used for the statistical analysis.
Bardet-Biedl Syndrome ciliopathy is linked to altered hematopoiesis and dysregulated self-tolerance (2020)
Figure 5

Figure 5. Blood homeostasis is altered in BBS patients and Bbs4-deficient mice

(A-G) (A, C, D, F) Results of the blood tests of BBS patients from the Guy's Hospital and Great Ormond Street Hospital were extracted from the medical records and compared to two sets of healthy controls obtained from the UK Biobank. The BMI-random controls were age- and gender-matched to the set of BBS patients, with random BMIs. The BMI-matched controls were matched for age, gender, and BMI. BBS patients n=43 for parameters White blood cell count, Mean cell volume, Hemoglobin and Platelets; n=41 for parameters Neutrophils, Lymphocytes, Monocytes, Eosinophils; n=39 for parameters Red Blood cells, Mean cell Hemoglobin and red cell distribution width (RDW). Both data sets of healthy controls were selected as 10-fold larger than the set of BBS patients. Median is shown. Kruskal-Wallis test was used for the statistical analysis in (A, D and F). In (C), the percentage of patients or controls having CRP≥5 were compared using Fisher's exact test with post-hoc Sidak correction for multiple comparisons, BBS patients n=42. (B, E, G) Indicated parameters were measured using the blood from 20-21 weeks old Bbs4+/+ (WT) (n=15), Bbs4KO/KO (n=7), and Bbs4GT/GT (n=12) mice. Kruskal-Wallis test with Dunn's Multiple Comparison Post-tests was used for the statistical analysis. Median is shown.

Expanded View Figure Legends
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 1

Figure 1. Partial epigenetic and transcriptional activation of the Prdm1 locus in B cells.

(A) Mapping of open chromatin as well as active and repressive histone modifications at upstream regulatory regions of the Prdm1 locus in pro-B cells and plasmablasts. Open chromatin was determined by ATAC-seq (Buenrostro et al., 2013), and active (H3K4me2, H3K4me3, H3K9ac) and repressive (H3K27me3) histone marks were mapped by ChIP-seq analysis in ex vivo sorted pro-B cells (ATAC-seq and H3K27me3, this study), short-term in vitro cultured pro-B cells (H3K4me2, H3K4me3, H3K9ac) (Revilla-i-Domingo et al., 2012) and in vitro LPS-induced plasmablasts (Minnich et al., 2016) (Table EV4). The indicated upstream regions were previously shown by 3C-analysis to interact with the Prdm1 promoter (Wöhner et al., 2016). The mm9 genomic coordinates of mouse chromosome 10 and the respective positions of two human SNPs (rs658431 and rs548234) are indicated. RPM, reads per million mapped sequence reads. (B) Mapping of nascent transcripts or mature mRNA at the Prdm1 and Atg5 loci by GRO-seq or RNA-seq analyses of ex vivo sorted pro-B and FO B cells, respectively (Table EV4). Strand-specific GRO-seq reads are indicated in blue and red, respectively. (C) Quantification of nascent transcript and mRNA levels. The nascent transcription or mRNA expression of the Atg5 and Prdm1 genes is shown as mean expression value (TPM) with SEM based on two different GRO-seq or RNA-seq experiments for each B cell type, respectively. TPM, transcripts per million (Wagner et al., 2012).
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 2

Figure 2. Premature expression of the Blimp1 protein in Prdm1ihCd2/+ lymphocytes.

(A) Schematic diagram of the 3' end of the Prdm1ihCd2 allele (Minnich et al., 2016). The frt-flanked IRES-hCd2 (ihCD2) reporter gene, which was linked to a 258-bp long terminal repeat (LTR) sequence of the Moloney murine leukemia virus (MoMLV, green), was inserted between the stop codon and 3' UTR of the Prdm1 gene. C-terminal sequences of exon 8 contained in-frame tag sequences encoding the Flag and V5 epitopes, two TEV protease cleavage sites and a biotin acceptor sequence. The inserted MoMLV enhancer sequence is shown below together with its regulatory elements (Speck et al., 1990). GRE, glucocorticoid response element; LV, leukemia virus factor-binding site; NF1, nuclear factor 1; polyadenylation site, pA. (B) Expression of Prdm1 and Atg5 mRNA in ex vivo sorted pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ or wild-type (WT) mice. mRNA expression is shown as mean expression value (TPM) with SEM based on two independent RNA-seq experiments for each cell type and genotype. (C) RT-qPCR analysis of nascent Prdm1 and Atg5 transcripts in ex vivo sorted pre-B cells and in in vitro differentiated plasmablasts (PB) of the indicated genotypes. Plasmablasts were generated by stimulation of splenic FO B cells for 4 days with LPS. The data were normalized to those of the ubiquitously expressed control gene Tbp and are presented relative to those of the wild-type pre-B cells (set as 1). Nascent transcripts were PCR-amplified with primers located in the indicated introns (Table EV3). Statistical data are shown as mean value with SEM and were analyzed by the Student's t-test; *P < 0.05, **P < 0.01. Each dot corresponds to one mouse. (D,E) Flow cytometric analysis of hCD2 cell surface expression and intracellular Blimp1 expression of the indicated cell types. Bone marrow of Prdm1ihCd2/+ (red) or wild-type (WT, gray) mice was used to analyze ALPs, BLPs, pro-B, pre-B, immature B cells, NK cells and granulocytes, whereas the spleen was used for flow cytometric analysis of FO B, MZ B and plasma cells as well as naïve CD4 T and naïve CD8 T cells of both genotypes. The histograms show hCD2 (top row) and Blimp1 (bottom row) expression for the different cell types, which were defined as described in the Appendix Supplementary Methods. Wild-type FO B cells (dashed line) were used as negative control for the Blimp1 staining in plasma cells. The difference in mean fluorescence intensity (ΔMFI) between the two genotypes is shown for each cell type. (F) Immunoblot analysis of Blimp1 and the TATA-binding protein (TBP) in nuclear extracts prepared from short-term cultured wild-type or Prdm1ihCd2/+ pro-B cells. Marker proteins of the indicated size (in kilodaltons) are shown to the right.
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 3

Figure 3. Impaired B cell development in Prdm1ihCd2/+ mice.

(A,B) Flow cytometric analysis of total B cells (A) as well as pro-B, pre-B, immature (imm) B and recirculating (recirc) B cells (B) from the bone marrow of wild-type (WT, gray), Prdm1ihCd2/+ (red) and Prdm1ihCd2/ihCd2 (white) mice at the age of 6-8 weeks. (C) Flow cytometric analysis of total B, FO B and MZ B cells from the spleens of wild-type, Prdm1ihCd2/+ and Prdm1ihCd2/ihCd2 mice. Bar graphs show absolute cell numbers for each cell type and indicated genotype. The different cell types were defined as described in detail in the Appendix Supplementary Methods. The total numbers of bone marrow cells and splenocytes are shown in Fig S5A. (D) Determination of cell death of ex vivo pro-B and pre-B cells from the bone marrow of Prdm1ihCd2/+ (red) and wild-type (gray) mice by flow cytometric analysis of the loss of plasma membrane asymmetry (F2N12S ratiometric dye staining) and the loss of cell membrane integrity (SYTOX staining). Representative flow cytometry plots (left) and the quantification of dead and apoptotic (apop) cells (right) are shown. (E,F) BrdU labeling of immature (imm) and FO B cells in the spleen of 3-month-old Prdm1ihCd2/+ mice (red dots) and control wild-type littermates (gray dots). The percentage of BrdU+ B cells was determined for each B cell type by flow cytometric analysis after 10 days of continuous BrdU labeling (day 10, white bar) or after a subsequent 15-day chase period (day 25; hatched bar) without BrdU in the drinking water (E). A diagram (below) indicates the design of the BrdU labeling and chase experiments. Flow cytometric data obtained with FO B cells from one mouse of each genotype are shown in (F). The percentage of BrdU+ FO B cells is shown above the indicated gates (F). Statistical data (A-E) are shown as mean value with SEM and were analyzed by the Student's t-test; *P < 0.05, ***P < 0.001, ****P < 0.0001. Each dot corresponds to one mouse.
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 4

Figure 4. Blimp1-dependent deregulation of the B cell gene expression program.

(A,B) Scatter plot of gene expression differences in ex vivo sorted Prdm1ihCd2/+ and wild-type (WT) pro-B (A) and pre-B (B) cells, based on two RNA-seq experiments for each cell type and genotype. The expression data of individual genes (indicated by dots) were plotted as normalized (norm) rlog values. Genes with an expression difference of > 3-fold, an adjusted P value of < 0.05 and a TPM value of > 5 (in one of the two pro-B or pre-B cell types, respectively) are colored in blue or red, corresponding to activation or repression by Blimp1. (C) Expression of commonly activated (blue) and commonly repressed (red) genes in pro-B and pre-B cells. The log2-fold expression change observed between Prdm1ihCd2/+ and wild-type pro-B cells (horizontal axis) as well as between Prdm1ihCd2/+ and wild-type pre-B cells (vertical axis) is plotted for each gene. Lines indicated 2-fold (2x) and 3-fold (3x) expression differences. (D) Identification of 762 Blimp1 peaks in short-term cultured Prdm1ihCd2/+ pro-B cells, as detected by ChIP-seq with an anti-V5 antibody and MACS peak calling with a P value of < 10-10. The 9320 Blimp1 peaks identified in plasmablasts (PB; Minnich et al., 2016) are shown for comparison. Common (black) and unique (white) peaks are shown for both cell types. (E) Densities of Blimp1 binding. Average read density profiles aligned at the center of the Blimp1 peak are shown for the common Blimp peaks of both cell types. (F) Blimp1 binding at activated and repressed genes in Prdm1ihCd2/+ pro-B and pre-B cells. Regulated genes, which are bound by Blimp1 in Prdm1ihCd2/+ pro-B cells, are shown in black. (G) Blimp1 binding and regulation of the commonly repressed target genes Sell (CD62L) and B3gnt5. The ChIP-seq data (left) were obtained with Prdm1ihCd2/+ pro-B cells (this study) and Prdm1Bio/Bio Rosa26BirA/BirA plasmablasts (PB; Minnich et al., 2016). The RNA-seq data (right) were determined in pro-B and pre-B cells of the Prdm1ihCd2/+ (red) and wild-type (gray) genotype and in Blimp1-deficient (Prdm1Gfp/∆, blue) and wild-type (grey) pre-plasmablasts (Pre-PB; Minnich et al., 2016). Cell types expressing Blimp1 (+) are indicated. (H, I) Expression of selected Blimp1-activated and Blimp1-repressed genes coding for transcriptional regulators (H) and intracellular signal transducers (I) in Prdm1ihCd2/+ (red) and wild-type (gray) pre-B cells. Blimp1-bound genes are underlined. The mRNA expression of the indicated genes is shown as mean expression value (TPM) with SEM, based on two different RNA-seq experiments for pre-B cells of each genotype.
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 6

Figure 6. Cell-autonomous B cell phenotype except for the T cell-dependent loss of GC

B cells in Prdm1ihCd2/+ mice.

(A) Schematic diagram describing the generation of mixed bone marrow chimeras. Lineage-depleted bone marrow of B cell-deficient JHT Prdm1+/+ mice was mixed at ratio of 1:15 with lineage-depleted bone marrow of T-cell deficient Eβ-/- Prdm1ihCd2/+ or control Eβ-/- Prdm1+/+ mice prior to injection into lethally irradiated Rag2-/- recipient mice and subsequent analysis 4 months after transplantation. (B-F) Flow cytometric analysis of total B cells and different B cell subsets in the bone marrow (B,D) and spleen (C-F) of non-immunized Eβ-/- Prdm1ihCd2/+ (red) and Eβ-/- Prdm1+/+ (gray) chimeric mice. Bar graphs indicate absolute numbers of the different B cell types in these chimeric mice. (F) Histograms display the expression of hCD2 (Blimp1) in splenic FO B, GC B and naïve CD4 T cells from the chimeric mice of the indicated genotypes. Statistical data (B-E) are shown as mean value with SEM and were analyzed by the Student's t-test; ****P < 0.0001. Each dot corresponds to one mouse.
Early expression of Blimp1 in B cells causes autoimmunity with increased self-reactive plasma cells (2018)
Figure 7

Figure 7. Precocious Blimp1 expression causes autoimmunity in Prdm1ihCd2/+ mice with

progressing age.

(A) Presence of IgG antibodies detecting dsDNA, cardiolipin, SSA (Ro-52) and SSB (La) in the serum of Prdm1ihCd2/+ (red dots) and wild-type (gray dots) mice at the age of 2, 4 and 12 months. The titers of the different IgG antibodies were determined in the serum of male and female mice by ELISA and are displayed as arbitrary units (AU). The serum of 6-month-old Faslgld/gld mice was used as positive control. (B) Representative images of anti-nuclear antibody (ANA) staining obtained with serum from 12-month-old Prdm1ihCd2/+ or wild-type mice (right), as detected by indirect immunofluorescence assay using HEp-2 cells and an Alexa488-conjugated anti-mouse IgG detection antibody. The serum of Faslgld/gld mice was used as positive control. The presence of ANA-IgG antibodies was analyzed for 22 Prdm1ihCd2/+ and 12 wild-type mice, as summarized in the pie-chart (right). (C) Periodic acid-Schiff (PAS) staining of paraffin-embedded kidney sections of Prdm1ihCd2/+ and wild-type mice at 12 months of age. The mean score of kidney pathology was determined for each mouse by evaluating 40 individual glomeruli to assess the severity of glomerulonephritis as shown in Table EV2 and described in the Appendix Supplementary Methods. Representative pictures of glomeruli of Prdm1ihCd2/+ kidneys with different pathology scores are shown to the left, and dot plots with average scores for male and female mice (Table EV2) are shown to the right. Arrows denote intracapillary hyaline 'thrombi', asterisks indicate mesangial sclerosis with obscured capillary loops, and the arrowhead points to an obsolescent glomerulus with collapse of the glomerular tuft architecture. Statistical data (A,C) are shown as mean value with SEM and were analyzed by the Mann-Whitney test (A) or the Student's t-test (C); *P < 0.05, **P < 0.01, ***P < 0.001. Each dot corresponds to one mouse. (D) Proposed model explaining the autoimmune phenotype of Prdm1ihCd2/+ mice. Precocious Blimp1 expression in Prdm1ihCd2/+ mice results in decreased numbers of developing B cells (red arrows) and increased differentiation of immature and mature B cells to plasmablasts (blue arrows) and plasma cells (green arrow). As a consequence, autoreactive immature B cells may differentiate to plasmablasts prior to their elimination by central tolerance mechanisms (receptor editing or clonal deletion; Nemazee, 2017), and autoreactive mature B cells may escape their anergic state, imposed by anergy-induced BCR desensitization (peripheral tolerance; Theofilopoulos et al., 2017), by premature differentiation to plasmablasts, which leads to an increase of autoantibody-secreting plasma cells in Prdm1ihCd2/+ mice.
ABCA12 regulates insulin secretion from beta-cells (2020)
Figure 1

Figure 1. Expression profiling of ABCA12 and generation of mice lacking ABCA12 in β-cells.

(A) Immunofluorescent staining of ABCA12 (green) and Insulin (red) in mouse islets showing extensive co-localisation in β-cells as well as expression in other islet populations (arrowheads, scale bar=25μm).

(B) Co-expression of Abca12 (green) and Insulin (red) in MIN6 cells (scale bar = 10μm).

(C) LacZ staining (blue) to detect gene expression of the knocked-in LacZ cassette from the Abca12 locus in islets (islet outlined, scale bar = 25μm).

(D,E) ABCA12 immunostaining and the development of hyperkeratotic skin disease in E18.5 Abca12tm1a/tm1a mouse embryos (brackets indicate thickened epidermis, dashed line indicates epidermal basement membrane, scale bar = 50μm).

(F, G) Immunohistochemical detection of cre expression (green) in pancreatic islets from conditional mouse lines showing co-expression with Insulin (red)(scale bar = 50μm).

(H) Detection of recombination of the Abca12 locus by PCR, indicating different alleles (tm1c, wild type and tm1d).

(I-K) Detection of ABCA12 expression (green) and deletion in pancreas sections from mice of indicated genotypes (islets outlined, scale bar = 25μm).

(L) Quantitation of ABCA12 expression by densitometry of Western blots from purified pancreatic islets (values relative to wild type, n=3-8 animals per genotype, mean±SEM, *p=<0.05, Students t-test), showing representative blot with Actin loading control (right).

(M,N) (M) Average daily food intake of conditionally modified mice and (N) animal body weight (n=4-10 animals/genotype, mean±SEM).