GIPR-Ab/GLP-1 peptide-antibody conjugate requires brain GIPR and GLP-1R for additive weight loss in obese mice

Abstract

Glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide 1 receptor (GLP-1R) are expressed in the central nervous system (CNS) and regulate food intake. Here, we demonstrate that a peptide-antibody conjugate that blocks GIPR while simultaneously activating GLP-1R (GIPR-Ab/GLP-1) requires both CNS GIPR and CNS GLP-1R for maximal weight loss in obese, primarily male, mice. Moreover, dulaglutide produces greater weight loss in CNS GIPR knockout (KO) mice, and the weight loss achieved with dulaglutide + GIPR-Ab is attenuated in CNS GIPR KO mice. Wild-type mice treated with GIPR-Ab/GLP-1 and CNS GIPR KO mice exhibit similar changes in gene expression related to tissue remodelling, lipid metabolism and inflammation in white adipose tissue and liver. Moreover, GIPR-Ab/GLP-1 is detected in circumventricular organs in the brain and activates c-FOS in downstream neural substrates involved in appetite regulation. Hence, both CNS GIPR and GLP-1R signalling are required for the full weight loss effect of a GIPR-Ab/GLP-1 peptide-antibody conjugate.

Fig. 1. Anti-GIPR antibody administration to the brain reduces body weight and food intake in obese mice.

a, Timeline of ICV and ICV versus IP dosing studies in obese mice (created with BioRender.com). To examine the dose response from central administration of mGIPR-Ab, male DIO mice received ICV cannula targeting the third ventricle in the brain. Mice were randomized into one of six groups: (1) vehicle/aCSF (1 µl per mouse, n = 11), (2) IgG1 (15 µg per µl per mouse, n = 11), (3) IgG1 (30 µg per µl per mouse, n = 11), (4) mGIPR-Ab (7.5 µg per µl per mouse, n = 12), (5) mGIPR-Ab (15 µg per µl per mouse, n = 11) or (6) mGIPR-Ab (30 µg per µl per mouse, n = 12). All mice were dosed every 2 days for ten treatments. b–d, Change in body weight (b), body weight (c) and cumulative food intake (d) were measured daily. To directly compare central versus systemic administration of mGIPR-Ab, all male DIO mice received ICV cannula and were randomized into one of four groups: (1) central IgG1 (15 µg per µl per mouse), (2) central mGIPR-Ab (15 µg per µl per mouse), (3) systemic IgG1 (5 mg kg⁻¹) or (4) systemic mGIPR-Ab (5 mg kg⁻¹). All mice were dosed every 2 days for seven treatments. e–g, Change in body weight (e), body weight (f) and food intake (g) were measured daily. h–m, Pharmacokinetic exposure to mGIPR-Ab treatment from central (ICV) and systemic (IP) dosing in forebrain (h), hindbrain (i), total brain (j), plasma (k), inguinal WAT (l) and epididymal WAT (m) was measured 48 h after the last dose. The forebrain and hindbrain were separated at around −3.87 mm from bregma. For b,c, aCSF (1 µl per mouse, n = 11), IgG1 (15 µg per µl per mouse, n = 11), IgG1 (30 µg per µl per mouse, n = 11), mGIPR-Ab (7.5 µg per µl per mouse, n = 12), mGIPR-Ab (15 µg per µl per mouse, n = 11) and mGIPR-Ab (30 µg per µl per mouse, n = 12); for d, aCSF (1 µl per mouse, n = 9), IgG1 (15 µg per µl per mouse, n = 10), 3) IgG1 (30 µg per µl per mouse, n = 11), mGIPR-Ab (7.5 µg per µl per mouse, n = 11), mGIPR-Ab (15 µg per µl per mouse, n = 8) and mGIPR-Ab (30 µg per µl per mouse, n = 12); for e–m, n = 15 per group. In b–g, one-way or two-way repeated measures ANOVA with Tukey’s test for multiple comparisons; in h–m, two-tailed unpaired t-test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus aCSF (b–d) or versus IgG1 from respective dosing route or as noted by brackets (e–g); +P < 0.05, central IgG1 versus systemic IgG1; #P < 0.05, central mGIPR-Ab versus systemic mGIPR-Ab. Data represent means; error bars, s.e.m. aCSF, artificial cerebrospinal fluid. Source data

Fig. 2. Mice with CNS KO of Gipr are resistant to diet-induced obesity.

a, Breeding schematic for generation of CNS Gipr KO mice (created with BioRender.com). Male Gipr^fl/fl and Gipr^Syn−/− littermates were fed HFD for 12 weeks. b,c, Gipr RNA expression from isolated pancreatic islets (b) and brainstem, hypothalamus and hippocampus (c) were assessed to confirm CNS Gipr KO. d, Body weight, e, food intake, f, fat mass and g, lean mass were measured throughout HFD feeding. For b, n = 6 mice per genotype; for c, Gipr^fl/fl (n = 5 per brain region), Gipr^Syn−/− (n = 6 brainstem, n = 7 hypothalamus and hippocampus); for d–g, Gipr^fl/fl (n = 11) and Gipr^Syn−/− (n = 10). In b, multiple unpaired t-tests with a 1% false discovery rate for multiple comparisons; in c–f, two-way ANOVA with Šidák’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Data represent mean; error bars, s.e.m. Source data

Fig. 3. Gipr KO in the CNS extends the body weight reduction and anorectic effect of GLP-1R agonism in DIO mice.

Male Gipr^fl/fl and Gipr^Syn−/− littermates were fed HFD for 14 weeks and then treated IP with vehicle (2 ml kg⁻¹ every 3 days) + vehicle (2 ml kg⁻¹ every 6 days), dulaglutide (0.3 mg kg⁻¹ every 3 days) + vehicle (2 ml kg⁻¹ every 6 days) or dulaglutide (0.3 mg kg⁻¹ every 3 days) + mGIPR-Ab (25 mg kg⁻¹ every 6 days) for 27 days (all mice received the same number of injections each dosing day). a, Day 27 body weight per cent change. b–e, Body weight over time in Gipr^fl/fl (b) and Gipr^Syn−/− (c) littermates and average daily food intake measured every 3 days in Gipr^fl/fl (d) and Gipr^Syn−/− (e) littermates. f, Fat mass measured by MRI on days −3 and 25. g–i, Day 27 necropsy tissue weights inguinal of WAT(g), epididymal WAT (h) and liver (i). j,k, Day 27 4 h fasted blood glucose (j) and plasma insulin (k). For a–k, Gipr^fl/fl / vehicle + vehicle (n = 8) Gipr^fl/fl / dulaglutide + vehicle (n = 8), Gipr^fl/fl / dulaglutide + mGIPR-Ab (n = 7), Gipr^Syn−/− / vehicle + vehicle (n = 9), Gipr^Syn−/− / dulaglutide + vehicle (n = 9), Gipr^Syn−/− / dulaglutide + mGIPR-Ab (n = 9). In a–c and f–k, one-way or two-way repeated measures ANOVA with Tukey’s test for multiple comparisons; in d and e, mixed-effects analysis with genotype and treatment as main factors followed by Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 versus vehicle + vehicle within genotype or comparison indicated by brackets; #P < 0.05, ##P < 0.01 versus vehicle + vehicle within genotype (Gipr^fl/fl); ^P < 0.05, ^^P < 0.01 versus vehicle + vehicle within genotype (Gipr^Syn−/−); +P < 0.05, Gipr^fl/fl / dulaglutide + vehicle versus Gipr^fl/fl / dulaglutide + mGIPR-Ab. Data represent means; error bars, s.e.m. MRI, magnetic resonance imaging. Source data

Fig. 4. Weight loss pursuant to treatment with the mGIPR-Ab/GLP-1 peptide–antibody conjugate requires GIPR activity in the CNS.

Male Gipr^fl/fl and Gipr^Syn−/− littermates were fed HFD for 13 weeks and then treated with vehicle (2 ml kg⁻¹ every 6 days) or mGIPR-Ab/P1 (1.5 mg kg⁻¹ every 6 days) for 18 days. a, Day 18 body weight per cent change, b, per cent body weight change from day 0 and c, change in body weight over time in Gipr^fl/fl and Gipr^Syn−/− littermates. d, Average daily food intake in Gipr^fl/fl and Gipr^Syn−/− littermates. e, Fat mass measured by MRI on day 17. f, Day 18 necropsy tissue weights for inguinal WAT, g, epididymal WAT and h, liver. For a–h, Gipr^fl/fl / vehicle (n = 8), Gipr^fl/fl / mGIPR-Ab/P1 (n = 8), Gipr^Syn−/− / vehicle (n = 8), Gipr^Syn−/− / mGIPR-Ab/P1 (n = 7). In a and e–h, two-way ANOVA with Tukey’s multiple comparisons test; b–d, two-way repeated measures ANOVA or mixed-effects analysis with genotype and treatment as main factors followed by a Tukey’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 vs vehicle within genotype or comparison indicated by bracket; # indicates P < 0.05 versus vehicle within genotype (Gipr^fl/fl), ^ indicates P < 0.05 versus vehicle within genotype (Gipr^Syn−/−), + (purple) indicates P < 0.05 versus Gipr^fl/fl within mGIPR-Ab/P1 treatment, + (black) indicates P < 0.05 versus Gipr^fl/fl within vehicle treatment. Data represent means; error bars, s.e.m. Source data

Fig. 5. Weight loss generated by the mGIPR-Ab/GLP-1 peptide–antibody conjugate is dependent on GLP-1R activity in the CNS.

a, Breeding schematic for generation of CNS Glp1r KO mice (created with BioRender.com). Male (blue) and female (red) Glp1r^fl/fl and Glp1r^Wnt1−/− littermates were fed HFD for 13 weeks and then treated with vehicle (2 ml kg⁻¹ every 6 days) or mGIPR-Ab/P1 (1.5 mg kg⁻¹ every 6 days) for 18 days. b,j, Day 18 body weight per cent change, c,k, per cent body weight change from day 0 and d, change in body weight over time in male and female Glp1r^fl/fl and Glp1r^Wnt1−/− littermates. e, Average daily food intake in male Glp1r^fl/fl and Glp1r^Wnt1−/− littermates. l, Day 18 body weight change in grams in female Glp1r^fl/fl and Glp1r^Wnt1−/− littermates. Average body weight of female mice at study start (day 0) for Glp1r^fl/fl vehicle-treated mice was 29.07 g, Glp1r^Wnt1−/− vehicle-treated mice was 32.44 g, Glp1r^fl/fl mGIPR-Ab/P1-treated mice was 34.07 g and Glp1r^Wnt1−/− mGIPR-Ab/P1-treated mice was 33.57 g. f,m, Fat mass measured by MRI on day 17. Day 18 necropsy tissue weights for g,n inguinal WAT, h,o, gonadal WAT and i,p, liver. For b–i (males), Glp1r^fl/fl / vehicle (n = 10), Glp1r^fl/fl / mGIPR-Ab/P1 (n = 11), Glp1r^Wnt1−/− / vehicle (n = 10) and Glp1r^Wnt1−/− / mGIPR-Ab/P1 (n = 10). For j–p (females), Glp1r^fl/fl / vehicle (n = 7), Glp1r^fl/fl / mGIPR-Ab/P1 (n = 4), Glp1r^Wnt1−/− / vehicle (n = 6) and Glp1r^Wnt1−/− / mGIPR-Ab/P1 (n = 7). In b, f–i, j and l–o, two-way ANOVA with Tukey’s multiple comparisons test; in c–e and k, two-way repeated measures ANOVA with genotype and treatment as main factors or mixed-effects analysis followed by a Tukey’s multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 where comparison indicated by bracket; # indicates P < 0.05 versus vehicle within genotype (Glp1r^fl/fl), ^ indicates P < 0.05 versus vehicle within genotype (Glp1r^Wnt1−/−), + (blue or red) indicates P < 0.05 versus Glp1r^fl/fl within mGIPR-Ab/P1 treatment, + (black) indicates P < 0.05 versus Glp1r^fl/fl within vehicle treatment. Data represent means; error bars, s.e.m. ns, not significant. Source data

Fig. 6. mGIPR-Ab/P1 peptide–antibody conjugate treatment and CNS GIPR KO result in gene expression profile changes in WAT.

Transcriptomic analysis was conducted on WAT from male DIO Gipr^fl/fl, Gipr^Syn−/−, Glp1r^fl/fl and Glp1r^Wnt1−/− mice treated with either vehicle or mGIPR-Ab/P1. Samples were analysed for differential gene expression using DESeq2, and Ingenuity Pathway Analysis was used to identify the most significantly affected pathways. a,c, Bubble plot representing pathways enriched for DEGs in Gipr^Syn−/− vehicle-treated and Gipr^fl/fl mGIPR-Ab/P1-treated mice (a), and Glp1r^Wnt1−/− vehicle-treated and Glp1r^fl/fl mGIPR-Ab/P1-treated mice (c). Bubble size represents P value and colour shade represents z-score/enrichment score for each pathway. b,d, Heatmap for top five genes per enriched pathway in Gipr^Syn−/− vehicle-treated and Gipr^fl/fl mGIPR-Ab/P1-treated mice (b) and Glp1r^Wnt1−/− vehicle-treated and Glp1r^fl/fl mGIPR-Ab/P1-treated mice (d). Top five genes were selected based on biological relevance and statistical significance. In a and c, right-tailed Fisher’s exact test; n = 5 per group. Activ. of gene express., activation of gene expression; ECM, extracellular matrix; superpath., superpathway; veh, vehicle. Source data

Fig. 7. mGIPR-Ab/P3 peptide–antibody conjugate is measurable in CVOs in the brain and induces c-Fos in the central nucleus of the amygdala, parabrachial nucleus and nucleus of the solitary tract.

DIO male mice were treated with vehicle or mGIPR-Ab/P3 4 h before IgG1 measurement and c-FOS analysis. a, Horizontal cross-section of brain showing IgG1, a marker for mGIPR-Ab/GLP-1 peptide–antibody conjugate, biodistribution in vascular organ of the lamina terminalis, subfornical organ, median eminence and AP. b, Heatmap showing log₂(fold change) in IgG1 across five mice. c, Bar chart demonstrating brain regions significantly greater log₂(fold change) in average IgG1 staining in mGIPR-Ab/P3 group than in average vehicle, ranked by P value. d, Horizontal cross-section of brain showing group average c-Fos activity signature in response to mGIPR-Ab/P3 compared with vehicle. Scale bar, 500 µm. e, Heatmap showing log₂(fold change) in c-Fos following mGIPR-Ab/P3 treatment across five mice. f, Bar chart demonstrating brain regions significantly greater fold change in average mGIPR-Ab/P3 group in comparison to average vehicle, ranked by P value. g–i, Representative c-Fos activity in the central amygdalar nucleus (g), parabrachial nucleus (h) and nucleus of the solitary tract (i) in vehicle or mGIPR-Ab/P3 treated group. For a–i, vehicle (n = 8) and mGIPR-Ab/P3 (n = 5); in c and f–i, negative binomial generalized linear model, followed by Dunnett’s test for multiple comparisons. *P < 0.05, **P < 0.01, ***P < 0.001 compared to vehicle. Data represent means; error bars, s.e.m. AP, area postrema; BST, bed nuclei of the stria terminalis; CEA, central amygdalar nucleus; CVO, circumventricular organs; DIO, diet-induced obese; ME, median eminence; NTS, nucleus of the solitary tract; OV, vascular organ of the lamina terminalis;PB, parabrachial nucleus; PSTN, parasubthalamic nucleus; PVT, paraventricular nucleus of the thalamus; SFO, subfornical organ. Source data

Extended Data Fig. 1. In vivo pharmacokinetic profile and in vitro activity of mGIPR-Ab.

To determine dose level and dose frequency of central administration of mGIPR-Ab, pharmacokinetic profile of (a) brain and (b) plasma exposure was assessed at 4, 24, 72, 120, and 168 h following a single ICV injection of 15 µg/µL/mouse dose of mGIPR-Ab. To evaluate and compare weight loss from ICV vs. IP mGIPR-Ab treatment, (c) change in body weight was normalized to change in body weight in IgG1 control antibody treatment group with corresponding route of administration. To define meaningful levels of exposure in vivo, (d) mGIPR-Ab antagonist inhibition of GIP-induced cAMP production was measured in neuro2A cells (mGIPR-Ab run in technical quadruplicates and GIP run in technical duplicates). In vitro IC₅₀ value of mGIPR-Ab could then be used to compare with exposure levels in brain, plasma, and peripheral tissues following central or systemic administration of mGIPR-Ab in diet-induced obese male mice. (A-B) 4 h (n=3), 24 h (n=3), 72 h (n=3), 120 h (n=3), 168 h (n=2). (A-B) Data represent mean ± SEM. (c) Data represent mean normalized to Control-Ab for the corresponding route of administration. (d) Assay was run in technical quadruplicate (mGIPR-Ab) and triplicate (GIP). cAMP = cyclic AMP; EC₅₀ = half-maximal effective concentration; IC₅₀ = half-maximal inhibitory concentration; ICV = intracerebroventricular. Source data

Extended Data Fig. 2. mGIPR-Ab/P1 and mGIPR-Ab/P3 have similar in vitro potency and comparable effects on cAMP activity.

Potency and relative activity of mGIPR-Ab/P1 and mGIPR-Ab/P3 were compared in vitro. (a) GIPR IC₅₀ and GLP-1R EC₅₀ are comparable with both molecules, despite different GLP-1 analogs used. (b) mGIPR-Ab/P1 and mGIPR-Ab/P3 have identical curves in a GIPR cAMP antagonist assay in 293 T hGIPR-expressing cells, as well as in (c) GLP-1R cAMP agonist assay in CHOK1 hGLP-1R-expressing cells. Assay was run in technical duplicates. cAMP = cyclic AMP; CHO = Chinese hamster ovary. Source data

Extended Data Fig. 3. Central administration of mGIPR-Ab/P3 peptide-antibody conjugate induces sustained weight loss in obese mice.

ICV-cannulated DIO mice were randomized into groups to receive 25 µg/µL/mouse IgG1 or 1, 5, 10, or 25 µg/µL/mouse mGIPR-Ab/P3. Mice were dosed ICV every 4 days for 5 treatments. (a) Percent change in body weight from day 0, (b) body weight, and (c) food intake was measured up to 44 days. (A-C) 25 µg/µL/mouse IgG1 (n=5), 1 µg/µL/mouse mGIPR-Ab/P3 (n=6), 5 µg/µL/mouse mGIPR-Ab/P3 (n=6), 10 µg/µL/mouse mGIPR-Ab/P3 (n=6), or 25 µg/µL/mouse mGIPR-Ab/P3 (n=6). (A-B) Two-way ANOVA with Tukey’s test for multiple comparisons, (C) mixed-effects analysis with Tukey’s test for multiple comparisons, *p < 0.05 vs. IgG1 control. Data represent mean ± SEM. ANOVA = Analysis of Variance; DIO = diet-induced obese; ICV = intracerebroventricular. Source data

Extended Data Fig. 4. Characterization of CNS GIPR Knockout mice.

Plasma biomarker levels were assessed in two separate cohorts of mice. Plasma (a) total cholesterol, (b) triglycerides, (c) high density lipoprotein (HDL) cholesterol, and (d) low density lipoprotein (LDL) cholesterol were measured in Gipr^fl/fl (n=8) and Gipr^Syn−/− mice (n=8). In a separate cohort (given sample constraints), plasma levels of (e) leptin, (f) adiponectin, (g) resistin, (h) Interleukin-6 (IL-6), (i) tumor necrosis factor alpha (TNF), and (j) monocyte chemoattractant protein-1 (MCP-1) were measured in Gipr^fl/fl (n=8) and Gipr^Syn−/− (n=14) mice. (A-J) Two-tailed unpaired t test. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. Gipr^fl/fl control. Source data

Extended Data Fig. 5. Gipr CNS KO mice are resistant to mGIPR antibody treatment.

Male Gipr^fl/fl and Gipr^Syn−/− littermates were fed HFD for 13 weeks and then treated with vehicle (2 mL/kg) or mGIPR-Ab (25 mg/kg) every 6 days for 48 days. (a) Day 48 % change in body weight, (b) body weight over time, and (c) average daily food intake measured every 3 days. Body composition measured by MRI (d) fat mass and (e) lean mass on days -3 and 46. Day 48 necropsy tissue weights (f) epididymal WAT, (g) inguinal WAT, and (h) liver. Day 48 4-hour fasted (i) blood glucose, (j) plasma insulin, and (k) plasma total cholesterol. (A-K) Gipr^fl/fl / Vehicle (n=8), Gipr^fl/fl / mGIPR-Ab (n=7), Gipr^Syn−/− / Vehicle (n=14), Gipr^Syn−/− / mGIPR-Ab (n=14). (A, D-K) One-way ANOVA with Tukey’s test for multiple comparisons, (B-C) Two-way repeated measures ANOVA or mixed-effects analysis with Tukey’s test for multiple comparisons. *p <0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 vs. vehicle within genotype or comparison indicated by bracket; + (black) vs. Gipr^fl/fl within vehicle; + (red) vs. Gipr^fl/fl within mGIPR-Ab treatment. Data represent mean ± SEM. Source data

Extended Data Fig. 6. mGIPR-Ab/P1 treatment and CNS GIPR KO result in gene expression profile changes in liver.

Transcriptomic analysis was conducted on liver tissues from Gipr^fl/fl, Gipr^Syn−/−, Glp1r^fl/fl and Glp1r^Wnt1−/− mice treated with either vehicle or mGIPR-Ab/P1. Samples were analyzed for differential gene expression (DEG) using DESeq2 and Ingenuity Pathway Analysis was used to identify most significantly affected pathways. Bubble plot representing pathways enriched for DEGs in (a) Gipr^Syn−/− vehicle-treated and Gipr^fl/fl mGIPR-Ab/P1-treated mice, and (c) Glp1r^Wnt1−/− vehicle-treated and Glp1r^fl/fl mGIPR-Ab/P1-treated mice. Size of bubble represent p-value and color shade of bubble represent z-score/enrichment score for each pathway. Heat map for top 5 genes per enriched pathway in (b) Gipr^Syn−/− vehicle-treated and Gipr^fl/fl mGIPR-Ab/P1-treated mice, and (d) Glp1r^Wnt1−/− vehicle-treated and Glp1r^fl/fl mGIPR-Ab/P1-treated mice. Top 5 genes were selected based off biological relevance and statistical significance. (A,C) Right-tailed Fisher’s Exact Test. n=5/group. Chrom = chromosomal, activat. = activation, metab. = metabolism, superpath. = superpathway, biosyn. = biosynthesis. Source data

Extended Data Fig. 7. Pre-treatment with mGIPR-Ab and mGIPR-Ab/P3 blocks DA-GIP- induced c-FOS in the mouse brain.

GIPR and GLP-1R expressions were visualized in the area postrema by in situ hybridization. (a) mGIPR (pink) and mGLP1R (teal) were localized on different cells, but within proximity of each other, in the area postrema. Relative expression levels of mGIPR and mGLP1R were scored on a scale of 0-4 based on ACD scoring criteria, where level 2 indicates ‘4-9 dots/cell, no or very few dot clusters’. The effect of mGIPR-Ab and mGIPR-Ab/P3 on DA-GIP induced c-Fos in the area postrema was analyzed with fluorescent immunohistochemistry. (b, e) DA-GIP significantly increases c-Fos expression in the area postrema following IgG1 pre-treatment. (c, e) DA-GIP expression does not increase c-Fos expression in the area postrema with mGIPR-Ab pre-treatment. (d, e) Moreover, DA-GIP does not increase c-Fos expression in the area postrema following mGIPR-Ab/P3 pre-treatment, however, mGIPR-Ab/P3 alone induces high levels of c-Fos expression in the area postrema. (A) n = 2, (B-E) IgG1/A52Su (n=5), IgG1/DA-GIP (n=4), mGIPR-Ab/A52Su (n=6), mGIPR-Ab/DA-GIP (n=6), mGIPR-Ab/P3/A52Su (n=6), mGIPR-Ab/DA-GIP (n=6). One-way ANOVA with Sidak’s test for multiple comparisons; *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001 comparison indicated by bracket. Data represent mean ± SEM. Scale bar 100 µm. Source data