EMBO/soda-nel huggingface dataset:
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 3

Figure 3. JunD was involved in menin-mediated Pbk suppression

A, B. Menin knock down (KD) in INS-1 cells using a Men1-specific CRISPR sgRNA upregulated Pbk protein levels (A) and mRNA levels (B). qPCR data was from three independent experimets (n=3). **P = 0.0093, ***P = 0.0004 (two-tailed unpaired student's t-test).

C, D. Men1 knock out (KO) in PIME cells through Cre-mediated gene editing upregulated Pbk protein level (C) and mRNA level (D). qPCR data was from three independent experimets (n=3).**P = 0.0059 (two-tailed unpaired student's t-test).

E, F. JunD KD in INS-1 cells using shRNA upregulated Pbk protein levels (E) and mRNA levels (F). qPCR data was from three independent experimets (n=3).***P = 0.0001 (shRNA-#1), ***P = 0.0007 (shRNA-#2) (two-tailed unpaired student's t-test).

G, H. JunD KD in PIME cells using shRNA increased Pbk protein levels (G) and mRNA levels (H). qPCR data was from three independent experimets (n=3).*P = 0.0236 (shRNA-#1), *P = 0.0175 (shRNA-#2) (two-tailed unpaired student's t-test).

I, J. JunD KD in menin null PIME cells moderately suppressed Pbk protein levels (I) and mRNA levels (J). qPCR data was from three independent experimets (n=3).*P = 0.0030 (shRNA-#1), *P = 0.0065 (shRNA-#2) (two-tailed unpaired student's t-test).

K. The predicted JunD binding sites within Pbk promoters of mouse, human, and rat genomes via software PROMO3.0.

L. HEK293T cells were co-transfected with a JunD-expressing plasmid and luciferase reporter constructs driven by different wild-type mouse JunD binding sites (JBSs) for 36 h, followed by luciferase assay. Three independent experimets (n=3). **P = 0.0011 (site 2), **P = 0.0032 (site 3), ***P = 0.0001 (site 4) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

M. Luciferase reporters driven by WT or mutant JBS4 were transfected into HEK293T cells with a JunD expression plasmid or with or without menin expressing plasmid. Luciferase assays were performed 36h after transfection. Three independent experimets (n=3).

N. JunD overexpression in PIME cells suppressed Pbk protein levels (M, Lane 2 vs Lane 1), but did not affect Pbk expression in menin-null PIME cells (M, Lane 4 vs Lane 3).

O. JunD KD suppressed PIME cell growth, but not menin-null PIME cells. Two independent experimets (n=2). **P = 0.0025 (Two-way ANOVA). ns, not statistically significant difference.

Data information: Data are represented as mean ± SEM.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 4

Figure 4. JunD recruits menin-HDAC3 complex to repress Pbk expression

A. Co-IP experiments in HEK293T cells transfected with vector and flag-HDAC3 expression plasmids. Anti-flag antibody was used to put down HDAC3 protein. WB data showed the level of menin and HDAC3 of input samples and output samples.

B, C. Different concentrations of vorinostat were used to treat PIME cells (B) and menin null PIME cells (C) for 48 hrs. Pbk expression was examined using WB.

D. The control INS-1cells and JunD KD INS-1 cells were subjected to ChIP assay to detect for the binding of menin, HDAC3, JunD, and histone3 acetylation level at the Pbk locus. 1% input denotes the signal level normalized by the equivalent total input DNA (i.e. 1% of sonicated genome DNA) for ChIP assay. Three independent experimets (n=3). *P = 0.0430 (JunD), *P = 0.0480 (Menin), ***P = 0.0008 (HDAC3), *P = 0.0480 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

E, F. The control INS-1cells and menin KD INS-1 cells induced by CRISPR/Cas9 system were subjected to ChIP assay to detect for the binding of menin, HDAC3, JunD, and histone3 acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0196 (E, Menin), *P = 0.0164 (E, HDAC3), *P = 0.0323 (F, Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

G. Control and JunD KD PIME cells were subjected to ChIP assay to detect the binding of JunD, menin, HDAC3, and histone acetylation at the Pbk locus. Three independent experimets (n=3). ***P = 0.0007 (JunD), *P = 0.0486 (Menin), *P = 0.0117 (HDAC3), **P = 0.0012 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

H, I. PIME cells and menin-null PIME cells were subjected to ChIP assay to detect the binding of menin, HDAC3, JunD (H) as well as histone acetylation level (I) at the Pbk locus. Three independent experimets (n=3). *P = 0.0226 (H, Menin), **P = 0.0084 (H, HDAC3), **P = 0.0041 (I, Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

J, K. The effect of T5224 on the binding of JunD, menin, HDAC3, and acetylated histone3 at the Pbk locus in PIME cells (J) or INS-1 cells (K). Three independent experimets (n=3). *P = 0.0490 (J, JunD), *P = 0.0479 (J, Menin), *P = 0.0154 (J, HDAC3), **P = 0.0029 (J, Ac-H3), *P = 0.0104 (K, JunD), **P = 0.0024 (K, Menin), ***P = 0.0002 (K, HDAC3), ***P = 0.0001 (K, Ac-H3), (two-tailed unpaired student's t-test). ns, not statistically significant difference.

L. PIME cells were treated with various doses of T5224 for 48 hours, followed by detecting Pbk expression using WB.

Data information: Data are represented as mean ± SEM.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 5

Figure 5. Pharmacological inhibition of menin decreases the binding of menin at the Pbk locus

A. Crystal structure of the menin-JunDMBM complex (Adapted from (Huang et al., 2012)). MI potentially blocks the interaction of menin and JunD.

B. Co-IP in HEK293T cells transfected with vector or a flag-JunD expressing plasmid upon treatment with DMSO, MI-503 (30, 100, 300 nM from lane 3 to lane 5, respectively), or MI-463 (30, 100, 300 nM from lane 6 to lane 8, respectively).

C, D. ChIP assay to detect the binding of menin, HDAC3, JunD, and histone3 acetylation at the Pbk locus in vehicle or MI-503 treated PIME cells. Three independent experimets (n=3). *P = 0.0132 (Menin), *P = 0.0153 (HDAC3), **P = 0.0092 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

E. ChIP assay to detect the binding of menin, HDAC3, JunD, and histone3 acetylation at the Pbk locus in vehicle or MI-503 treated INS-1 cells. Three independent experimets (n=3). ***P = 0.0008 (Menin), *P = 0.0183 (HDAC3), *P = 0.0485 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

F. Mouse primary islets treated with MI-503 for 3 days were subjected ChIP assay to detect the binding of menin, HDAC3, JunD, as well as histone acetylation level at the Pbk locus. Three independent experimets (n=3). *P = 0.0218 (Menin), *P = 0.0145 (HDAC3), *P = 0.0184 (Ac-H3) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

Data information: Data are represented as mean ± SEM.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 6

Figure 6. Menin inhibitor (MI) increased Pbk expression and correlates with beta cell proliferation in vitro

A, B. PIME cells were treated with various doses of MI-463 for 48 hours, followed by detecting Pbk expression using WB (A) and qPCR (B). qPCR data was from three independent experimets (n=3). **P = 0.0022 (25 nM), *P = 0.0479 (50 nM), *P = 0.0233 (100 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

C, D. Menin-null PIME cells were treated with various concentrations of MI-463 for 48 hrs, followed by detecting Pbk expression with WB (C) and qPCR (D). qPCR data was from three independent experimets (n=3). ns, not statistically significant difference (two-tailed unpaired student's t-test).

E, F. MI-503 and MI-463 treatment promotes growth of PIME cells (E) and INS-1 cells (F). Three independent experimets (n=3). ns, not statistically significant difference (Two-way ANOVA).

G. MI-503 increases proliferation of menin-expressing PIME cells but not menin-null PIME cells. Three independent experimets (n=3). ***P < 0.0001 (Two-way ANOVA).

H-J. 5 days of MI-463 treatment increases the expression of Pbk at the protein level (H) and mRNA level (I), as well as the proliferation marker ki67 mRNA levels (J) in mouse primary islets. qPCR data was from three independent experimets (n=3). *P = 0.0105 (I, 50 nM), *P = 0.0287 (I, 100 nM), *P = 0.0150 (J, 25 nM), *P = 0.0273 (J, 50 nM), *P = 0.0314 (J, 100 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

K-M. 5 days of MI-503 treatment impacts the expression of Pbk at protein level (K) and mRNA level (L), as well as ki67 mRNA levels (M) in human primary islets (donor number is four).

qPCR data was from three independent experimets (n=3). **P = 0.0036 (L, 10 nM), **P = 0.0024 (L, 50 nM), **P = 0.0073 (L, 100 nM), *P = 0.0237 (M, 50 nM) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

Data information: Data are represented as mean ± SEM.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 7

Figure 7. Pbk is essential for MI-increased beta cell proliferation and improved GT in vivo

A. A diagram of experimental design. 8 to 12-week-old PbkKI/KI and PbkWT/WT mice were treated with MI-463 at 70mg/Kg (n=4) or vehicle (n = 4) by gavage daily.

B, C. GTT data from PbkKI/KI and PbkWT/WT mice (n=4 for each group) after 8 weeks of MI-463 or vehicle treatment (B) (***P < 0.0001 (Two-way ANOVA), ns, not statistically significant difference) and AUC of the GTT results (C) (***P = 0.0005 (two-tailed unpaired student's t-test). ns, not statistically significant difference). The mice were starved overnight prior to the test (16h). Dot line, fasting blood glucose number >150 was designated as hyperglycemia.

D. In vivo GSIS test on 11-week-old male PbkKI/KI and PbkWT/WT mice (n=4 for each group). The mice were starved overnight prior to the test (16h). Y-axis represents the ratio of insulin level at 15 min over insulin level at 0 min with glucose challenge. **P = 0.0042 (two-tailed unpaired student's t-test). ns, not statistically significant difference.

E-L. Representative images of insulin (green) and Ki67 (red) double staining on mouse islets with 8 wks MI treatment (E-H), and insulin (green) or Pbk (red) double staining on mouse islets (I-L). Nuclei were labeled by DAPI (blue). Islet area was circled by white dashed line. Ki67 and Pbk staining are denoted by white arrows. Scale bars: 50 μm.

M, N. Quantification of the percentages of Ki67 staining-positive beta cells (M) and Pbk staining-positive beta cells (N). Four mice for each group, and 5-10 islet images per mouse were analyzed. **P = 0.0026 (M, WT/WT-Mock : WT/WT-MI-463), **P = 0.0013 (M, KI/KI-Mock : KI/KI-MI-463), **P = 0.0011 (N, WT/WT-Mock : WT/WT-MI-463), ***P = 0.0009 (N, KI/KI-Mock : KI/KI-MI-463) (two-tailed unpaired student's t-test). ns, not statistically significant difference.

O. Beta cell mass comparison between MI-463- and vehicle-treated PbkKI/KI mice and PbkWT/WT mice (n=4 for each group). *P = 0.0174 (two-tailed unpaired student's t-test). ns, not statistically significant difference.

Data information: Data are represented as mean ± SEM.
Menin-regulated Pbk controls High fat diet-induced compensatory beta cell proliferation (2021)
Figure 8

Figure 8. The crucial role of Pbk in MI-mediated hyperglycemia reversion and the amelioration of IGT in HFD-induced diabetic mice

A. A diagram for treating mice with MI. HFD-fed mice (18 weeks old) were treated with MI-463 at 70mg/Kg (n=5) or vehicle (corn oil, n=5) by gavage daily.

B. Fasting blood glucose levels of MI-463 or vehicle-treated mice were measured at the indicated times during administration. n=5 for each group of mice. Dot line, fasting blood glucose number >150 was designated as hyperglycemia. **P = 0.0026 (Two-way ANOVA).

C. AUC comparison of IPGTT data from MI-463 or Vehicle treated mice during 12 weeks administration. n=5 for each group of mice. **P = 0.0024 (Two-way ANOVA).

D, E. Double staining for insulin (green) and BrdU (red) of mice islets after 12 weeks of vehicle (D) or MI (E) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). BrdU staining is indicated by arrows. Scale bar:50 μm.

F, G. Quantification of BrdU positive beta cells (F) and beta cell mass (G) of MI-463 treated HFD-fed mice or control mice. Five mice for each group, and at least 10 islet images per mouse were analyzed. **P = 0.0059, *P = 0.0103 (two-tailed unpaired student's t-test).

H, I. Double staining for insulin (green) and Pbk (red) of mice islets after 12 weeks of vehicle (H) or MI (I) administration. Islet area was circled by white dashed line. Nuclei were labeled by DAPI (blue). Pbk staining is indicated by arrows. Scale bar: 50 μm.

J. Quantification of Pbk staining-positive β-cells. Five mice for each group, and at least 10 islet images per mouse were analyzed. *P = 0.0120 (two-tailed unpaired student's t-test).

K. Quantification of the percentage with pbk and BrdU co-staining cells among total Pbk positive cells in HFD-fed mouse islets. The mean values of five mice from 5 sections for each mouse were presented.

L. MI-463 treatment schedule of PbkKI/KI and PbkWT/WT mice with HFD feeding. Seven-week-old male PbkKI/KI and PbkWT/WT mice were fed with HFD (n=6 for each group). Four weeks later, HFD fed PbkKI/KI and PbkWT/WT mice were started to treat with MI-463 or vehicle (i.e., n=3 for each group) for 8 weeks.

M, N. GTT data of PbkKI/KI and PbkWT/WT mice (n=6 for each group) with 3 weeks HFD feeding (M) ( ***P < 0.0001 (Two-way ANOVA) ) and AUC comparison of GTT results (N) (***P < 0.0001(WT/WT-Chow : WT/WT-HFD), ***P = 0.0007 (KI/KI-Chow : KI/KI-HFD) (two-tailed unpaired student's t-test)).

O, P. GTT data for MI-463 or vehicle treated PbkKI/KI or PbkWT/WT mice (n=3 for each group) on 8 weeks HFD (O) (*P = 0.0133 (Two way ANOVA), ns, not statistically significant difference.) and AUC of GTT data (P) (*P = 0.0442 (two-tailed unpaired student's t-test), ns, not statistically significant difference.).

Data information: Data are represented as mean ± SEM.

Expanded View Figure legends:
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 1

Figure 1. ASM deficiency alters microglia number and morphology.

(A) Immunofluorescence staining against Iba-1 in cortex (Cx), hippocampus (Hip) and cerebellum (Cb) of NPA affected and control 3 year-old children. Scale bars, 50 μm.

(B) Magnified image showing ramified (left) versus amoeboid (right) morphology in control and NPA microglia. Scale bars, 10 μm.

(C) Mean ± SEM area of Iba-1 positive cells in different brain regions from a NPA affected and a control child (n = 20 cells, Student´s t-test).

(D) Immunofluorescence staining against Iba-1 in Cx, Hip and Cb of 3 month-old ASMko and wt mice. DAPI staining shows cell nuclei. Scale bars, 50 μm.

(E) Mean ± SEM number of Iba-1 positive cells in different brain regions from ASMko and wt mice (n = 5 mice per group, Student´s t-test).

(F) 3D rendered images obtained with Imaris Surface software from high magnification images of immunofluorescence staining against Iba-1 showing representative microglial morphology in Cx, Hip and Cb of ASMko and wt mice. Scale bars, 10 μm.

(G) Mean ± SEM area of Iba-1 positive cells in the different brain regions from ASMko and wt mice (n = 5 mice per group, >10 cells per mouse, Student´s t-test). Scale bars, 15 μm.

(H) Example of projection detection by the Filament Imaris software.

(I) Mean ± SEM branch point number per process and process length in iba1+ cells obtained with the Imaris Filament software (Student´s t-test).

Data information: P**<0.005; P***<0.001.
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 2

Figure 2. Deleterious effects of microglia ablation in ASMko mice.

(A) Immunofluorescence staining against Iba-1 in Cb of ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 100 μm.

(B) Magnified images (from A) showing ramified (left) versus amoeboid (right) morphology in wt and ASMko microglia treated or not with PLX. Scale bars, 30 μm.

(C) Mean ± SEM number of Iba-1 positive cells in the Cb of the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).

(D, E) Performance in the Rotarod test of ASMko and wt mice treated or not with PLX for 2 months expressed as the mean ± SEM time spent in the rotating rod on each of four trials (D) or the mean ± SEM time of the four trials (E) (n = 7 mice per group, Two-way Anova, Games-Howell (D) and Bonferroni (E) post hoc).

(F) Mean ± SEM body weight of ASMko and wt mice treated or not with PLX at the indicated time during long-term treatment (n= 7 mice per group, Two-way Anova, Bonferroni post hoc).

(G) Survival curve of ASMko and wt mice with or without long-term PLX treatment (n = 7 mice per group).

(H) Immunofluorescence staining against the PC marker Calbindin in mid and posterior lobules of Cb in ASMko and wt mice treated or not with PLX for 2 months. DAPI staining shows cell nuclei. Scale bar, 500 μm.

(I) Mean ± SEM number of Calbindin positive cells in the anterior (lobules I-V), middle (lobules VI-VIII) and posterior zone (lobules IX-X) of the Cb from the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).

Data information: P*<0.05; P**<0.005; P***<0.001.
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 3

Figure 3. Coexistence of different microglia populations and myelin debris in ASMko mice.

(A) Immunofluorescence staining against CD16/CD32 or Arg-1 in Cb of ASMko mice (red). Microglia were identified by F4/80 or Iba-1 staining (green). DAPI staining shows cell nuclei. Scale bar, 50 μm.

(B) Mean ± SEM number of Arg-1 and CD16/CD32 positive microglia per area in Cb from ASMko and wt mice (n = 6 mice per group).

(C) Mean ± SEM levels of TNFa protein in the Cb from ASMko and wt mice measured by ELISA (n = 7 mice per group, Student´s t-test).

(D) Mean ± SEM levels of TNFa mRNA in Cb of ASMko and wt mice determined by qRT-PCR (n = 7 mice per group, Student´s t-test).

(E) Survival after LPS challenge in ASMko and wt mice treated or not with dexamethasone (n = 8 mice per group).

(F) Immunofluorescence staining against MBP in Cb of ASMko and wt mice treated or not with PLX for 2 months. Scale bar, 50 μm.

(G) Mean ± SEM MBP intensity in the different mouse groups (n = 7 mice per group, Two-way Anova, Bonferroni post hoc).

(H) High magnification of immunofluorescence staining against MBP in ASMko mice treated or not with PLX for 2 months. White arrows show MBP aggregates indicative of myelin debris. Scale bar, 50 μm.

(I) Mean ± SEM number of MBP aggregates per cell area in the different mouse groups (n = 7 mice per group, Student´s t-test).

(J) High magnification images and their 3D rendered replicates from cerebellar wt and ASMko microglia identified by Iba-1 and of the myelin marker MBP. Scale bars, 10 μm.

(K) Amplified image and its 3D rendered replicate of amoeboid microglia containing myelin debris (arrowheads) in the ASMko cerebellum. The orthogonal projection of this cell in the lower panels confirms the presence of the myelin debris inside the cell and underneath the plasma membrane (arrowhead). DAPI staining shows cell nuclei. Scale bars, 10 μm.

(L) Mean ± SEM percentage of microglia showing internalised myelin debris in wt and ASMko mice (n = 7 mice per group).

Data information: P*<0.05.
Lipid induced lysosomal damage after demyelination corrupts microglia protective function in Lysosomal Storage Disorders (2018)
Figure 4

Figure 4. SM mediated lysosomal damage and CathB accumulation in Arg-1 positive microglia of ASMko mice.

(A) Transmission electron microscopy image of microglia in the Cb of ASMko mice showing accumulation of multillamelar vacuoles and other lysosome related vesicles filling the whole cytoplasm of these cells. Arrowhead identifies the nuclei. Scale bar, 5 μm. On the right, higher magnification showing a multillamelar body (arrowhead) with the characteristic concentric lamellar inclusions. Scale bar, 0.5 μm.

(B, C) Immunofluorescence staining against Galectin3-GFP in BV2 microglial cells treated with or without SM (B) or Siramesine (C). Scale bar, 10 μm.

(D) Mean ± SEM number of Galectin3-GFP positive vesicles in BV2 microglial cells in the different conditions (n = 4 independent cultures, >50 cells per culture, Student´s t-test).

(E) Lysotracker-Red staining in BMDMs from wt and ASMko mice. Scale bar, 10 μm.

(F) Western blot of CathB levels and the cytosolic protein GAPDH in the cellular and cytosolic fractions of wt and ASMko BMDMs extracted with digitonin (left). Mean ± SEM pro-CathB and CathB levels in the cytosolic fractions (right) (n=2 cultures per genotype, Student´s t-test).

(G) Immunofluorescence staining against Iba-1 (green) and CathB (red) in Cb of ASMko and wt mice. Arrowheads show double positive cells. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm.

(H) Immunofluorescence staining against CathB (green), Arg-1 (red, anti-inflammatory microglia) and F4/80 (yellow, all microglia) in Cb of ASMko mice. DAPI staining shows cell nuclei. Scale bar, 50 μm. Below, magnified images of the selected areas. Scale bar, 20 μm.

(I) Table showing mean ± SEM percentage of microglia presenting CathB staining (CathB +/Iba-1+), and the number of CathB positive microglia co-expressing Arg-1 (CathB+/Arg-1+) in Cb of ASMko and wt mice (n = 5 mice, >10 cells per mouse).

Data information: P*<0.05; P***<0.001.