Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 1. TNF- and LMP1 enhance LTR-mediated viral transcrip- tion. (A) XMRV-infected HEK293 cells were incubated with TNF- at the indicated concentrations. After 24 h, the conditioned media were harvested and ultracentrifuged (100,000 g for 1 h), and each pellet was subjected to Western blot analysis for Gag protein (p30CA) by using the rat MAb R187. The bar graph reflects quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. (B) TNF- (5 ng/ml) was added to HEK293 cells transiently cotrans- fected with the XMRV LTR construct fused to firefly luciferase (FL) and with a renilla luciferase (RL) plasmid (pTK-RL). After a 16-h incubation, FL and RL activities were measured. FL activity was nor- malized to RL activity. The results are presented as fold increases ( standard deviations [SD] for triplicate cultures) in normalized FL in cultures with TNF- relative to that in medium only. (C) HEK293 cells were cotransfected with the pCMV-LMP1 plasmid at the indicated concentrations and the XMRV LTR FL reporter. For normalization, we also transfected with pTK-RL. FL and RL activities were measured from cells harvested 16 h after transfection. The results are presented as fold increases ( SD for triplicate wells) of normalized FL induced by LMP1 transfection relative to that without LMP1 transfection.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Infection, Incubation, Western Blot, Construct, Luciferase, Plasmid Preparation, Activity Assay, Transfection

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 2. Identification of NF-B binding sites B-1 and B-2 in the XMRV LTR and functional analysis of NF-B binding to the XMRV LTR. (A) DNA sequences of consensus and NF-B binding sites in the immunoglobulin (Ig) chain enhancer and in the XMRV LTR. Mutated (mut) sequences are listed below the WT sequences; complementary sequences (c) are noted. Lowercase letters represent mutations. (B) Representative immunoblotting results from DNA affinity binding assays. Individual biotinylated double-stranded DNA probes (sequences shown in panel A) were incubated with nuclear extracts from the EBV-infected lymphoblastoid IB4 cell line as a source of p65/RelA. After precipitation with streptavidin Sepharose, the binding of p65/RelA to biotinylated DNA was revealed by Western blotting (WB) with a specific antibody to p65/RelA. NE, nuclear extracts only. (C) Effects of TNF and LMP1 on WT and mutant XMRV LTR transcriptions as measured in luciferase reporter assays. Mutations in the B-1 and B-2 sites (shown in panel A) were introduced in the XMRV LTR reporter construct. Each firefly luciferase (FL) LTR reporter construct (the WT is XLTR, and mutants 1 and 2 are XLTR mut1 and mut2, respectively) was transiently transfected into HEK293 cells; the cells were cultured in medium alone or with 5 ng/ml of TNF-. The cells were also cotransfected with pCMV-LMP1 (0.1 g/well). After 16 h of incubation, FL activity was measured and normalized for renilla luciferase (from cotransfection with pTK-RL). The results are presented as relative luciferase activities ( SD for triplicate wells) in comparison to HEK293 cells transfected with XLTR and cultured in medium alone. R, repeat sequence.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Binding Assay, Functional Assay, Western Blot, Incubation, Infection, Mutagenesis, Luciferase, Construct, Transfection, Cell Culture, Activity Assay, Cotransfection, Comparison, Sequencing

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 3. Mutation of the B-1 site reduces XMRV replication. (A) Schematic representation of the XMRV provirus constructs [pcDNA3.1()VP62 and pcDNA3.1()VP62mB-1]; the WT B-1 site and the mutant mB-1 site are expanded. The mutated bases (G to C at position 7959 and C to G at position 7967) are shown. CMV, human cytomegalovirus immediate early promoter; , packaging signal for XMRV. Lowercase letters represent mutations. (B) Conditioned medium from 293T/17 cells transfected with pcDNA3.1()VP62 or pcDNA3.1()VP62mB-1 was spun at 100,000 g for 1 h, and virus-like particles were subjected to Western blotting for Gag protein by using the rat MAb R187. (C) Duplicate semiconfluent cultures of the prostate carcinoma cell lines LNCaP and DU145 were infected with equal amounts (500 l [top and bottom panels] and50 l [middle panel] viral preparation shown in panel B) of WT XMRV (VP62) or mutant virus (VP62mB-1). After culture for the indicated periods (2 to 6 days), individual conditioned media were ultracentrifuged and the pellets analyzed by Western blotting for Gag protein (p30CA) by using the rat MAb R187. The bar graphs reflect quantitative analysis of duplicate band intensities from the membranes shown above; the results are expressed as relative band intensities, and error bars reflect the ranges of measurements. U, sample from uninfected cells. Each experiment was repeated at least three times, and representative results are shown. (D) Reverse transcriptase (RT) activity in the culture supernatants of LNCaP cells left uninfected or infected (50 l) with VP62 or VP62mB-1. The results reflect RT activity (ng/ml; SD for triplicate cultures) in the precipitates from conditioned media (0.5 ml) tested in duplicate. Exp., experiment. (E) Relative luciferase activity after transfection of the pIL-6-Luc plasmid into the cell lines HEK293, LNCaP, and DU145, with or without the addition of TNF- (20 ng/ml) to the cultures. For normalization, each cell line was cotransfected with pTK-RL. FL and RL activities were measured from cells harvested 18 h after transfection.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Mutagenesis, Construct, Transfection, Virus, Western Blot, Infection, Reverse Transcription, Activity Assay, Luciferase, Plasmid Preparation

Journal: Journal of Virology

Article Title: NF-κB Activation Stimulates Transcription and Replication of Retrovirus XMRV in Human B-Lineage and Prostate Carcinoma Cells

doi: 10.1128/jvi.02333-10

Figure Lengend Snippet: FIG. 5. Relative effects of dexamethasone and NF-B on XMRV production. (A) HEK293 cells were infected with WT XMRV (pcDNA- VP62). Ten days after infection (75% of cells expressed Gag protein, per immunostaining), cells were harvested, washed, and seeded onto 6-well plates in fresh medium supplemented with dexamethasone (Dex) at the indicated concentrations. After 24 h, the conditioned media (0.5 ml) were harvested and ultracentrifuged (100,000 g for 1 h), and each pellet was analyzed by Western blotting for Gag protein (p30CA) by using the rat MAb R187. (B) HEK293 cells were infected with WT or B-1 mutant XMRV (VP62 or VP62mB-1) with or without Dex (1.5 M). After 6 days of incubation, the conditioned media (0.5 ml) were harvested and ultracentrifuged, and each pellet was analyzed by Western blotting for Gag protein (p30CA). The bar graphs below the nitrocellurose membrane images in panels A and B reflect quantitative analysis of duplicate band intensities; the results are expressed as relative band intensities, and the error bars reflect the ranges of measurements. The dotted line in panel B demarks the mean relative band intensity from culture supernatants of XMRV VP62-infected HEK293 cells cultured in medium. Each experiment was repeated three times, and representative results are shown. (C) Schematic representation of XMRV LTR-mediated replication regulated by the Dex-glucocorticoid receptor (GR)-GRE and TNF-/LMP1–NF-B–B-1 pathways. TNFR, tumor necrosis factor receptor; IKK, IB kinase.

Article Snippet: Human embryonic kidney-derived HEK293 and HEK293T/17 cells (American Type Culture Collection [ATCC]) were propagated in Dulbecco’s modified Eagle’s medium (DMEM)-Glutamax medium (Invitrogen) with 10% fetal bovine serum (FBS) (Atlanta Biologicals).

Techniques: Infection, Immunostaining, Western Blot, Mutagenesis, Incubation, Membrane, Cell Culture

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a Adenoviral Neurog3 , Pdx1 and Mafa in islets (inset, endogenous gene expression) ( n = 5 animals; paired t -test). b Islets transduced with Ad-M3C (β-cell mature; B-MAT) lose β-cells occupying the bottom 15 percentile for PDX1 compared to controls (β-cell normal; B-NORM) (inset, non-normalized polynomial-fitted B-NORM distribution) ( n = 6 islets/3 animals; two-way ANOVA, Bonferonni’s multiple comparison) (F = 18.75, DF = 20). c As for b , but showing the frequency distribution for MAFA ( n = 8 islets/3 animals; two-way ANOVA, Bonferonni’s multiple comparison) (F = 3.03, DF = 20). d Images showing more homogenous PDX1/MAFA fluorescence in B-MAT islets (scale bar = 60 µm). e – g INS-PDX1 ( e ), INS-MAFA ( f ) and MAFA-PDX1 ( g ) are positively correlated ( n = 137 cells, linear regression). h The linear correlation between PDX1 and BFP in Pdx1-BFP islets is lost following Ad-M3C transduction (B-MAT) ( n = 465 cells/3 animals). i BFP LOW cells (prior immature) become PDX1 HIGH in B-MAT islets, while BFP HIGH cells (prior mature) remain PDX1 HIGH ( n = 93 cells/3 animals; one-way ANOVA with Sidak’s multiple comparison) (F = 52.12, DF = 3). j Images from Pdx1-BFP islets showing cells that underwent PDX1 LOW - > PDX1 HIGH conversion (arrow shows a cell that remained PDX1 HIGH ) (scale bar = 50 µm) ( n = 5 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (F = 2.80, DF = 18). k – q No differences are detected in the ratios of α- to β-cells ( n = 23 islets/3 animals) and δ- to β-cells ( n = 18 islets/3 animals) ( k – n ), or the proportion of PDX1 + /INS− or PDX1 + /GLU + cells ( n = 10 islets/4 animals) ( o – q ) in B-MAT islets (unpaired t-test) (scale bar = 40 µm). r No difference in TUNEL+ cell numbers is detected in B-MAT islets ( n = 18 islets/4 animals; unpaired t-test) (scale bar = 42.5 µm). s Cell proliferation is similar in B-NORM and B-MAT islets, as shown by PCNA staining ( n = 24 islets/4 animals; unpaired t-test) (scale bar = 42.5 µm). t Transition to high PDX1/MAFA content occurs in PDX1 LOW /MAFA LOW cells (1), whereas PDX1 HIGH /MAFA HIGH cells remain unaffected (2), with PDX1/MAFA levels never surpassing those in B-NORM islets (3). Bar graphs show the mean ± SEM. Violin plot shows median and interquartile range. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant. BFP-blue fluorescent protein; INS-insulin; GLU-glucagon; SST-somatostatin; TUNEL-terminal deoxynucleotidyl transferase dUTP nick-end labeling; PCNA-proliferating cell nuclear antigen.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Gene Expression, Transduction, Comparison, Fluorescence, TUNEL Assay, Staining

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a Binding of multiple transcription factors to enhancer clusters (boxed in red) regulates expression of key β-cell transcription factors in human islets. For reference, RNA-seq, H3K27ac (enhancer mark) and H3K4me3 (promoter mark) are also shown. All scales are set to 20 RPKM for ChIP-seq and 20 or 60 RPKM for RNA-seq (TF strand to 60, other to 20). b Expression of MAFA and PDX1 correlate over 64 human islet samples. The axes represent normalized expression values (−3 to 3) for each gene used for the co-expression network analysis . c Correlation of expression of mRNA for PDX1 and NEUROD1 , NKX6-1 , GAPDH and GLIS3 across 64 human islet samples. The axes represent normalized expression values (−3 to 3) for each gene used for the co-expression network analysis . d Single cell gene expression levels for MAFA , MAFB , NKX6-1 and PDX1 in cells with high and low mRNA levels for PDX1. Analysis was performed using Monocle, the y-axis representing gene expression levels in log10 scale. Datasets were obtained from , .

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Binding Assay, Expressing, RNA Sequencing, ChIP-sequencing, Gene Expression

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a – c Ca 2+ fluxes ( a ) in response to glucose ( b ) or glucose + KCl ( c ) are impaired in B-MAT islets, also shown by representative images ( d ) ( n = 34 islets/4 animals; unpaired t-test) (scale bar = 40 µm). Inset in ( a ) shows an inverse correlation between glucose-stimulated Ca 2+ amplitude and BFP expression in individual β-cells (Pdx1-BFP; n = 6 islets/3 animals; R 2 = 0.21, P < 0.0001) (G11, 11 mM glucose; KCl, 10 mM). e No diffeences in the % glucose-responsive β-cells are detected in B-MAT islets ( n = 34 islets/4 animals; unpaired t-test). f – h As for ( a – c ), but using Fura2 ( n = 33 islets/4 animals; unpaired t-test). i Expression of genes encoding CACNA1D and CACNB2 Ca 2+ channel subunits is reduced in B-MAT islets ( n = 8 animals; paired t-test). j , k Ca 2+ pulse duration is reduced in B-MAT islets, as shown by summary bar graph ( j ) and traces ( k ) ( n = 8 islets/4 animals; unpaired t -test). l , m ATP/ADP ratios are reduced in B-MAT islets, as shown by mean traces ( l ) and summary bar graph ( m ) ( n = 40 islets/4 animals; unpaired t -test). n , o GCK expression ( n ) tends to be reduced in B-MAT islets ( n = 10 islets/2 animals; paired t-test), although Gck levels are normal ( o ) ( n = 7 animals; paired t-test) (scale bar = 15 µm). p , q Ca 2+ ( p ) and ATP/ADP ( q ) responses to increasing glucose concentration are decreased in B-MAT islets (Ca 2+ : n = 11 islets/5 animals; two-way ANOVA F = 20.36, DF = 4) (ATP/ADP: n = 37 islets/5 animals, two-way ANOVA; F = 6.10, DF = 4) (Bonferroni’s multiple comparison). r , s Mean traces ( r ) and bar graph ( s ) showing reduced cAMP levels in response to glucose and forskolin (FSK, 100 μM) in B-MAT islets ( n = 13 islets; unpaired t-test). t Adcy8 expression remains unchanged in B-MAT islets ( n = 6 animals; paired t-test). u G6pc2 and Ascl1 are up- and down-regulated, respectively, in B-MAT islets ( n = 6 animals; paired t-test). Color scale shows Ca 2+ as min (0%) to max (100%) value. Bar graphs and traces show the mean ± SEM. All tests are two-sided where relevant. CTCF-corrected total cell fluorescence.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Expressing, Concentration Assay, Comparison, Fluorescence

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a , b Ad-M3C increases Neurog3 , Pdx1 and MafA expression ( a ), while no differences are detected in native NEUROG3 , PDX1 and MAFA expression ( b ) ( n = 4–8 donors). c Ad-M3C increases the proportion of cells expressing high PDX1 levels (B-hMAT) (inset is the non-normalized B-hNORM distribution fitted with a polynomial) ( n = 13 islets/4 donors; two-way ANOVA, Bonferroni’s multiple comparison) (F = 4.14, DF = 20). d Representative images showing loss of PDX1 LOW cells in B-hMAT islets (detected using a PDX1 antibody with reactivity against mouse and human protein) (scale bar = 42.5 µm). e PDX1 and INS1 are positively correlated in individual cells from B-hNORM islets ( n = 220 cells). f – h Ca 2+ traces ( f ) showing decreased responsiveness to glucose ( g ) and KCl ( h ) in B-hMAT islets ( n = 16 islets/3 donors; unpaired t -test). i , j as for ( f – h ), but representative images (scale bar = 25 µm) showing loss of glucose-stimulated Ca 2+ rises in B-hMAT but not B-hNORM islets ( i ), despite no differences in the proportion of responsive cells ( j ) ( n = 16 islets/3 donors; unpaired t -test). k The VDCC and Na + channel subunits CACNA1G , CACNA1C , CACNA1D , SCN1B , SCN3A and SCN8A are all downregulated in B-hMAT islets ( n = 4–6 donors; paired t -test). l – o GJD2 expression ( l ) is decreased in B-hMAT islets ( n = 6 donors; paired t-test), which is associated with a decrease in the number of hubs (circled in red) ( m ) and coordinated β-cell-β-cell activity (connectivity) ( n and o ) (representative traces are from ‘connected’ cells; raster plots show intensity over time) ( n = 7–8 islets/3 donors; unpaired t -test). p – r Non-normalized Insulin secretion is similar in B-hMAT and B-hNORM islets ( p ), although B-hMAT islets only release a fraction of their total insulin ( q and r ) (% insulin content = secreted insulin / total insulin) ( n = 17–18 replicates/5 donors; unpaired t -test and two-way ANOVA, Bonferroni’s multiple comparison). s Schematic showing proposed changes occurring in β-cells in B-hMAT islets. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant. Color scale shows Ca 2+ as min (0%) to max (100%) value. GCaMP6-genetically-encoded Ca 2+ indicator; VDCC-voltage-dependent Ca 2+ channels; VGSC-voltage-gated Na + channels; GJD2 -Gap junction delta-2 protein encoding Connexin-36.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Expressing, Comparison, Activity Assay

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a sh Pdx1 increases the proportion of β-cells in the islet with low levels of PDX1 and MAFA (β-cell immature; B-IMMAT) (scale bar = 60 µm). b Quantification of PDX1 and MAFA expression intensity shows an increase in β-cells occupying the bottom 15 percentile in B-IMMAT islets ( n = 13–14 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (PDX1: F = 2.38, DF = 20) (MAFA: F = 3.20, DF = 20). c RT-qPCR showing a decrease in Pdx1 expression levels in B-IMMAT islets ( n = 5; paired t -test). d Induction of homogenous β-cell immaturity does not alter the α- to β-cell ratio (scale bar = 42.5 µm) ( n = 18 islets/ 2–3 animals; unpaired t-test). e – g B-IMMAT islets display decreased insulin content ( e ), increased basal insulin release and absence of significant glucose-stimulated insulin secretion ( f and g ) ( n = 10–12 replicates/4 animals; paired t -test and one-way ANOVA, Sidak’s multiple comparison) (G3, 3 mM glucose; G16.7, 16.7 mM glucose; Ex4, 20 nM Exendin-4). h – j Ca 2+ traces ( h ) and bar graphs ( i and j ) showing impaired responses to glucose and glucose + KCl in B-IMMAT islets ( n = 49–51 islets/4–5 animals; unpaired t-test) (representative images shown above bar graph, scale bar = 75 µm). k mRNA for the L-type VDCC subunits Cacnb2 and Cacna1d are significantly downregulated in B-IMMAT islets ( n = 5–6; paired t -test). l Schematic showing the proposed changes in B-IMMAT islets. Color scale shows Ca 2+ as min (0%) to max (100%) value. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant. sh Pdx1 - short hairpin RNA against Pdx1 ; VDCC-voltage-dependent Ca 2+ channels.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Expressing, Comparison, Quantitative RT-PCR, shRNA

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a–c Islet dissociation (B-NORM DISS.) leads to loss of β-cells in the bottom 15 percentile for PDX1 ( a ) and MAFA ( b ), also shown by representative images ( c ) (B-NORM data are superimposed for comparison) ( n = 6 islets/4 animals; two-way ANOVA, Bonferroni’s multiple comparison) (PDX1: F = 7.23, DF = 19) (MAFA: F = 4.69, DF = 20) (scale bar = 42.5 µm). d PDX1 LOW β-cells are present 3 h following islet dissociation ( n = 80 islets/10 coverslips; two-way ANOVA; Bonferonni’s multiple comparison test) (PDX1: F = 9.54, DF = 40) (MAFA: F = 5.22, DF = 20). e , f sh Gjd2 decreases Gjd2 expression ( e ) ( n = 5 animals; paired t-test), but this does not alter the proportion of PDX1 LOW β-cells ( f ) ( n = 8 islets/2 animals; two-way ANOVA, Bonferroni’s multiple comparison) (F = 12.85, DF = 20). g – i h4MDi is expressed at the β-cell membrane ( g ) ( n = 3 islets) (scale bar = 85 µm), allowing silencing of Ca 2+ activity in D-MAT but not D-NORM (control) islets ( h , i ) ( n = 7 islets/3 animals; paired t -test). j 3 h CNO incubation decreases Ca 2+ levels in D-MAT islets (vehicle, DMSO) ( n = 16 islets/5 animals; Mann-Whitney U -test). k , l CNO decreases Ca 2+ oscillation frequency ( k ) in D-MAT islets, also shown by traces ( l ) ( n = 6 islets/2 animals; unpaired t -test). m – o 48 h CNO incubation induces β-cell loss in the bottom 15 percentile for PDX1 ( m ) and MAFA ( n ) in D-MAT islets, also shown by representative images ( o ) ( n = 8 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (PDX1: F = 5.34, DF = 20) (MAFA: F = 4.63, DF = 20) (scale bar = 60 µm). p 2 h washout restores Ca 2+ levels in CNO-treated islets ( n = 21 islets/3 animals; unpaired t -test). q , r Ca 2+ traces ( q ) showing blunted responses to 11 mM glucose (G11) and KCl (10 mM) ( r ) in D-MAT islets (following CNO washout) ( n = 21 islets/3 animals; unpaired t -test). s , t D-MAT islets display decreases in β-cell–β-cell connectivity ( s ), associated with hub loss (red circles) ( t ) ( n = 7 islets/4 animals; Mann Whitney U-test). u Schematic showing effects of altering Ca 2+ signaling patterns. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Comparison, Expressing, Membrane, Activity Assay, Control, Incubation, MANN-WHITNEY

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a Recombination of RIP7rtTA and TetO/M3C mice allows doxycycline-inducible changes in β-cell Neurog3 , Pdx1 and Mafa expression in Tet-MAT but not Tet-NORM (control) islets. b Pdx1 , Mafa and Neurog3 expression increases following incubation of Tet-MAT islets with 100 ng/ml doxycycline for 48 h ( n = 3 animals; paired t -test). c , d A significant decrease in the number of PDX1 LOW β-cells is seen in doxycycline-treated Tet-MAT islets, as shown by representative images ( c ), and shown also by the loss of cells in the lowest fluorescence intensity bins ( d ) ( n = 6 islets/3 animals; two-way ANOVA, Bonferroni’s multiple comparison) (scale bar = 20 µm) (F = 41368, DF = 20). e – g Mean traces ( e ) and bar graphs ( f and g ) showing impaired glucose- and KCl-stimulated Ca 2+ rises in Tet-MAT but not Tet-NORM islets ( n = 33 islets/4 animals; unpaired t -test). h Volcano plot of differential gene expression between Tet-NORM and Tet-MAT islets. Fold-change (Log2, x -axis) gene expression is plotted against adjusted p -value for differential gene expression (normalized by GLM, -Log10, y-axis). Colored dots represent Ensembl genes that are differentially regulated at an adjusted p -value < 0.05 ( n = 5 animals). i Gene ontology analysis of differentially regulated genes in Tet-MAT islets. A set of 83 genes were functionally annotated using DAVID (adjusted p-value of < 0.05). j Gene set enrichment analysis (GSEA) suggests that genes belonging to the gene set “hallmark β-cells” are upregulated in Tet-MAT islets. Normalized enrichment score (NES) and nominal p-value is presented in the top right corner of the graph. k GSEA analysis shows enrichment of genes belonging to glucose and carbohydrate derivative metabolic processes amongst the upregulated genes in Tet-MAT islets. l RT-qPCR analyses confirming upregulation of Ucn3 , G6pc2 , Cox6a2 and Rgs4 but not Pkib in Tet-MAT islets ( n = 3 animals; paired t -test). Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Expressing, Control, Incubation, Fluorescence, Comparison, Gene Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: PDX1 LOW MAFA LOW β-cells contribute to islet function and insulin release

doi: 10.1038/s41467-020-20632-z

Figure Lengend Snippet: a – c A significant decrease in the proportion of PDX1 HIGH β-cells is detected in palmitate-treated islets ( a ), and this can be reversed using Ad-M3C ( b ), as shown by representative images ( c ) ( n = 7 islets/4 animals; two-way ANOVA, Bonferroni’s multiple comparison) (Palm: F = 4.28, DF = 20) (Palm + Ad-M3C: F = 0.90, DF = 20) (BSA, bovine serum albumin; Palm, 0.5 mM palmitate for 48 h) (scale bar = 42.5 µm). Note that the same BSA-only (control) PDX1 fluorescence intensity distribution is shown in both graphs ( a ) and ( b ) to allow cross-comparison (the experiments were performed in parallel). d–f Ca 2+ responses to glucose ( d ) and KCl ( e ) are blunted in palmitate-treated, but not palmitate + Ad-M3C-treated islets (n = 27 islets/4 animals; one-way ANOVA, Sidak’s multiple comparison) (G11: F = 18.80, DF = 2) (KCl: F = 23.13, DF = 2), as shown by mean traces ( f ). g Schematic showing that a decrease in the proportion of PDX1 LOW /MAFA LOW β-cells leads to altered islet Ca 2+ fluxes, decreased expression of Ca 2+ -dependent genes such as Ascl1 , and broader changes to β-cell function, including impaired ATP/ADP and insulin responses to glucose. Bar graphs and traces show the mean ± SEM. Box-and-whiskers plot shows median and min-max. All tests are two-sided where relevant.

Article Snippet: Membranes were then incubated in antibodies against PDX1 (Iowa DSHB Cat#F6A11, RRID:AB_1157904, dilution 1:1000) and GAPDH (Cell Signaling Technology Cat# 5174, RRID:AB_10622025, dilution 1:2000), diluted in TBS-T containing 3% (w/v) bovine serum albumin (BSA) overnight at 4 °C.

Techniques: Comparison, Control, Fluorescence, Expressing, Cell Function Assay

Journal: International Journal of Molecular Sciences

Article Title: Expression and Regulation of a Novel Decidual Cells-Derived Estrogen Target during Decidualization

doi: 10.3390/ijms24010302

Figure Lengend Snippet: Cstb expression is associated with angiogenesis in mouse uterine stromal cells Angptl7 . ( A ) Gene ontology (GO) functional classification of the DEGs. ( B ) The expressions of Cstb and ZO1 were detected by immunofluorescence in the uterus on day 6. Bar = 300 μm. ( C ) The expression of Angptl7 in stromal cells of the con NC group, dc NC group, and dc si Cstb group was detected by RT-qPCR. ( D ) The expression of Angptl7 in mouse uteri from days 5 to 8 of pregnancy was detected by in situ hybridization. Bar = 300 μm. * p < 0.05. NC, negative control; si Cstb , siRNA of Cstb ; dc, in vitro decidualization.

Article Snippet: Briefly, frozen sections were fixed in 4% paraformaldehyde solution for 10 min and then soaked in 0.1% Triton X-100 in PBS for 15 min. After blocking with 5% donkey serum (Zhongshan Jinqiao, Beijing, China) in a 37 °C oven for 60 min, the sections were incubated with mouse anti- Cstb (1:500, Santa Cruz, sc-166561), ZO1 (1:200, Cell Signaling Technology, Boston, MA, USA, #13663) diluted in PBS overnight in a 4 °C refrigerator.

Techniques: Expressing, Functional Assay, Immunofluorescence, Quantitative RT-PCR, In Situ Hybridization, Negative Control, In Vitro

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267R

Figure Lengend Snippet: Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1 ) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2 ) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade desmosomal and corneodesmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3 ) The findings in the present study suggest that inhibition of CtsB by Cst6 protects HF maintenance in mice. 4 ) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse Cst6.

Article Snippet: Each well was measured for mouse Cst6 at an absorbance of 450 nm with an ELISA microplate reader (Bio-Rad, Hercules, CA, USA).

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Molecular Metabolism

Article Title: Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA

doi: 10.1016/j.molmet.2019.12.012

Figure Lengend Snippet: GAS5 KD with or without GC treatment affects the expression of key proteins in glucocorticoid signaling (GR and SGK1) and beta cell function (PDX1, NKX6-1, and SYT13). A. Expression of GAS5. B. Protein expression of GR. C. Protein expression of SGK1. D. Expression of PDX1. E. Protein expression of NKX6-1. F. Protein expression of SYT13. The data are presented as mean ± SEM. n = 4, *p < 0.05 vehicle vs Dexa; # p < 0.05 scramble vs GAS5 KD.

Article Snippet: Probe-based TaqMan Assays (Applied Biosystems) were used to measure the expression levels of human GAS5 (Hs03464472_m1), PDX1 (Hs00236830_m1), NKX6-1 (Hs00232355_m1), SYT13 (Hs00951871_m1), SGK1 (Hs00985033_g1), and GR (Hs00353740_m1).

Techniques: Expressing, Cell Function Assay

Journal: Molecular Metabolism

Article Title: Glucocorticoid induces human beta cell dysfunction by involving riborepressor GAS5 LincRNA

doi: 10.1016/j.molmet.2019.12.012

Figure Lengend Snippet: GAS5 and gene expression changes in islets from diabetic donors, GK islets, and beta cells under glucotoxic conditions. A. Expression of GAS5 in islets from control (ND) (n = 10) and T2D donors (n = 9) as measured by qPCR assay. B. GAS5 expression in islets vs HbA1c levels in all of the donors (n = 19). C. GAS5 expression in the islets of T2D model GK rats. D. Glucose regulation of GAS5 in EndoC-βH1 cells (n = 4) exposed to glucotoxic conditions. E. GR protein expression. F. Protein expression of PDX1. G. Protein expression of NKX6-1 and SYT13. The data are presented as mean ± SEM; *p < 0.05, **p < 0.01, ***p < 0.001.

Article Snippet: Probe-based TaqMan Assays (Applied Biosystems) were used to measure the expression levels of human GAS5 (Hs03464472_m1), PDX1 (Hs00236830_m1), NKX6-1 (Hs00232355_m1), SYT13 (Hs00951871_m1), SGK1 (Hs00985033_g1), and GR (Hs00353740_m1).

Techniques: Gene Expression, Expressing, Control

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 1. Phenotype of rescued Tg(INV-Cst6)Cst6ichq/ichq mice. A) Tg(INV-Cst6)Cst6ichq/ichq mice survived and showed periodic hair loss. After 4 mo the progenies became completely bald. Keratitis and thickening of the cornea were observed in Tg(INV-Cst6)Cst6ichq/ichq mice from 4 to 5 mo. The mice shown are 9 and 32 wk old. Inset: magnified view of an affected eye. B) Keratitis and metaplasia of the corneal epithelium in Tg(INV- Cst6)Cst6ichq/ichq mice. H&E staining of the eye and the cornea in WT and Tg(INV-Cst6)Cst6ichq/ichq mice. C) Immunofluores- cence staining for the expression of loricrin (LOR) and filaggrin (FLG) in the cornea. Scale bars, 100 mm.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 2. Destruction of the HFs in Tg(INV-Cst6)Cst6ichq/ichq mice. A) Immunofluorescence double labeling of INV and Cst6 in the epidermis and the HF of WT mice. Cst6 was expressed in the stratum granulosum and halfway up the HF, including the area around the bulge, whereas INV expression was also seen in the stratum granulosum, but remained only in the proximal part of the HF. B) Immunohistological labeling in WT mice to detect the location of the bulge area (arrows) using the stem cell markers CD34 and keratin 15 and the proliferation marker Ki67. C) H&E staining of Tg(INV-Cst6)Cst6ichq/ichq mice from 11, 13, and 16 wk showed disappearance of the HFs. Scale bar, 100 mm.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Labeling, Expressing, Marker, Staining

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 3. Inhibition of mouse CtsB by mouse Cst6. A) BMV109 labels active cysteine cathepsins, such as CTSB and CTSX (14) in RAW cell lysates (murine Mf cell line). Lane 1, no inhibitor; lane 2, JPM-OEt (pan-cathepsin inhibitor, 50 mM); lane 3, CA-074 (CtsB inhibitor, 10 mM); lane 4, Z-FY (t-BU)DMK (CtsL inhibitor, 10 mM); lane 5, mouse Cst6 (18 mM); and lane 6, heat inactivation. Note that CA-074 and Cst6 inhibited CtsB (lane 3 and 5). M = precision plus protein dual-color marker. B) The Ki for the inhibition of CtsB by Cst6 was determined by measuring the residual enzymatic activity of a fixed concentration of enzyme, incubated with increasing concentrations of the inhibitor. An Easson-Stedman plot (inset) was used to calculate the Ki, according to the following equation: [I]/1 2 a = (Ki/a) + E0, were I is the inhibitor concentration, E0 is the enzyme concentration at time 0, and a is the fractional activity. The plot yielded a straight line with a Ki slope of 0.98 nM.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Inhibition, Chromosome Transmission Fidelity Colony Color Assay, Activity Assay, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 4. CtsB and Cst6 colocalize in the mouse HF. A) Immunofluorescence double staining for Cst6 (green) and CtsB (red) in WT mice revealed that colocalization of both proteins was found in the proximal part of the HF as well as the lower region where the bulge area resides. Cst6 is also expressed in the epidermis, whereas no CtsB is observed. Scale bar, 100 mm. B) Cst6 and CtsB expression in mouse epidermis and HFs of Tg(INV-Cst6)Cst6ichq/ichq mice. No immunofluorescence staining for Cst6 was observed in the lower region of the HF (close to the bulge, arrowheads). Scale bar, 100 mm. C) Schematic presentation of INV, Cst6, and CtsB localization in the HF and epidermis of WT mice and the situation in rescued transgenic Tg(INV-Cst6)Cst6ichq/ichq mice.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Double Staining, Expressing, Staining, Transgenic Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 5. Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade (corneo)-desmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3) The findings in the present study suggest that inhibition of CtsB by CST6 protects HF maintenance in mice. 4) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse CST6.

Article Snippet: Protease inhibitor activity of recombinant mouse Cst6 (R&D Systems) against recombinant mouse CtsB (R&D Systems) was determined by measuring the hydrolysis of the fluorescent Z-Leu-Arg-AMC substrate (R&D Systems) (4).

Techniques: Activity Assay, Control, Inhibition, Functional Assay

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a,b) Adenovirus-mediated GFP or MST1 overexpression in human islets for 96h. (a) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at GFP control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively and insulin content analyzed after GSIS and normalized to whole islet protein. (b) MST1 and PDX1 immunoreactivity were analyzed by Western blotting. Lower panel shows densitometry analysis from at least 3 independent experiments normalized to actin. Right panel shows PDX1 target genes including SLC2A2, GCK and Insulin analyzed by RT-PCR. (c-d) HEK293 cells were transfected with plasmids encoding Myc-MST1 and GFP-PDX1. (c) A kinase-dead MST1 (dn-MST1: K59R) was co-transfected with GFP-PDX1 (left panel). At 48 h after transfection, HEK293 cells were treated with cycloheximide (CHX) for 8h (middle panel). At 36h after transfection, HEK293 cells were treated with the proteasome inhibitor MG-132 for 6h (right panel). PDX1 and MST1 were analyzed by western blotting. (d) In vivo ubiquitination assay in HEK293 cells transfected with GFP-PDX1 and HA-ubiquitin, alone or together with Myc-MST1 or MST1-K59 expression plasmids for 48h (left) and human islets transfected with HA-ubiquitin and infected with Ad-GFP or Ad-MST1 for 48h (right; 2 different donors). MG-132 was added during the last 6h of the experiment. Cell lysates were immunoprecipitated with an anti-PDX1 antibody followed by immunoblotting with ubiquitin antibody to detect ubiquitinated PDX1. (e) HEK293 cells were transfected with GFP-PDX1 alone or together with Myc-MST1 for 48h. Reciprocal co-immunoprecipitations performed using anti-GFP and anti-Myc antibodies and western blot analysis performed with precipitates and input fraction using anti-Myc and anti-GFP antibodies, respectively. (f) In vitro kinase assay was performed by incubating recombinant MST1 and PDX1 proteins and analyzed by NuPAGE followed by western blotting using pan-phospho-threonine specific, PDX1 and MST1 antibodies. (g) Lysates of HEK293 cells transfected with PDX1-WT or PDX1-T11A expression-plasmids were immunoprecipitated with PDX1 antibody and subjected to an in vitro kinase assay using recombinant MST1. Phosphorylation reactions were analyzed by Western blotting using p-T11-PDX1 specific and pan-phospho threonine antibodies (left panel). HEK293 cells were transfected with PDX1-WT or PDX1-T11A alone or together with MST1 expression-plasmids for 48h. MST1 and PDX1 were analyzed by western blotting (middle panel). PDX-1-WT or PDX1-T11A co-transfected with MST1 in HEK293 cells for 36h and treated with CHX, western blot analysis for PDX1 and densitometry analysis of bands (right panel). (h) Human islets transfected with GFP, PDX1-WT or PDX1-T11A expression-plasmids and western blot analysis for PDX1. (i,j) human islets were infected with Ad-GFP or Ad-MST1 for 72h. (i) Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content and basal secretion at control. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (j) PDX1 target genes in human islets analyzed by RT-PCR and levels normalized to tubulin and shown as change from PDX1-WT transfected islets. All western blots show representative results from at least 3 independent experiments from 3 different donors (human islets). Tubulin/Actin was used as loading control. RT-PCR (b,j) and GSIS (a,i) show pooled results from 3 independent experiments from 3 different donors. Results shown are means ±SE. *p<0.05 MST-OE compared to control, **p<0.05 PDX-1T11A-MST1 compared to PDX-1WT-MST1.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Over Expression, Incubation, Control, Western Blot, Reverse Transcription Polymerase Chain Reaction, Transfection, In Vivo, Ubiquitin Proteomics, Expressing, Infection, Immunoprecipitation, In Vitro, Kinase Assay, Recombinant, Phospho-proteomics

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-d) Human islets transfected with MST1 siRNA or control siScr and treated with the cytokines mixture IL/IF, 33.3 mM glucose or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72h. (a) Beta-cell apoptosis analyzed by double staining of TUNEL and insulin. An average number of 11390 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments from 3 different donors. (b) Western blotting confirmed successful (~80%) MST1 depletion in human islets. MST1, pMST1, BIM, pH2B, caspase-9 and caspase-3 cleavage analyzes by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (c) RT-PCR for BCL2L11 performed in human islets and levels normalized to tubulin shown as change from siScr control transfected islets. (d) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (e,f) Islets were isolated from Mst1 −/− mice and their WT littermates and exposed to the cytokines mixture IL/IF or the mixture of 33.3 mM glucose and 0.5 mM palmitate (33.3Pal) for 72 hours. (e) beta-cell apoptosis analyzed by double staining for TUNEL and insulin. An average number of 24180 insulin-positive beta-cells were counted for each treatment condition in 3 independent experiments. (f) Insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 1h-incubation with 2.8 mM glucose. (g-i) Stable INS-1E clones were generated by transfection of vectors for shMst1 and shScr control and treated with the cytokines mixture IL/IF or 22.2 or 33.3 mM glucose for 72h. (g) Mst1, Bim, Pdx1, caspase-3 and PARP cleavage were analyzed by western blotting. Right panel shows densitometry analysis from at least 3 independent experiments normalized to actin. (h) Insulin stimulatory index. (i) PDX1 target genes in shMst1 and shScr control INS-1E cells normalized to tubulin and shown as change from shScr control INS1-E clones. Western blots (b,g) show representative results from 3 independent experiments from 3 different donors (human islets). Actin was used as loading control. TUNEL data (a,e), GSIS (d,f,h) or RT-PCR (c,i) show pooled results from 3 independent experiments. Results shown are means ±SE. *p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr untreated controls (g,h,i) , **p<0.05 compared to siScr (a,b,c,d), WT (e,f) or shScr (g,h,i) at the same treatment conditions.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Transfection, Control, Double Staining, TUNEL Assay, Western Blot, Reverse Transcription Polymerase Chain Reaction, Incubation, Isolation, Clone Assay, Generated

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-g) Mst1 −/− mice (n=15) and their WT littermates (n=14) were injected with 40 mg/kg streptozotocin or citrate buffer for 5 consecutive days. (a) Random fed blood glucose measurements after last STZ injection (day 0) over 21 days and intraperitoneal glucose tolerance test (ipGTT) performed at day 17. (b) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). (c) The ratio of secreted insulin and glucose is calculated at fed state. (d-g) Mice were sacrificed at day 22. (d) Beta-cell mass and quantitative analyses from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. (e) The pancreatic area of alpha- (stained in red) and beta-cells (stained in green) are given as percentage of the whole pancreatic section from 10 sections spanning the width of the pancreas . (f,g) Representative double-staining for Bim (red, f ) or Pdx1 (red, g ) and insulin (green) is shown from STZ-treated Mst1 −/− mice and controls. White arrows indicate areas of cytosolic Pdx1 localization and its total absence in WT-STZ mice. (h-j) b Mst1 −/− mice with specific deletion in the beta-cells using the Cre-Lox system (n=5) and their Rip-Cre (n=3) and fl/fl controls (n=3) were injected with 40 mg/kg STZ for 5 consecutive days. (h) Random fed blood glucose measurements after last STZ injection (day 0) over 32 days and ipGTT at day 32. (i) Insulin secretion during an ipGTT measured before (0 min) and 30 min after glucose injection and data are expressed as ratio of secreted insulin at 30 min/0 min (stimulatory index). The ratio of secreted insulin and glucose is calculated at fed state (right panel). (j) Mice were sacrificed at day 32. Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. Data show means ± SE. *p<0.05 WT-STZ compared to WT saline injected mice, **p<0.05 MST1 −/− -STZ compared to WT-STZ mice. #p<0.05 b MST -STZ compared to fl/fl-STZ or Cre-STZ mice.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Injection, TUNEL Assay, Staining, Double Staining, Saline

Journal: Nature medicine

Article Title: MST1 is a novel regulator of apoptosis in pancreatic beta-cells

doi: 10.1038/nm.3482

Figure Lengend Snippet: (a-e) bMST1 −/− mice (fl/fl-Cre; n=12) and the Cre control mice (n=12) were fed a normal (ND) or high fat/ high sucrose diet (“Surwit”; HFD) for 20 weeks. (a) Random fed blood glucose measurements, (b) intraperitoneal glucose tolerance test (ipGTT) and (c) insulin secretion during an ipGTT measured before (0 min), 15 and 30 min after glucose injection. (d,e) Mice were sacrificed at week 21. (d) Islets were isolated from all 4 treatment groups, cultured overnight and subjected to an in vitro GSIS assay. Insulin secretion during 1h-incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content. The insulin stimulatory index denotes the ratio of secreted insulin during 1h-incubation with 16.7 mM and 2.8 mM glucose, respectively. (e) Beta-cell mass analysis and results from triple stainings for TUNEL or Ki67, insulin and DAPI expressed as percentage of TUNEL- or Ki67-positive beta-cells ±SE. The mean number of beta-cells scored was 23121 for each treatment condition. *p<0.05 Cre HFD compared Cre ND mice. **p<0.05 b Mst1 −/− -HFD compared to Cre HFD mice. (f) Our view on how diabetic stimuli lead to activation of MST1. Active MST1 triggers cytochrome c release and mitochondrial-dependent apoptosis by modulating Bim/Bax/Bcl2/Bcl-xL through JNK/AKT signaling. Active caspase-9 then triggers cleavage of caspase-3, which triggers the caspase-3-dependent cleavage of MST1 to its constitutively active fragment, which leads to further MST1 activation and processing of caspase-3 by a positive feedback mechanism, and acceleration of beta-cell death occurs. Cleaved MST1 translocates to the nucleus and directly phosphorylates PDX1 (we do not exclude the possibility that MST1 targets PDX1 also in cytoplasm) and histone H2B. PDX1 then shuttles to cytosol, where it marks for ubiquitination and subsequent degradation by proteasome machinery and beta-cell function is impaired. Histone H2B phosphorylation by MST1 also induces chromatin condensation, one of the characteristic features of apoptosis.

Article Snippet: TaqMan(R) Gene Expression Assays were used for pdx1 (Hs00426216_m1), SLC2A2 (Hs01096905_m1), GCK (Hs01564555_m1), insulin (Hs02741908_m1), PPIA (Hs99999904_m1), BCL2L11 (Hs01083836_m1) and tubulin (Hs00362387_m1) for human and PDX1 (Rn00755591_m1), SLC2A2 (Rn00563565_m1), GCK (Rn00688285_m1), INS1 (Rn02121433_g1), INS2 (Rn01774648_g1), PPIA (Rn00690933_m1) and tuba1a (Rn01532518_g1) for rat.

Techniques: Control, Injection, Isolation, Cell Culture, In Vitro, Incubation, TUNEL Assay, Activation Assay, Ubiquitin Proteomics, Cell Function Assay, Phospho-proteomics

Journal: Molecular Metabolism

Article Title: Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

doi: 10.1016/j.molmet.2018.01.011

Figure Lengend Snippet: Efficient differentiation of XM001 iPSCs into pancreatic progenitors . (A) Schematic of iPSC-derived pancreatic progenitor differentiation protocol. (B) Immunostaining for FOXA2 and SOX17 on day 3. Scale bar indicates 50 μm. (C) Representative FACS plot of SOX17 + cells at DE stage. A differentiated sample stained with only the secondary antibody and XM001 iPSCs were used as negative controls. (D) FACS quantification of the percentage of SOX17 + cells at DE stage (n = 3). (E) Immunostaining for PDX1 on day 10. Scale bar indicates 50 μm. (F) Representative FACS plot of PDX1 + cells at the PP stage. A differentiated sample stained with only the secondary antibody and XM001 iPSCs were used as negative controls. (G) FACS quantification of the percentage of PDX1 + cells at PP stage (n = 3).

Article Snippet: The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, #sc-17320, Santa Cruz), mouse anti-TRA-1-60 (1:1000, #4746, Cell Signaling), mouse anti-TRA-1-81 (1:50, MAB4381, Millipore), mouse anti-SSEA4 (1:500, #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, #8186, Cell Signaling), goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus), goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems).

Techniques: Derivative Assay, Immunostaining, Staining

Journal: Molecular Metabolism

Article Title: Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

doi: 10.1016/j.molmet.2018.01.011

Figure Lengend Snippet: Characterization of PDX1 binding in XM001 PP cells . (A) ChIP-seq data tracks showing the enrichment of H3K27ac (blue) and PDX1 (red) at the loci of important pancreatic genes. (B) Average ChIP-seq Signal of H3K27ac (blue) and PDX1 (red) at PDX1 binding sites shows enrichment of H3K27ac at PDX1-bound sites. (C) Distribution of PDX1 binding sites among genomic features. PDX1 binds predominantly to intergenic, intronic, and promoter regions. (D) Meta-genomic plot of the enrichment of PDX1 at the transcriptional start sites (TSS) of its target genes displayed as binding sites per base pair (bp) per gene over the genomic regions of all RefSeq genes. (E) Most enriched motif discovered by de novo motif analysis resembles the known PDX1 consensus sequence and is identified in 62.2% of all PDX1-bound sequences.

Article Snippet: The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, #sc-17320, Santa Cruz), mouse anti-TRA-1-60 (1:1000, #4746, Cell Signaling), mouse anti-TRA-1-81 (1:50, MAB4381, Millipore), mouse anti-SSEA4 (1:500, #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, #8186, Cell Signaling), goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus), goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems).

Techniques: Binding Assay, ChIP-sequencing, Sequencing

Journal: Molecular Metabolism

Article Title: Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

doi: 10.1016/j.molmet.2018.01.011

Figure Lengend Snippet: Functional characterization of PDX1-bound genes . (A) Venn diagram depicting the overlap of PDX1-bound genes and the differentially expressed genes identified by microarray analysis. (B) MA plot showing the log2 fold change over the mean log2 expression of PDX1-bound genes. Differentially expressed genes are displayed in color. Green indicates enrichment in PPs, whereas brown indicates enrichment in iPSCs. (C) Bar chart of log10 p-values from enriched GO terms and KEGG and Reactome pathways of PDX1-bound genes upregulated in PPs. (D) Venn diagram showing the overlap of high confidence PDX1 binding sites from adult human islets and PPs and some annotated genes. (E) Bar chart of log10 p-values from enriched gene ontology and KEGG/Reactome pathways from genes bound in adult islets and/or PPs.

Article Snippet: The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, #sc-17320, Santa Cruz), mouse anti-TRA-1-60 (1:1000, #4746, Cell Signaling), mouse anti-TRA-1-81 (1:50, MAB4381, Millipore), mouse anti-SSEA4 (1:500, #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, #8186, Cell Signaling), goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus), goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems).

Techniques: Functional Assay, Microarray, Expressing, Binding Assay

Journal: Molecular Metabolism

Article Title: Genome-wide analysis of PDX1 target genes in human pancreatic progenitors

doi: 10.1016/j.molmet.2018.01.011

Figure Lengend Snippet: Analysis of T2DM SNPs in XM001 PPs and adult islets . (A) Venn diagram depicting the overlap of T2DM SNPs found in active regions of adult islets and XM001 PPs. (B) P-values of T2DM SNPs near known T2DM-associated genes found in active regions of adult islets , and XM001 PPs. P-values of SNPs bound by PDX1 in PPs and islets are shown as black circles and triangles, respectively. (C) ChIP-seq data tracks showing H3K27ac and PDX1 from PP and islet , , cells at the HNF1B locus. The SNP rs11263763 is located in a PP specific PDX1 binding site in the first intron of HNF1B. The PP specific enhancer regions are shaded in gray.

Article Snippet: The following antibodies and dilutions were used: goat anti-OCT-3/4 (1:500, #sc-8628, Santa Cruz), goat anti-SOX2 (1:500, #sc-17320, Santa Cruz), mouse anti-TRA-1-60 (1:1000, #4746, Cell Signaling), mouse anti-TRA-1-81 (1:50, MAB4381, Millipore), mouse anti-SSEA4 (1:500, #4755, Cell Signaling), rabbit anti-FOXA2 (1:250, #8186, Cell Signaling), goat anti-SOX17 antibody (1:500, #GT15094, Acris/Novus), goat anti-PDX1 antibody (1:500, #AF2419, R&D Systems).

Techniques: ChIP-sequencing, Binding Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 1. Phenotype of rescued Tg(INV-Cst6)Cst6ichq/ichq mice. A) Tg(INV-Cst6)Cst6ichq/ichq mice survived and showed periodic hair loss. After 4 mo the progenies became completely bald. Keratitis and thickening of the cornea were observed in Tg(INV-Cst6)Cst6ichq/ichq mice from 4 to 5 mo. The mice shown are 9 and 32 wk old. Inset: magnified view of an affected eye. B) Keratitis and metaplasia of the corneal epithelium in Tg(INV- Cst6)Cst6ichq/ichq mice. H&E staining of the eye and the cornea in WT and Tg(INV-Cst6)Cst6ichq/ichq mice. C) Immunofluores- cence staining for the expression of loricrin (LOR) and filaggrin (FLG) in the cornea. Scale bars, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Staining, Expressing

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 2. Destruction of the HFs in Tg(INV-Cst6)Cst6ichq/ichq mice. A) Immunofluorescence double labeling of INV and Cst6 in the epidermis and the HF of WT mice. Cst6 was expressed in the stratum granulosum and halfway up the HF, including the area around the bulge, whereas INV expression was also seen in the stratum granulosum, but remained only in the proximal part of the HF. B) Immunohistological labeling in WT mice to detect the location of the bulge area (arrows) using the stem cell markers CD34 and keratin 15 and the proliferation marker Ki67. C) H&E staining of Tg(INV-Cst6)Cst6ichq/ichq mice from 11, 13, and 16 wk showed disappearance of the HFs. Scale bar, 100 mm.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Labeling, Expressing, Marker, Staining

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 3. Inhibition of mouse CtsB by mouse Cst6. A) BMV109 labels active cysteine cathepsins, such as CTSB and CTSX (14) in RAW cell lysates (murine Mf cell line). Lane 1, no inhibitor; lane 2, JPM-OEt (pan-cathepsin inhibitor, 50 mM); lane 3, CA-074 (CtsB inhibitor, 10 mM); lane 4, Z-FY (t-BU)DMK (CtsL inhibitor, 10 mM); lane 5, mouse Cst6 (18 mM); and lane 6, heat inactivation. Note that CA-074 and Cst6 inhibited CtsB (lane 3 and 5). M = precision plus protein dual-color marker. B) The Ki for the inhibition of CtsB by Cst6 was determined by measuring the residual enzymatic activity of a fixed concentration of enzyme, incubated with increasing concentrations of the inhibitor. An Easson-Stedman plot (inset) was used to calculate the Ki, according to the following equation: [I]/1 2 a = (Ki/a) + E0, were I is the inhibitor concentration, E0 is the enzyme concentration at time 0, and a is the fractional activity. The plot yielded a straight line with a Ki slope of 0.98 nM.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Inhibition, Chromosome Transmission Fidelity Colony Color Assay, Activity Assay, Concentration Assay, Incubation

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 4. CtsB and Cst6 colocalize in the mouse HF. A) Immunofluorescence double staining for Cst6 (green) and CtsB (red) in WT mice revealed that colocalization of both proteins was found in the proximal part of the HF as well as the lower region where the bulge area resides. Cst6 is also expressed in the epidermis, whereas no CtsB is observed. Scale bar, 100 mm. B) Cst6 and CtsB expression in mouse epidermis and HFs of Tg(INV-Cst6)Cst6ichq/ichq mice. No immunofluorescence staining for Cst6 was observed in the lower region of the HF (close to the bulge, arrowheads). Scale bar, 100 mm. C) Schematic presentation of INV, Cst6, and CtsB localization in the HF and epidermis of WT mice and the situation in rescued transgenic Tg(INV-Cst6)Cst6ichq/ichq mice.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Double Staining, Expressing, Staining, Transgenic Assay

Journal: The FASEB Journal

Article Title: Cathepsin B as a potential cystatin M/E target in the mouse hair follicle

doi: 10.1096/fj.201700267r

Figure Lengend Snippet: Figure 5. Regulation of epidermal protease activity by CST6. Model of the regulatory role of CST6 in processes that control epidermal cornification, desquamation and HF maintenance. 1) Inhibition of CTSL activity by CST6 is important in the cornification process, as CTSL is the elusive processing and activating enzyme for (TGM)-3. CTSL is also able to process CTSD, which in turn can activate TGM-1. 2) Inhibition of CTSV regulates desquamation, as CTSV is able to degrade (corneo)-desmosomal proteins, such as desmoglein-1, desmocollin-1, and corneodesmosin. As CTSV is expressed only in humans, murine CTSL probably controls the specific functional enzymatic activities of both human CTSL and CTSV. 3) The findings in the present study suggest that inhibition of CtsB by CST6 protects HF maintenance in mice. 4) Inhibition of human LGMN regulates the processing of (pro)-cathepsins; however mouse LGMN is not inhibited by mouse CST6.

Article Snippet: Subsequently, standards, controls, and samples (undiluted up to 323 diluted) were incubated for 1 h, followed by incubation with monoclonal rat anti-mouse Cst6 (R&D Systems) in PBS/1% normal rabbit serum/0.1% bovine serum albumin/0.05% Tween-20 for 30 min. Next, wells were incubated with goat anti-rat biotinylated antibody (Vector Laboratories) for 30min, followed by a final incubation with avidin-biotinylated horseradish peroxidase complex (Vector Laboratories) for 30 min.

Techniques: Activity Assay, Control, Inhibition, Functional Assay