Journal: Nature Communications

Article Title: Branched-chain α-ketoacids are preferentially reaminated and activate protein synthesis in the heart

doi: 10.1038/s41467-021-21962-2

Figure Lengend Snippet: a Correlation of 13 C-valine concentration with Bcat1 , Bcat2, and Slc25a44 mRNA levels across rat heart ( n = 6; red), liver ( n = 5; blue), kidney ( n = 6; green), and gastrocnemius ( n = 5; orange). Data for tissue labeling of 13 C valine was obtained in rats injected with [U- 13 C] KIV, as summarized in Fig. . b Experimental design for the study of AAV-mediated overexpression of SLC25A44 in mouse heart. c Relative mRNA expression of Slc25a44 in isolated perfused hearts following treatment with AAV9-CMV-SLC25A44 ( n = 7; purple) versus AAV9-CMV-GFP ( n = 7; navy). The concentration of 13 C-labeled valine in the heart ( d ), the total rate of formation of 13 C-labeled valine ( e ), perfusate concentration of 13 C-labeled valine ( f ), and concentrations of 13 C-labeled TCA cycle intermediates from [U- 13 C]KIV (100 μM) ( g ) following treatment with AAV9-CMV-SLC25A44 or AAV9-CMV-GFP. h Relative levels of transcripts encoding BCAA transporters in AAV9-CMV-SLC25A44 versus AAV9-CMV-GFP-treated hearts N = 7 per group. i Correlations of Scl25a44 with Slc7a5 mRNA levels in rat heart, liver, kidney, or gastrocneminus (gastroc) muscle. N = 5–6 per group. Data represent mean ± SEM. Statistical differences indicated by Pearson correlation ( a, i ) or a two-way, paired Student’s t -test ( c–h ): * P < 0.05, *** P < 0.0005.

Article Snippet: RNA was reverse transcribed using the Bio-Rad iScript cDNA synthesis kit or High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed with Applied Biosystems TaqMan® gene expression assays for Bcat1 (Rn00568471_m1), Bcat2 (Rn00574455_m1), Slc25a44 (Rn01769831_m1) and rPLP0 (Rn03302271_gH) and PowerUp SYBr Green Master Mix (Applied Biosystem) for all BCAA transporters, 60S acidic ribosomal protein P0 ( Rplp0 ), and Peptidylprolyl Isomerase A ( Ppia ) on a QuantStudio 6 Flex Real-Time PCR system (Applied Biosystems).

Techniques: Concentration Assay, Labeling, Injection, Over Expression, Expressing, Isolation

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A–D) Representative western blots (A and C) and quantitative densitometry analysis (B and D) of INS-1E cells (A and B; n = 6) or isolated human islets (C and D; n = 6) treated with high glucose (22 mM) for 2 days. (E and F) Representative western blots (E) and quantitative densitometry analysis (F) of isolated islets from normal diet (ND) or high-fat/high-sucrose diet (HFD)-fed mice for 16 weeks (n = 8). (G) Representative images shown double immunostaining for PHLPP2 in red and insulin in green in pancreatic sections from ND- and HFD-treated mice. (H and I) Representative western blots (H) and quantitative densitometry analysis (I) of isolated islets from 10-week-old diabetic db/db mice and their heterozygous db /+ littermates (n = 5). (J–L) Human islets transduced with LacZ control or PHLPP1 and PHLPP2 adenoviruses for 48 h. (J) Pooled TUNEL analysis (n = 4; an average of 18,718 β cells were counted from each treatment condition). (K) Insulin secretion during 1 h of incubation with 2.8 mM (basal) and 16.7 mM (stimulated) glucose, normalized to insulin content. (L) Insulin stimulatory index denotes the ratio of stimulated and basal (n = 5). (M) Scheme of the in vivo experimental strategy. 8-week-old male C57BL/6 mice were intraperitoneally (i.p) administrated a mixture of in vivo jetPEI-PHLPP1/2 or −GFP control constructs, one of five injections on every alternate day, and sacrificed after 10 days, one day after the last injection. (N and O) Pooled TUNEL analysis (N) and double staining for TUNEL (red) and insulin (green) (O) of isolated pancreases from GFP- or PHLPP1/2-transfected mice (n = 3; an average of 13,618 β cells were counted from each treatment condition). Data are expressed as means ± SEM. *p < 0.05 compared with untreated or nondiabetic or LacZ or GFP control. White scale bars depict 10 μm.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Western Blot, Isolation, Double Immunostaining, Transduction, Control, TUNEL Assay, Incubation, In Vivo, Construct, Injection, Double Staining, Transfection

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A–D) Representative western blots of isolated human islets (A) and INS-1E cells (B) transduced with LacZ control or PHLPP1 and PHLPP2 adenoviruses for 48 h or (C) transfected with PHLPP1 and/or PHLPP2 siRNA or control siScr for 2 days. (D) Representative western blots of islets isolated from WT and PHLPP1-KO mice (A–D: n = 3). (E–N) Schematic cartoons and representative western blots of INS-1E cells overexpressed with adenoviruses for LacZ (control) or PHLPP1/2 and transfected with GFP or siScr (control), Myr-AKT1 (E and F), HA-tagged AKT-S473D (G and H), kinase dead MST1-K59R (I and J), siRNA to MST1 (K and L), or MST1-T387E (M and N) plasmids (all n = 2). (O and P) Representative images of triple staining for TUNEL (black), insulin (green) and DAPI (blue) (O; scale bar depicts 10 μm) and pooled TUNEL analysis (P) of isolated islets from MST1-KO mice and their WT littermates after transduction with adenoviruses for LacZ (control) or PHLPP1/2 (n = 5–6; an average of 15,301 β cells were counted from each treatment condition). (Q) Pooled TUNEL analysis of isolated human islets overexpressed with adenoviruses for LacZ (control) or PHLPP1/2 and transfected with GFP (control) or Myr-AKT1 or HA-tagged AKT-S473D, or MST1-T387E plasmids (n = 3; an average of 14,034 β cells were counted from each treatment condition). Data are expressed as means ± SEM. *p < 0.05 compared with LacZ control. **p < 0.05 MST1-KO-PHLPP or PHLPP-AKT-S473D or PHLPP- MST1-T387E compared with WT-PHLPP or PHLPP-GFP.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Western Blot, Isolation, Transduction, Control, Transfection, Staining, TUNEL Assay

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A and B) Representative western blots of INS-1E cells (A) and isolated human islets (B) pre-treated with 100 nM rapamycin and cultured with 22.2 mM glucose for 2 (INS-1E) and 3 (human islets) days. n = 3. (C and D) Representative western blots of INS-1E cells transfected with siS6K1 (C), siRaptor (D) or siScr and then exposed to 22.2 mM glucose for 2 days. n = 3 (E) Representative western blots of INS-1E cells transfected with active S6K1 or GFP control plasmids for 2 days. n = 3. (F) Experimental strategy of the AHARIBO-based isolation of active polyribosomes and associated RNAs. (G and H) qPCR measurement of PHLPP1 or PHLPP2 mRNA associated with polysomes of INS-1E cells treated with high glucose (G) (n = 3) or of isolated islets from mice fed for 16 weeks with a ND or HFD (H) (n = 2 independent experiments; each pooled from 8–10 mice/condition). Data are expressed as means ± SEM. *p < 0.05 compared with untreated controls.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Western Blot, Isolation, Cell Culture, Transfection, Control

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A and B) TUNEL analysis of isolated islets from PHLPP1-KO mice and their WT littermates (A) and of isolated human islets transfected with PHLPP1 and/or PHLPP2 siRNA or control siScr (B) and then treated with 22.2 mM glucose plus 0.5 mM palmitate (HG/Pal) or the mixture of 2 ng/mL IL1β plus 1,000 U/mL IFN-γ (IL/IF) for 3 days. n = 4–9. (C–R) PHLPP1-KO and WT control mice injected with streptozotocin (STZ; 40 mg per kg body weight) or saline for 5 consecutive days (n = 6–7). (C) Random-fed blood glucose measurements after first saline or STZ injection (day 0) over 30 days and (D) respective area-under-the curve (AUC) analyses. (E) i.p. glucose tolerance test (GTT) and (F) respective AUC analyses in PHLPP1-KO and WT mice. (G) Insulin levels during an i.p. GTT measured before (0 min) and 15 min after glucose injection and expressed (H) as the ratio of secreted insulin at 15 to 0 min (stimulatory index). (I) Ratio of secreted insulin and glucose calculated at the fed state. (J) Insulin-positive area and (K) β-cell mass (given as the percentage of insulin-positive to the entire pancreatic section area from 10 sections spanning the width of the pancreas). (L–O) Quantitative analyses and representative images from triple staining for Ki67 (L and M; an average of 11,609 β cells were counted from each treatment condition) or TUNEL (N and O; an average of 12,733 β cells were counted from each treatment condition), insulin, and DAPI; expressed as the percentage of TUNEL- or Ki67-positive β cells ± SEM (P and Q) Quantitative analyses (P) and representative images (Q) of the percentage of α cells (red) and β cells (green). (R) Representative double-staining for Glut2 (green), and insulin (red). Data are expressed as means ± SEM. + p < 0.05 versus untreated control. # p < 0.05 PHLPP1-KO or siPHLPP1/2 versus WT or siScr at the same treatment conditions. *p < 0.05 WT-STZ compared with WT saline-injected mice, **p < 0.05 PHLPP1-KO-STZ versus WT-STZ mice. White scale bars depict 10 μm.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: TUNEL Assay, Isolation, Transfection, Control, Injection, Saline, Staining, Double Staining

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A–C) PHLPP1-KO and WT control mice were fed an ND or an HFD (“Surwit”) for 17 weeks. (A) Random-fed blood glucose, (B) i.p. GTT, and (C) respective AUC analyses. n = 8–22. (D and E) Insulin secretion during an i.p. GTT measured before (0 min), 15 and 30 min after glucose injection and expressed as (E) the ratio of secreted insulin at 15 to 0 min (stimulatory index) (n = 7–21). (F and G) Islets were isolated from all four treatment groups, cultured overnight, and subjected to an in vitro GSIS (F). Insulin secretion during 1 h of incubation with 2.8 mM (basal) and 16.7 mM glucose (stimulated), normalized to insulin content, and (G) the stimulatory index denotes the ratio of stimulated to basal insulin secretion (n = 10–11). (H) Islets isolated from HFD-fed groups transduced with LacZ control or PHLPP1 adenoviruses for 1 day and subjected to an in vitro GSIS (n = 4–6). (I–K) Insulin-positive area (I) and β-cell mass (J) (given as percentage of the insulin-positive area to the entire pancreatic section from 10 sections spanning the width of the pancreas) and (K) respective representative images (n = 3–4; yellow scale bar depicts 50 μm). (L–N) Quantitative analyses from double/triple staining for Ki67 (L) or TUNEL (M) (and representative images: N; white scale bar scale depicts 10 μm), insulin, and DAPI expressed as percentage of Ki67- or TUNEL-positive β cells (n = 3–4; an average of 7,648 [Ki67] or 9,009 [TUNEL] β cells were counted from each treatment condition). (O and P) Representative western blots (O) and quantitative densitometry analysis (P) of isolated islets from WT and PHLPP1-KO mice fed an ND or an HFD (n = 3–4). Islet samples isolated from mice under ND or HFD were run on different gels. For each cohort, protein expression signal is normalized to the corresponding WT mice and quantitative densitometry analysis as a fold of the change is presented separately. (Q and R) Representative western blots (Q) and quantitative densitometry analysis (R) of islets isolated from ND- and HFD-fed mice treated with 100 nM rapamycin (PHLPP1/PHLPP2, n = 7; pMST1, n = 3). Data are expressed as means ± SEM. *p < 0.05 WT-HFD versus WT-ND mice, **p < 0.05 PHLPP1-KO-HFD versus WT-HFD mice, ***p < 0.05 PHLPP1-KO-ND versus WT-ND, + p < 0.05 HFD-PHLPP1-KO-AdPHLPP1 versus HFD-PHLPP1-KO-LacZ, # p < 0.05 rapamycin-HFD versus control-HFD.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Control, Injection, Isolation, Cell Culture, In Vitro, Incubation, Transduction, Staining, TUNEL Assay, Western Blot, Expressing

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet: (A and B) Representative western blots (A) and quantitative densitometry analysis (B) of human isolated islets from non-diabetic controls (n = 9) and patients with T2D (n = 8). (C) Representative images of double immunostaining for PHLPP2 in red and insulin in green of pancreatic autopsy sections from non-diabetic controls (n = 4) and patients with T2D (n = 4; scale bar depicts 10 μm). (D–F) Isolated human islets from non-diabetic individuals (D) and patients with T2D (E and F) were transfected with PHLPP1 and/or PHLPP2 siRNA or control siScr for 2 days. (D and E) Insulin secretion during 1 h of incubation with 2.8 mM (basal) and 16.7 mM (stimulated) glucose, normalized to insulin content (n = 3 controls; n = 5 T2D; each from three independent replicates, respectively). (F) Pooled TUNEL analysis (n = 4; each from three independent replicates, an average of 2,515 β cells were counted from each treatment condition). (G and H) Representative western blots (G) and quantitative densitometry analysis (H) of human isolated islets from patients with T2D transfected with raptor siRNA or control siScr for 2 days (n = 3). Data are expressed as means ± SEM. *p < 0.05 T2D versus control islets. **p < 0.05 siPHLPP1/2-transfected stimulated versus siPHLPP1/2-transfected basal. # p < 0.05 siPHLPP1/2- or siRaptor-transfected compared with siScr-transfected T2D islets.

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Western Blot, Isolation, Double Immunostaining, Transfection, Control, Incubation, TUNEL Assay

Journal: Cell reports

Article Title: Inhibition of PHLPP1/2 phosphatases rescues pancreatic β-cells in diabetes

doi: 10.1016/j.celrep.2021.109490

Figure Lengend Snippet:

Article Snippet: TaqMan® Gene Expression Assays were used for PHLPP1 (#Hs01597875_m1), PHLPP2 (#Hs00982295_m1), PPIA (#Hs99999904_m1), and TUBA1A (#Hs00362387_m1) for human, Phlpp1 (#Mm01295850_m1), Phlpp2 (#Mm01244267_m1), Ppia (#Mm03024003_g1), and Tuba1a (#Mm00846967_g1) for mouse, and Phlpp1 (#Rn00572211_m1), Phlpp2 (#Rn01431647_m1), Ppia (#Rn00690933_m1) and Tuba1a (#Rn01532518_g1) for rat. qPCR was performed and analyzed by the Applied Biosystems StepOne Real-Time PCR system.

Techniques: Virus, Plasmid Preparation, Recombinant, Western Blot, Reverse Transcription, Transfection, In Vivo, Bicinchoninic Acid Protein Assay, Enzyme-linked Immunosorbent Assay, In Situ, Isolation, Knock-Out, Gene Expression, Mutagenesis, Software, Real-time Polymerase Chain Reaction