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

Journal: EMBO Molecular Medicine

Article Title: LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy

doi: 10.15252/emmm.202114511

Figure Lengend Snippet: A The JAK inhibitor baricitinib (Ba) blocked LIF‐induced STAT3 phosphorylation. BCE cells were preincubated with DMSO, baricitinib (2 μM), cobimetinib (Co) (150 nM), or BEZ235 (BE) (5 nM) for 1 h and were then treated with vehicle or LIF (10 ng/ml, Sigma) for 15 min. Ctrl, no preincubation with inhibitors. B Baricitinib suppressed LIF‐induced BCE cell growth. BCE cells were preincubated with DMSO, baricitinib, cobimetinib, or BEZ235 for 1 h and then treated with vehicle, LIF (10 ng/ml), or VEGF (10 ng/ml). Cell proliferation was analyzed after 6 days, n = 3. C, D STAT3 knockdown in BCE cells. BCE cells were transfected with siNegative and siRNAs targeting STAT3. qRT‐PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and are presented in C. In D, cells transfected with siRNAs were treated with LIF (10 ng/ml) or vehicle for 15 min. Whole‐cell lysates were subjected to Western blotting with the indicated antibodies. E LIF‐induced BCE cell growth was abolished by STAT3 knockdown. BCE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) or vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading at 590 nm for each vehicle group was set as 1, n = 3. siNegative, negative control siRNA not targeting any known genes. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. Two‐way ANOVA was used as statistical test. Source data are available online for this figure.

Article Snippet: TaqMan gene expression assay probes were used: bovine RPLP0 (Bt03218086_m1), STAT3 (Bt03259865_m1), CTSL1 (Bt03257307_m1 and Bt03257309_m1), CTSB (Bt03259161_m1), MYC (Bt03260377_m1), JunB (Bt03246919_s1), CCNA2 (Bt03240503_g1), CCNB1 (Bt03237853_g1), and PIM1 (Bt03212957_m1).

Techniques: Phospho-proteomics, Knockdown, Transfection, Quantitative RT-PCR, Western Blot, Cell Culture, Fluorescence, Negative Control

Journal: EMBO Molecular Medicine

Article Title: LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy

doi: 10.15252/emmm.202114511

Figure Lengend Snippet: A Recombinant human LIF inhibited growth of BAE cells in a dose‐dependent manner. BAE cells were cultured in the presence of vehicle and indicated concentrations of recombinant human LIF (rhLIF). Cell proliferation was analyzed after 6 days, n = 3. B JAK inhibitor baricitinib blocked activation of STAT3 by LIF. BAE cells preincubated with DMSO and inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml) for 15 min. Whole‐cell lysates were subjected to Western blotting with indicated antibodies. Ctrl, no preincubation with inhibitors; Ba, baricitinib (2 μM); Co, cobimetinib (150 nM); BE, BEZ235 (5 nM). C The JAK inhibitor baricitinib reversed LIF‐induced BAE growth inhibition. BAE cells preincubated with inhibitors for 1 h were treated with vehicle and LIF (10 ng/ml, Sigma). Cell proliferation was analyzed after 6 days using alamar blue, n = 3. D, E Knockdown of STAT3 in BAE cells. BAE cells were transfected with siRNAs targeting STAT3. qRT‐PCR was performed to examine STAT3 mRNA levels. STAT3 level in siNegative was set as 1. Data from three independent experiments were averaged and shown in D. In E, cells transfected with siRNAs were treated with LIF (10 ng/ml, Sigma) and vehicle for 15 min. Whole‐cell lysates were subjected to Western blotting with indicated antibodies. F LIF‐induced BAE cell growth inhibition was abolished by knockdown of STAT3. BAE cells with STAT3 knockdown were cultured with LIF (10 ng/ml, Sigma) and vehicle. Cell proliferation was analyzed after 3 days. Fluorescence reading for each vehicle group was set as 1, n = 3. Data information: Bars and error bars represent mean ± SD. All experiments were carried out in three independent studies. siNegative, negative control siRNA not targeting any known genes. Two‐way ANOVA was used as statistical test. Source data are available online for this figure.

Article Snippet: TaqMan gene expression assay probes were used: bovine RPLP0 (Bt03218086_m1), STAT3 (Bt03259865_m1), CTSL1 (Bt03257307_m1 and Bt03257309_m1), CTSB (Bt03259161_m1), MYC (Bt03260377_m1), JunB (Bt03246919_s1), CCNA2 (Bt03240503_g1), CCNB1 (Bt03237853_g1), and PIM1 (Bt03212957_m1).

Techniques: Recombinant, Cell Culture, Activation Assay, Western Blot, Inhibition, Knockdown, Transfection, Quantitative RT-PCR, Fluorescence, Negative Control

Journal: EMBO Molecular Medicine

Article Title: LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy

doi: 10.15252/emmm.202114511

Figure Lengend Snippet: A, B Intravitreal injection of LIF increases blood vessel density in the mouse eyes. Adult mice were intravitreally injected with VEGF (10 ng) or LIF (10–100 ng, Sigma). Seven days after injection, PFA‐fixed choroid–sclera complexes and retina were subjected to CD31 IF. Representative images of CD31‐positive vessels are shown in A. Scale bar = 100 µm. Vascular density, determined with ImageJ software, is shown in B), n = 5–8. C, D OCT‐A imaging of LIF‐treated mouse retina. Adult mice were intravitreally injected with 1 μl of LIF (50 ng, Sigma) or vehicle solution (PBS). Retinal OCT‐A images were obtained 7 days after the injection and representative images are shown. Blood vessel density was determined as percentage of vessel‐covered area/total area surface using ImageJ software and shown in D), n = 7–8. E STAT3 was phosphorylated in retinal ECs after LIF treatment. Fifty nanogram of our in‐house LIF with low endotoxin levels was injected. Phospho‐Stat3 was examined on cryosection of retina after 2 h by IHC. Magnification in the dashed box is shown in the lower panel. n = 5. Scale bar = 100 µm. F Five‐day‐old neonatal mice were intravitreally injected with LIF (50 ng) or vehicle solution (PBS). After 3 days, retinas were subjected to IF staining with Dyight‐488‐labeled lectin. Representative images for similar ocular loci and quantification of lectin‐labeled area using ImageJ software were shown, n = 4. Scale bar = 100 µm. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two‐way ANOVA was used as statistical test. Source data are available online for this figure.

Article Snippet: TaqMan gene expression assay probes were used: bovine RPLP0 (Bt03218086_m1), STAT3 (Bt03259865_m1), CTSL1 (Bt03257307_m1 and Bt03257309_m1), CTSB (Bt03259161_m1), MYC (Bt03260377_m1), JunB (Bt03246919_s1), CCNA2 (Bt03240503_g1), CCNB1 (Bt03237853_g1), and PIM1 (Bt03212957_m1).

Techniques: Injection, Software, Imaging, Staining, Labeling

Journal: EMBO Molecular Medicine

Article Title: LIF, a mitogen for choroidal endothelial cells, protects the choriocapillaris: implications for prevention of geographic atrophy

doi: 10.15252/emmm.202114511

Figure Lengend Snippet: Q‐PCR (Taqman) analysis of LIF expression in BCE cells and bovine pericytes (bPericyte). Bovine LIF ELISA of BCE cells and bovine pericyte. Cells were cultured for 48 h before harvesting the medium. LIFR expression in BCE cells and bovine pericyte by qPCR (Taqman). Image of FACS selection of CD13 + /CD45 − and CD31 + /CD45 − cells. Immunostaining for CD31, CD13, and DAPI in isolated primary mouse cells before and after FACS selection. Mouse LIF expression in FACS‐selected cells. Human choroidal EC (HCEC) and mouse retinal EC (MREC) were treated with LIF 10 ng and LIF 100 ng for 30 min. (PBS was used as control). STAT3 phosphorylation was detected by Western blot. H&E staining of LIFR and CD31 in mouse eye sections. The magnified images of the dashed line boxes, a and b, are shown on the right panel. Box a shows LIFR staining in retina and box b showed LIFR staining in RPE/choroid layer. Whole‐mount staining of LIFR and CD31 in mouse retina. The fluorescent staining for CD31 (green) and LIFR (red) is shown. White arrows indicate the co‐staining of CD31 and LIFR on retinal ECs. Data information: Bars and error bars represent mean ± SEM. All experiments were carried out in three independent studies. Two‐way ANOVA was used as statistical test.

Article Snippet: TaqMan gene expression assay probes were used: bovine RPLP0 (Bt03218086_m1), STAT3 (Bt03259865_m1), CTSL1 (Bt03257307_m1 and Bt03257309_m1), CTSB (Bt03259161_m1), MYC (Bt03260377_m1), JunB (Bt03246919_s1), CCNA2 (Bt03240503_g1), CCNB1 (Bt03237853_g1), and PIM1 (Bt03212957_m1).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Cell Culture, Selection, Immunostaining, Isolation, Control, Phospho-proteomics, Western Blot, Staining

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: RNA sequencing reveals that tomatidine modulates ATF4-dependent ER stress genes in pancreatic cancer cells Human and murine PDAC cell lines were treated for 40 h with 6.4 μg/mL tomatidine, RNA was isolated, and RNA sequencing was performed to analyze differences in gene regulation. N = 3 biological separate experiments. (A–D) (A) Heatmap of all genes and how they change in tomatidine-treated vs. untreated cells for Panc1 cells (FDR<0.05). Volcano plot highlighting ER stress-related genes in (B) Panc1 and (C) MT5 cells. (Upregulated genes on the right of the central axis and vice versa with higher fold change as we go away from the origin on y axis) (D) IPA upstream analysis of ATF4-related genes in Panc1 cells. (E) Heatmap elucidating targeting of ATF4-related genes in treated vs. untreated Panc1 cells. (Fold change: +2.5 to −1.5; FDR<0.03). (F) Top 10 pathways focusing on UPR, ATF4, and ER stress via Reactome analysis of the RNA-sequencing data of treated vs. untreated samples for Panc1 cells. (FDR< 0.03).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: RNA Sequencing, Isolation

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: ATF4 expression in PDAC (A) KM plotter was utilized to examine ATF4 expression in human PDAC specimens examining high vs. low expression compared to overall survival. (B) Data distribution elucidating significance of the overall survival curve prepared by KM Plotter. (C and D) (C) scRNA-seq datasets of pancreatic cancer tissue from metastatic patients were obtained from NIH dbGAP (accession phs002045.v1.p1) and (D) analyzed for ATF4 expression. (See also Table S1 ). (E) Pancreatic tumor tissue from KPC mice were stained by multiplex IF and imaged using Akoya Vectra Polaris and the Phenochart software to analyze ATF4 in multiple cellular compartments. Markers used to determine ATF4 (white), epithelial cells (CK19; red), stroma (αSMA; orange and PDGFRβ; Green), CD8 T+ cells (yellow), and macrophages (F4/80; turquoise). Scale bar = 100 μm.

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Staining, Multiplex Assay, Software

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine inhibits ATF4-dependent signaling in PDAC (A and B) (A) MiaPaca-2 tumor cells were treated with tomatidine for 72 h and cell lysates were immunoblotted for ATF4, 4EBP1, and phospho-4EBP1(p-4EBP1) protein expression and (B) p-4EBP1/4EBP1 levels quantified by densitometry. (C) Immunofluorescence (IF) was performed on vehicle (DMSO) or tomatidine-treated Panc1 cells to track ATF4 (FITC-Green) translocation from nucleus (DAPI-Blue) to cytoplasm. Scale bar = 50 μm. (D–G) (D) Nuclear to cytoplasmic translocation was quantified. Panc1 cells were treated with vehicle (DMSO) or tomatidine for 40 h and ATF4 transcriptional activity was analyzed by chromatin immunoprecipitation (ChIP) qPCR evaluating binding of ATF4 to the downstream promoter regions of (E) eIF4EBP1 (F) CHOP- B site and (G) ASNS. Data are reported as the means + SEMs. n = 3 or more independent biological replicates (4B, One-way ANOVA with Tukey’s test for pairwise comparisons was used to analyze the data; 4D-G, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Expressing, Immunofluorescence, Translocation Assay, Activity Assay, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: In vivo tomatidine treatment inhibits pancreatic tumor growth (A–C) (A) MT5 tumor-bearing C57BL/6 mice (5 mice/group) were treated with 5 mg/kg daily i.p. injections of tomatidine or vehicle control (40% HPBCD) and monitored for tumor growth. (n = 5/group). RNA isolated from the tumor tissues of MT5 tumor-bearing C57BL/6 mice treated with vehicle or 5 mg/kg daily i.p. injections of tomatidine were assessed for (B) ATF4 and (C) eIF4EBP1 expression via qPCR. Data are reported as the means + SEMs. n = 5 mice per group. (5A, mixed between-within subjects ANOVA shows a significant interaction between days of treatment and group; 5B-C, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: In Vivo, Control, Isolation, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet: Tomatidine mediated inhibition of ATF4 signaling can increase sensitivity to ferroptotic cell death in PDAC (A) Panc-1 tumor cells were treated with tomatidine (6.4 μg/mL) and analyzed by RNA sequencing. Ingenuity pathway analysis of the regulated genes suggested ferroptosis as a top hit for tomatidine-treated cells. (B–D) (B) Pancreatic cancer cells were treated with vehicle (DMSO), erastin (to induce ferroptosis), ferrostatin-1 (to inhibit ferroptosis), tomatidine, or in different combinations and lipid peroxidation of (C) Panc-1 and (D) MiaPaca-2 was analyzed by flow cytometry using Bodipy-11. (E) Panc-1 and MiaPaca-2 cells treated with vehicle (DMSO) or tomatidine and lysates collected after 24 h were immunoblot for GPX4 expression. (F) Panc1 cells were plated overnight and treated with tomatidine for 6 h and then assayed by Seahorse assay to analyze mitochondrial fitness. (G) Schematic showing how tomatidine can regulate ATF4-dependent signaling to induce ferroptosis in pancreatic cancer. Data are reported as the means + SEMs. n = 3 or more independent biological replicates. (7C-D, Two-tailed independent student’s test was used to analyze the data; 7F, Two-tailed independent student’s test was used to analyze the data, ∗p < 0.05, ∗∗p < 0.007).

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Inhibition, RNA Sequencing, Flow Cytometry, Western Blot, Expressing, Two Tailed Test

Journal: iScience

Article Title: Tomatidine targets ATF4-dependent signaling and induces ferroptosis to limit pancreatic cancer progression

doi: 10.1016/j.isci.2023.107408

Figure Lengend Snippet:

Article Snippet: The cDNA was evaluated for ATF4 and eIF4EBP1 mRNA expression using TaqMan Universal Master Mix II, no UNG (Life Technologies, Carlsbad, CA) and Taqman Gene expression assay primers for ATF4 (Hs00909569_g1), and eIF4EBP1 (Hs00607050_m1).

Techniques: Control, Recombinant, Viability Assay, Luciferase, ChIP-qPCR, Gene Expression, Software

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Elucidation of mechanisms of AC0010 resistance by RNAseq profiling. Total RNA was isolated and subjected to RNAseq profiling as detailed in the Materials and Methods section. Shown are genes in p53 and TGFβ pathways enriched in H1975-P1 cells (A), and apoptosis and NFκB pathways in H1975-AVR1 cells (B). Real-time PCR analysis to confirm increased expression of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells, respectively (C). Western blot analysis of the levels of c-MET and BCL-2 in H1975-P1-R1 and H1975-AVR1-R2 cells as well as the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) (D).

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Isolation, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in cell culture model. (A) H1975-P1-R1 cells were transfected with siRNA targeting c-MET, along with the control siRNA, and the sensitivity of transfected cells to AC0010 was determined by WST-1 assay. Western blot analysis was performed to show c-MET knockdown. (B) Growth curve of H1975-P1-R1 cells treated with various concentrations of crizotinib. (C) Growth curve of H1975-P1-R1 cells treated with various concentrations of AC0010 in combination with 2 μM crizotinib or 0.5 μM crizotinib ( n = 3). (D) Growth curve of H1975-P1-R1 cells treated with 1 μM AC0010 in combination with various concentrations of crizotinib ( n = 3). (E) Clonogenic survival assays of H1975-P1-R1 cells treated with AC0010, criztonib, or the combination with indicated concentrations ( n = 3). (F) Western blot analysis of the phosphorylation status of c-MET, EGFR, and their downstream molecules (AKT1 and ERK) in H1975-P1-R1 cells treated with indicated drugs and concentrations.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: Cell Culture, Transfection, Control, WST-1 Assay, Western Blot, Knockdown, Phospho-proteomics

Journal: Neoplasia (New York, N.Y.)

Article Title: Overcoming Resistance to AC0010, a Third Generation of EGFR Inhibitor, by Targeting c-MET and BCL-2

doi: 10.1016/j.neo.2018.11.004

Figure Lengend Snippet: Overcoming AC0010 resistance by targeting c-MET in in vivo xenograft model. (A) Inhibition of H1975-P1-R1 tumor growth by AC0010 or crizotinib, alone or in combination, for a period of 14 days. Tumor growth curves are plotted as mean ± SEM ( n = 8). (B) Tumor weights of each group were analyzed using SPSS 17.0 software; t test statistical analysis. (C) Mouse body weights of each group are plotted as mean ± SEM ( n = 8). (D) H1975-P1-R1 tumor issues from mice treated with vehicle, AC0010, crizotinib, or AC0010 combined with crizotinib for 14 days were isolated and subjected to Western blotting analysis using indicated Abs.

Article Snippet: The expression of c-MET, BCL-2, and CDH11 was measured using the TaqMan Gene Expression Assay (Hs01565584_m1, Hs00608023_m1, and Hs00901479_m1, Applied Biosystems).

Techniques: In Vivo, Inhibition, Software, Isolation, Western Blot

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: Downregulated genes in miR- 1 and miR-133a transfectants

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques:

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: PNP expression was suppressed by miR-1 and miR-133a transfection at both the mRNA and protein levels in PCa cell lines, PC3 and DU145. ( A ) PNP mRNA expression after 72 h transfection with miR-1 and miR-133a . GAPDH expression was used for normalisation. ( B ) PNP protein expression after 72 h transfection of miRNAs. GAPDH was used as a loading control. The expression ratio of PNP/GAPDH was evaluated using ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.htmL ).

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Expressing, Transfection, Control, Software

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: miR-1 and miR-133a binding sites in 3′UTR of PNP mRNA. Luciferase reporter assay using the four types of vector encoding the putative miR-1 and miR-133a target sites; Position 365–371(+):Position 407–413 (+), deletion of putative miR-1 target site; Position 365–371(−):Position 407–413 (+), deletion of putative miR-133a target site; Position 365–371(+):Position 407–413 (−), deletion of putative target sites of both miR-1 and miR-133a ; Position 365–371(−):Position 407–413 (−). The Renilla luciferase values were normalised to firefly luciferase values. * P <0.01.

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Binding Assay, Luciferase, Reporter Assay, Plasmid Preparation

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: PNP expression was suppressed by si-PNP-1 and si-PNP-2 transfection at both the mRNA and protein levels in PCa cell lines, PC3 and DU145. ( A ) PNP mRNA expression 72 h after transfection of the si-RNAs. GAPDH expression was used for normalisation. ( B ) PNP protein expression 72 h after transfection of the si-RNAs. GAPDH was used as a loading control. The expression ratio of PNP/GAPDH was evaluated using ImageJ software (ver. 1.43; http://rsbweb.nih.gov/ij/index.htmL ).

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Expressing, Transfection, Control, Software

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: Effects of PNP knockdown by si- PNP transfection on PCa cell lines, PC3 and DU145. ( A ) Cell proliferation determined by the XTT assay; ( B ) cell migration activity determined by the wound-healing assay; and ( C ) cell invasion activity determined by the Matrigel invasion assay. ** P <0.0001. * P <0.01.

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Knockdown, Transfection, XTT Assay, Migration, Activity Assay, Wound Healing Assay, Invasion Assay

Journal: British Journal of Cancer

Article Title: Tumour suppressors miR-1 and miR-133a target the oncogenic function of purine nucleoside phosphorylase ( PNP ) in prostate cancer

doi: 10.1038/bjc.2011.462

Figure Lengend Snippet: Immunohistochemical staining of PNP in PCa ( n =60), PIN ( n =10), and normal prostatic tissue ( n =10) by tissue microarray (left panel, original magnification × 100; right panel, original magnification × 400). ( A ) Strongly stained tumour lesion (Gleason Score 3+4, pT3N0); ( B ) weakly stained PIN lesion; and ( C ) negative staining in hyperplastic tissue without malignancy. Right side of the figure: Quantification of PNP expression. Expression of PNP was upregulated in PCa specimens compared with normal hyperplastic tissue ( P =0.0037).

Article Snippet: Thermal cycling conditions were as follows: 95°C for 10 min, 40 cycles of 95°C for 15 s and 63°C for 1 min. TaqMan probes and primers for PNP (assay ID: Hs00165367_m1) and human GAPDH (assay ID: Hs0329097_g1) were obtained from Applied Biosystems (Assay-On-Demand Gene Expression Products).

Techniques: Immunohistochemical staining, Staining, Microarray, Negative Staining, 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–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: Chronic metabolic stress leads to hyper-activation of mTORC1, promoting the PHLPP translational machinery, which leads to the triangle loop of PHLPP activity, AKT inhibition, and MST1 activation and, ultimately, to β-cell death and dysfunction.

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: Activation Assay, Activity Assay, Inhibition

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

Journal: Cell reports

Article Title: A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice

doi: 10.1016/j.celrep.2019.11.062

Figure Lengend Snippet: (A) Representative western blotting image of mouse RPS28 protein from Hepa 1–6 cells 24 h post-transfection (n = 4 independent experiments). GAPDH, loading control.

Article Snippet: 500 ng of total RNA was reverse transcribed with the superscript IV RT kit (Thermo Fisher) and subjected to gene expression analyses with gene-specific TaqMan probes (Mm99999915_g1 for GAPDH and Mm04203728_gH for mouse Rps28 ).

Techniques: Western Blot, Transfection, Control

Journal: Cell reports

Article Title: A tRNA-Derived Small RNA Regulates Ribosomal Protein S28 Protein Levels after Translation Initiation in Humans and Mice

doi: 10.1016/j.celrep.2019.11.062

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 500 ng of total RNA was reverse transcribed with the superscript IV RT kit (Thermo Fisher) and subjected to gene expression analyses with gene-specific TaqMan probes (Mm99999915_g1 for GAPDH and Mm04203728_gH for mouse Rps28 ).

Techniques: Recombinant, Proliferation Assay, Northern Blot, Mutagenesis, Software

Journal: Emerging Microbes & Infections

Article Title: Modelling Toxoplasma gondii infection in human cerebral organoids

doi: 10.1080/22221751.2020.1812435

Figure Lengend Snippet: Distribution of Toxoplasma gondii in the human cerebral organoids. (A) Schematic representation of the life cycle of T. gondii . (B) 3D images of a cerebral organoid infected with T. gondii (green). (C – F) Representative fluorescence images of cerebral organoids infected with 2 strains of T. gondii : ME49 (top) and RH (bottom) infected cerebral organoids are shown stained for (C) TUJ1, a neuronal marker; (D) GFAP, an astrocyte marker; (E) O1, an oligodendrocyte marker; and (F) SOX2, a radial glial cell marker. Scale bars, as indicated.

Article Snippet: Tachyzoites of the T. gondii PTG-GFP 5 S65 T, haplogroup 2, ME49 strain (50941, ATCC, Manassas, VA, USA) and those of the T. gondii RH-GFP strain were maintained in vitro using Vero cells (CCL-81, ATCC) cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .

Techniques: Infection, Fluorescence, Staining, Marker

Journal: Emerging Microbes & Infections

Article Title: Modelling Toxoplasma gondii infection in human cerebral organoids

doi: 10.1080/22221751.2020.1812435

Figure Lengend Snippet: . T. gondii cyst formation in human cerebral organoids. Representative fluorescence image of cyst-like structures in an organoid infected with (A) ME49 and (B) RH. Images of transmission electron microscopy of (C–D) ME49- and (E) RH-infected cerebral organoids. Scale as indicated with the bar. PVM, parasitophorous vacuole membrane; Nu, nucleus; Rh, rhoptry; Co, conoid; Mt, mitochondrion; Dg, dense granule; and Am, amylopectin.

Article Snippet: Tachyzoites of the T. gondii PTG-GFP 5 S65 T, haplogroup 2, ME49 strain (50941, ATCC, Manassas, VA, USA) and those of the T. gondii RH-GFP strain were maintained in vitro using Vero cells (CCL-81, ATCC) cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .

Techniques: Fluorescence, Infection, Transmission Assay, Electron Microscopy, Membrane

Journal: Emerging Microbes & Infections

Article Title: Modelling Toxoplasma gondii infection in human cerebral organoids

doi: 10.1080/22221751.2020.1812435

Figure Lengend Snippet: Virulence of T. gondii in the infected cerebral organoids . (A) Schematic representation of the experimental design: T. gondii isolated from infected cerebral organoids was injected into mice. The levels of T. gondii P30 protein were measured by ELISA ( n = 5, biologically independent mice) 2 months postinfection. (B) ME49 and (C) RH antibody titre presented as the optical density of ELISA . * p < 0.05. Quantitative data are expressed as the mean ± S.E.M. of at least 3 independent experiments.

Article Snippet: Tachyzoites of the T. gondii PTG-GFP 5 S65 T, haplogroup 2, ME49 strain (50941, ATCC, Manassas, VA, USA) and those of the T. gondii RH-GFP strain were maintained in vitro using Vero cells (CCL-81, ATCC) cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .

Techniques: Infection, Isolation, Injection, Enzyme-linked Immunosorbent Assay

Journal: Emerging Microbes & Infections

Article Title: Modelling Toxoplasma gondii infection in human cerebral organoids

doi: 10.1080/22221751.2020.1812435

Figure Lengend Snippet: Transcriptome analysis of ME49 post-infection. (A) Differentially expressed genes of ME49 post-infection. Noninfectious ME49 was used as a control (|fc| ≥ 2, raw p -value < 0.05). A heat map was generated using Cluster grammer ( http://amp.pharm.mssm.edu/clustergrammer/ ). (B) Volume plot showing differential expression of ME49 genes between 0 and 72 h post-infection (|fc| ≥ 2, raw p -value < 0.05). The top 5 gene names are indicated. (C) KEGG pathway analysis was performed using the differentially expressed genes (DEGs) of ME49 (|fc| ≥ 2, raw p -value < 0.05). (D) Gene Ontology (GO) annotation of T. gondii postinfection of cerebral organoids; p -value < 0.01. MF, molecular function; BP, biological process; and CC, cellular component.

Article Snippet: Tachyzoites of the T. gondii PTG-GFP 5 S65 T, haplogroup 2, ME49 strain (50941, ATCC, Manassas, VA, USA) and those of the T. gondii RH-GFP strain were maintained in vitro using Vero cells (CCL-81, ATCC) cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .

Techniques: Infection, Control, Generated, Quantitative Proteomics

Journal: Emerging Microbes & Infections

Article Title: Modelling Toxoplasma gondii infection in human cerebral organoids

doi: 10.1080/22221751.2020.1812435

Figure Lengend Snippet: . Transcriptome analysis of T. gondii -infected cerebral organoids. Heat map showing differentially expressed genes (DEGs) of (A) ME49- and (B) RH-infected cerebral organoids 72 h post-infection (|fc| ≥ 2, raw p -value < 0.05). Noninfected cerebral organoids were used as controls ( n = 3, biologically independent samples). A heat map was generated using Cluster grammer ( http://amp.pharm.mssm.edu/clustergrammer/ ). Gene Ontology analysis of DEGs in the (C) ME49- and (D) RH-infected organoids. Top five Gene Ontology terms ( p -value < 0.005). Volume plot showing the top five genes (marked as red dots) in the (E) ME49- and (F) RH-infected organoids. Significantly upregulated and downregulated genes (|fc| ≥ 2, raw p -value < 0.05) are marked in blue. Noninfected organoids were used as controls. (G) Transcriptome data were analyzed using ingenuity pathway analysis (IPA) software ( p -value < 0.05).

Article Snippet: Tachyzoites of the T. gondii PTG-GFP 5 S65 T, haplogroup 2, ME49 strain (50941, ATCC, Manassas, VA, USA) and those of the T. gondii RH-GFP strain were maintained in vitro using Vero cells (CCL-81, ATCC) cultured in DMEM (Gibco) supplemented with 10% FBS (Gibco) at 37°C in a humidified incubator with an atmosphere of 5% CO 2 .

Techniques: Infection, Generated, Software

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Sequence analysis of tRNAs extracted from HeLa cells treated with DMSO or 1 μM DNMDP for 18 h ( n = 2 replicates), based on the mapping to the genomic tRNA database, GtRNAdb 35 . Scatter plot (left), tRNA abundance in DMSO-treated ( X axis) and DNMDP-treated cells ( Y axis). Red dots, tRNA Leu (TAA) isodecoders. b , Leucine tRNA abundance in a panel of cancer cell lines treated with DMSO or 1 μM DNMDP 3 . After demethylation of tRNA, cDNA was synthesized and leucine tRNA levels were measured using qPCR. tRNA Leu (TAA)-1 level is plotted relative to tRNA Leu (TAG)-2 as a control ( n = 3 replicates per condition). Data are represented as mean ± s.e.m. P values were calculated by unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Sequencing, Synthesized, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , b , Preferential digestion of tRNA Leu (TAA) by SLFN12. 0.5 μg of the indicated tRNAs were digested with ( a ) 2 μM SLFN12 ( n = 2 replicates for tRNA Leu (TAA)-1, tRNA Leu (TAA)-3, tRNA Ser (CGA) and tRNA Cys (GCA), and n = 3 replicates for others) or ( b ) 0.25 μM PDE3A and SLFN12 proteins preincubated with 1.25 μM DNMDP ( n = 3 replicates for tRNA Leu (TAA)-1 and tRNA Leu (TAA)-3, and n = 2 replicates for others). The relative amounts of intact tRNAs were measured and plotted. c , The variable loops or acceptor stems of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 were swapped to generate six different hybrid leucine tRNAs. Sequences of tRNA Leu (TAG)-2 and tRNA Leu (TAA)-3 are indicated by red and black colors, respectively. d , Each leucine tRNA synthesized by in vitro transcription was digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Cleaved tRNA was analyzed on 15% TBE-Urea gels. The results were reproducible and representative images are shown ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). e , The relative amount of intact tRNA, quantified using ImageJ software, is shown at the bottom of the gels and plotted ( n = 2 for hybrids 2, 3, 5 and 6; n = 3 replicates for others). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Software, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: a , Construction of resistant tRNA Leu (CAG:TAA) by mutating the anticodon of tRNA Leu (CAG)-1 to TAA. b , Indicated tRNAs were digested with 0.25 μM PDE3A and 0.25 μM SLFN12 proteins preincubated with 1.25 μM DNMDP. Intact tRNAs are marked with an arrow and were quantified by ImageJ (1.8.0), as reported at the bottom of each lane ( n = 2 replicates). c , HeLa cells were transfected with plasmids expressing tRNA Leu (CAG)-1, tRNA Leu (TAA)-3 or variant tRNA Leu (CAG:TAA) followed by DMSO or 0.5 μM DNMDP treatment. Forty-eight hours after drug treatment, cell viability was assessed by CellTiter-Glo ( n = 3 replicates). Data are represented as mean ± s.e.m. P values were calculated by the unpaired two-tailed Student’s t -test (NS, not significant).

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Expressing, Transfection, Variant Assay, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( a) Indicated tRNAs were synthesized using a T7 RNA polymerase-mediated transcription reaction. Synthetic tRNAs were treated with the indicated concentrations of SLFN12 at 37 °C for 40 minutes (n = 2 replicates for Leu-TAA-1, Leu-TAA-3, Ser-CGA and Cys-GCA; n = 3 replicates for others). ( b ) 0.25 μM PDE3A and SLFN12 proteins pre-treated with 1.25 μM DNMDP were incubated with synthetic tRNAs at 37 °C for 40 minutes (n = 3 replicates for Leu-TAA-1 and Leu-TAA-3; n = 2 replicates for others). ( c ) 0.5 μM ΔSLFN12 (amino acids 1–347) or PDE3A proteins pre-treated with DMSO, 2.5 μM DNMDP, 2.5 μM trequinsin or 2.5 μM estradiol were incubated with synthetic tRNAs at 37 °C for 40 minutes. After incubation, RNA samples were analyzed on a 15% denaturing polyacrylamide gel (n = 2 replicates). Representative figures are shown here. Intact tRNA is indicated with an arrow. The relative amounts of intact tRNA were quantified using ImageJ software and are shown at the bottom. ( d ) Leucine tRNA abundance was measured by qPCR in purified total RNA treated with 0.25 μM SLFN12 recombinant protein. tRNA-Leu-TAA-1 levels are plotted relative to control tRNA, tRNA-Leu-TAG-2 (n = 3 replicates). Data are represented as mean ± SEM. Statistical significance was calculated by a two-tailed unpaired Student’s t test.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: In Vitro, Synthesized, Incubation, Software, Purification, Recombinant, Control, Two Tailed Test

Journal: Nature chemical biology

Article Title: Velcrin-induced selective cleavage of tRNA Leu (TAA) by SLFN12 causes cancer cell death

doi: 10.1038/s41589-022-01170-9

Figure Lengend Snippet: ( a ) Sequences of the indicated tRNAs were aligned in R using the msa package. The consensus logo is shown at the top of the figure, with more conserved positions appearing larger. Positions where the sequences do not agree are blank in the consensus. Structural regions of the tRNA are labeled on the bottom of the alignment. ( b ) The computational model of the structure of SLFN12 bound to tRNA was generated using the HADDOCK software. Both the catalytic SLFN12 monomer (green) and the adaptor SLFN12 monomer (magenta) interact with a single molecule of tRNA-Leu-TAA-3 (orange). The catalytic SLFN12 monomer interacts with the tRNA variable loop region (yellow) and the adaptor SLFN12 monomer interacts with the tRNA acceptor stem (cyan). The active site residues of the catalytic SLFN12 monomer are close to the primary tRNA cleavage site in this model.

Article Snippet: RNAs were synthesized from PCR products using the Transcript Aid T7 High Yield Transcription Kit (ThermoFisher) and purified by extracting full-length tRNAs from 15% TBE-urea gels (ThermoFisher).

Techniques: Labeling, Generated, Software

Journal: Cell Reports Medicine

Article Title: Dominant CD4 + T cell receptors remain stable throughout antiretroviral therapy-mediated immune restoration in people with HIV

doi: 10.1016/j.xcrm.2023.101268

Figure Lengend Snippet:

Article Snippet: Library quantity, purity and size selection were assessed using Qubit fluorometer (Qubit DNA Assay kit, Invitrogen Q32854) and an Agilent Bioanalyzer (D5000 ScreenTape, Agilent 5067–5592) with acceptable library size distribution between 600 and 1150bp.

Techniques: Recombinant, Antibody Labeling, Staining, Cell Isolation, Control, Mass Cytometry, Sequencing, Software