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: 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: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Effect of EFEMP1 knockdown by small interfering RNA (siRNA) on chondrocyte phenotype. A The human chondrocyte cell line C20A4 was transfected with either EFEMP1 siRNA (siEFEMP1) or scrambled siRNA (siCtrl). EFEMP1 and MMP-13 protein expression was analyzed by Western blotting. B Chondrocyte-related gene expression, including SOX9, aggrecan (ACAN), MMP13, type X collagen (COL10A1), and type II collagen (COL2A1), was assessed by quantitative PCR in siEFEMP1- and siCtrl-treated C20A4 cells. C Phosphokinase protein arrays were used to profile phosphorylation changes in intracellular proteins extracted from C20A4 chondrocytes. D Spot intensities exhibiting changes greater than ± 25% between groups were quantified using ImageJ software. Red lines indicate baseline levels from the siCtrl group. E Phosphorylation of p70S6K at threonine 421/serine 424 (T421/T424) was further examined by Western blot analysis. Data are presented as mean ± standard error of the mean (SEM). Statistical significance was determined using an independent-samples t-test. * P < 0.05, ** P < 0.01, and *** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Knockdown, Small Interfering RNA, Transfection, Expressing, Western Blot, Gene Expression, Real-time Polymerase Chain Reaction, Phospho-proteomics, Software

Journal: Journal of Molecular Medicine (Berlin, Germany)

Article Title: Targeting EFEMP1 enhances chondrogenesis and inhibits hypertrophic differentiation in a spontaneous osteoarthritis mouse model

doi: 10.1007/s00109-026-02656-y

Figure Lengend Snippet: Immunofluorescence analysis of cartilage markers following EFEMP1 antibody treatment in STR/ort mice. A Representative immunofluorescence (IF) staining for EFEMP1, SOX9, MMP13, aggrecan fragments, COL2A1, and COL10A1 in the medial femoral condyle (MF) and medial tibial plateau (MT). Nuclei were counterstained with DAPI (blue), and articular cartilage regions are outlined by orange dashed lines. B Quantification of fluorescence intensity in articular cartilage regions of the femoral condyle and tibial plateau, with medial and lateral compartments combined. EFEMP1 and SOX9 signals were normalized to DAPI, whereas MMP13, aggrecan fragments, COL10A1, and COL2A1 signals were quantified as absolute FITC intensity. Data are presented as mean ± standard error of the mean (SEM). Statistical analysis was performed using an independent-samples t -test. P < 0.05, P < 0.01, and ** P < 0.001

Article Snippet: Samples were incubated overnight at 4 °C with primary antibodies against EFEMP1 (1:100, NBP1-77040; NOVUS, USA), SOX9 (1:50, sc-166505; Santa Cruz Biotechnology, USA), MMP-13 (1:100, GTX100665; GeneTex, USA), aggrecan fragments (1:100, AF1220; R&D Systems, USA), COL2A1 (1:100, ab34712; Abcam, UK), and COL10A1 (1:100, GTX37732; GeneTex, USA).

Techniques: Immunofluorescence, Staining, Fluorescence

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: Marveld3 is involved in the radiation-induced ferroptosis of skin cells. A . The viability of HaCaT and WS1 cells were assessed following exposure to varying doses of irradiation at multiple time points by CCK-8 kit. B . HaCaT and WS1 cells were subjected to a 24-hour treatment of Fer-1 following exposure to 10 Gy X-ray irradiation, and the CCK-8 assay was used to detect cell viabilities. C . The cells were treated with erastin for 24 h after 10 Gy X-ray irradiation, and reversal of erastin’s effect on skin cell viability after irradiation was observed upon knockdown of Marveld3 using specific-siRNA. D&E . The contents of ferric ions and MDA were significantly reduced in the Marveld3 knockdown group compared to the group transfected with a negative control siRNA, detected with corresponding kits. F . Confocal images of C11-BODIPY stained HaCaT and WS1 cells showed lipid peroxidation status. G . Western blot assay was used to analyze the expression of the crucial molecules involved in the signaling pathways associated with the defense against ferroptosis after X-ray irradiation. Student’s t tests were used to evaluate differences between groups; * P < 0.05, ** P < 0.01, and *** P < 0.001 compared to the control group

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques: Irradiation, CCK-8 Assay, Knockdown, Transfection, Negative Control, Staining, Western Blot, Expressing, Protein-Protein interactions, Control

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: mRNA expression profiling of skin cells samples subjected to 10 Gy X-ray irradiation. HaCaT and WS1 cells were transfected with Marveld3-targeting siRNA or negative control siRNA, followed by exposure to 10 Gy X-ray. A . The heatmap of RNA-seq data illustrates the differentially expressed genes, with red indicating upregulated genes and blue signifying downregulated genes. B&C . The number of differentially expressed genes between the treatment and control groups in both HaCaT and WS1 cells is shown. D . GO category enrichment of the differentially expressed mRNAs in irradiated skin cells following Marveld3-knockdown. E&F . KEGG pathway enrichment of the differentially expressed mRNAs from irradiated skin cells in response to Marveld3-knockdown

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques: Expressing, Irradiation, Transfection, Negative Control, RNA Sequencing, Control, Knockdown

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: Upregulated mRNAs by silencing Marveld3 in HaCaT cells after 10 gy X-ray irradiation

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques:

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: Upregulated mRNAs by silencing Marveld3 in WS1 cells after 10 gy X-ray irradiation

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques:

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: Downregulated mRNAs by silencing Marveld3 in WS1 cells after 10 gy X-ray irradiation

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques:

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: PRRX2 expression is upregulated in irradiated skin tissue, serving as a stress response. A . The volcano and radar plots of gene profiling between the Marveld3-targeting and negative control siRNA groups in HaCaT and WS1 cells. B&C . Confirmation of PRRX2 expression at both mRNA and protein levels was assessed using real-time PCR and Western blot techniques in Marveld3-knockdown skin cells. D . Immunohistochemical staining was used to detect the PRRX2 expression in both irradiated and nonirradiated rat skin tissues. Rat skin tissues were collected at days 1 and 7 following exposure to 45 Gy irradiation ( n = 3). The AI software was utilized to conduct a quantitative analysis of PRRX2 positive staining in rat skin tissues, encompassing parameters such as the count of positive cells, density of positive cells, and H-Score, in both irradiated and nonirradiated samples

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques: Expressing, Irradiation, Negative Control, Real-time Polymerase Chain Reaction, Western Blot, Knockdown, Immunohistochemical staining, Staining, Software

Journal: Molecular Medicine

Article Title: Upregulation of PRRX2 by silencing Marveld3 as a protective mechanism against radiation-induced ferroptosis in skin cells

doi: 10.1186/s10020-024-00958-w

Figure Lengend Snippet: Downregulated mRNAs by silencing Marveld3 in HaCaT cells after 10 gy X-ray irradiation

Article Snippet: To prevent nonspecific binding, the membranes were blocked with 5% BSA in TBS-Tween-20 (0.1% TBST) at room temperature for 1 h. Subsequently, the membranes were incubated overnight at 4 °C with antibodies against Marveld3 (25667-1-AP, 1:1000), GPX4 (67763-1-Ig, 1:1000), DHODH (14877-1-AP, 1:1000), FSP-1(20886-1-AP, 1:1000), GCH1(20886-1-AP, 1:1000) (all from Proteintech). α-Tubulin (1:1,000 dilution; Beyotime, Jiangsu, China) was employed as a loading control.

Techniques:

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Elevated extracellular and reduced cell associated Clusterin in Idiopathic Pulmonary Fibrosis. ( A ) Clusterin gene expression was quantitated using RT-PCR in lung tissue from healthy control lung tissue (n = 10), COPD patients (n = 19) and IPF patients (n = 54). ( B,C ) Circulating Clusterin protein levels were quantitated and compared between IPF (n = 60) and a cohort of age matched controls (n = 30) ( B ), and from COPD (n = 15) and a separate cohort of age matched controls (n = 25) ( C ). Levels were measured by Somascan analysis, each dot representing a different individual. ( D–J ) Clusterin expression was visualized (brown staining) by IHC analysis of three IPF lungs ( D – I ) and a representative normal lung ( J ) tissue, size bars are indicated on image. ( K ) The staining intensity of cell-associated Clusterin was quantified in airway epithelial cells using Aperio Scanscope software. Shown is the average Clusterin staining intensity in airway epithelial cells in normal, IPF and COPD lung tissue. Data are expressed as Mean ± SEM *P ≤ 0.05, ****P ≤ 0.001 significance to relevant control levels.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Gene Expression, Reverse Transcription Polymerase Chain Reaction, Control, Expressing, Staining, Software

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Induction of Clusterin in response to bleomycin-induced lung injury. ( A–H ) C57Bl/6 mice were given bleomycin intratracheally and CLU expression was visualized by IHC after 7 ( C , D ), 14 ( E , F ) and 21 ( G , H ) days of bleomycin instillation and compared to staining in the lungs of Day 21 saline treated mice ( A , B ). ( I ) Clusterin transcript expression was quantitated after 14, 21 and 28 days of bleomycin instillation by RT-PCR in whole lung samples. Data are expressed as Mean ± SEM*P ≤ 0.05, **P ≤ 0.01 significance to relevant control levels. ( J–Q ) Saline or bleomycin challenged C57Bl/6 mice were treated with 20 µg of recombinant Clusterin every three days starting at Day 2. Shown are representative histological images of the lungs stained with Masson’s trichrome from saline + PBS vehicle ( J , K ), saline + Clusterin ( L , M ), bleomycin + PBS ( N , O ) and bleomycin + Clusterin ( P , Q ) treated mice. The top images are of whole scans of the lungs and the bottom images are taken at 200x magnification. ( R ) Collagen content in the lungs of the recombinant Clusterin and vehicle treated mice was biochemically quantified using a hydroxyproline assay. Shown is the average hydroxyproline (µg) from right lung lobes. ( S , T ) Shown is the average total BAL cell number ( S ) and the total TGFß levels ( T ) in the lungs of vehicle or Clusterin treated mice 15 days after bleomycin challenge. ( U ) FN1, VEGFA, PDGFRA and MMP12 transcript expression was quantified in whole lung samples 15 days after bleomycin, vehicle and/or Clusterin treatment. Shown is the average expression from 3 Saline/PBS, 3 Saline/rClusterin, 7 Bleo/PBS and 7 Bleo/rClusterin treated mice.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Expressing, Staining, Saline, Reverse Transcription Polymerase Chain Reaction, Control, Recombinant, Hydroxyproline Assay

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Clusterin deficient mice show persistent fibrosis in response to bleomycin. WT or CLU −/− received bleomycin intratracheally on Day 0. ( A–H ) Depicted is Masson’s Trichrome histological staining of WT saline ( A , B ), WT bleomycin ( C , D ), CLU−/− saline ( E,F ) and CLU−/− bleomycin ( G , H ) treated lungs fourteen days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( I ) Collagen content in the lungs of the of bleomycin treated wildtype and CLU−/− mice was biochemically quantified using a hydroxyproline assay, 14 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( J ) Shown is the average total TGFβ levels in the BAL from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( K ) Shown is the average IL-1β levels in whole lung lysates from wildtype and CLU−/− mice, 14 days after bleomycin challenge. ( L , M ) The proliferative status ( L ) and Caspase3 expression ( M ) in live CD45 − EpCAM + epithelial cells was assessed via Ki67 and activated Caspase-3 staining, respectively, 5 days after bleomycin challenge. Depicted is the fold change in cell number in response to bleomycin, relative to its relevant saline control. ( N , O ) IHC analysis for cleaved Caspase3 was performed on fibrotic murine lungs and quantified using Aperio Scanscope software. Depicted is the fold change in clusters, as defined by three or more adjacent positive cells, staining for cleaved Caspase3, 14 ( N ) and 28 ( O ) days after bleomycin challenge, compared to relevant saline control. ( P – W ) Depicted is Masson’s Trichrome histological staining of WT saline ( P , Q ), WT bleomycin ( R , S ), CLU−/− saline ( T,U ) and CLU−/− bleomycin ( V , W ) treated lungs 28 days after instillation. Shown are whole lung images (top) and magnified images (bottom). ( X ) Collagen content in the lungs of the of wildtype and CLU−/− treated mice was biochemically quantified using a hydroxyproline assay 28 days after bleomycin instillation. Shown is the average hydroxyproline (µg) from right lung lobes. ( Y , Z ) Quantitative PCR analysis was performed on RNA purified from lungs of 28-day saline and bleomycin treated murine lungs. Shown is the average col3a1 ( Y ) and fn1 ( Z ) transcript expression. Data are Mean ± SEM., n = 8–13 mice/ group. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001 significance, or as stated. ns = not significant.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: Staining, Saline, Hydroxyproline Assay, Expressing, Control, Software, Real-time Polymerase Chain Reaction, Purification

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Loss of transcripts for Clusterin, MMR, BER & DSB DNA repair pathway components in IPF SSEA4 + basal-like epithelial cells. ( A ) SSEA4 + and SSEA4 − cells were sorted from normal and IPF stromal cultures, RNA was extracted and subjected to RNAseq analysis. Fold changes in IPF relative to normal were calculated and the results were uploaded onto ingenuity IPA for pathway analysis ( A ). ( B ) Shown is an representative sort plot depicting the forward and side scatter localization of SSEA4 + cells. ( C ) Normalized RNA sequencing FPKM values were mined for transcripts that were enriched in SSEA4 + relative to SSEA4 − cells. Shown are enriched basal cell lineage markers in normal lung and IPF SSEA4 + cells. ( D ) Shown is the average CLU transcript FPKM values normal and IPF SSEA4 + cells. ( E–G ) Transcripts encoding for components of MMR ( E ), BER ( F ) and DSB ( G ) pathways were mined from SSEA4 + RNAseq data, clustered using Euclidean distance with complete linkage and heat maps were generated using Morpheus (Broad Institute). ( H–M ) Normal and IPF lung biopsies were stained with anti-SSEA4 and BRCA1 antibodies followed by fluorescent microscopic analysis. Representative images were taken at 200x magnification from two normal and four IPF lung biopsies stained with BRCA1 ( H , K respectively), SSEA4 ( I , L respectively) and the merged composites ( J , M respectively). ( N ) The number of SSEA4 + cells showing both SSEA4 and BRCA1 staining was quantified, averaged, and the percentage of cells was calculated from 28 normal and 84 IPF SSEA4 + cells from 3 normal and 4 IPF lungs, respectively.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques: RNA Sequencing, Generated, Staining

Journal: Scientific Reports

Article Title: Divergent roles for Clusterin in Lung Injury and Repair

doi: 10.1038/s41598-017-15670-5

Figure Lengend Snippet: Pictorial summary for the potential role of Clusterin variants in the modulation of epithelial cell survival and lung fibrosis.

Article Snippet: After blocking, tissues were stained with goat anti-mouse Clusterin (R&D Systems), mouse anti human Clusterin (BD Pharmingen) diluted in PBST, FITC conjugated anti-8-hydroxyguanosine (Abcam), p-H2A.X (Cell Signaling) or rabbit anti-cleaved Caspase-3 (Cell Signaling) antibodies with background reducing components (DAKO) for 1 hour at room temperature.

Techniques:

Journal: Oncotarget

Article Title: Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

doi:

Figure Lengend Snippet: The raw data was exported from the TCGA CRC database, with representations of 22 samples from healthy control and 215 samples from CRC. A. Expression levels of NLRP1, NLRP3, NLRC3, NLRC4 and AIM2 were significantly decreased in human colorectal cancer; B. NOD1 and NOD2 had significantly higher levels of expression in CRC; C. NLRC5, NLRP6 and NLRP12 expression was slightly reduced in CRC but statistical significance was not reached. Data are expressed as mean ± SEM. Log2 median-centered ratio expression. Unpaired Mann-Whitney test was used to evaluate the significance of differential mRNA expression levels of candidate genes. ** indicates P < 0.01; **** indicates P < 0.0001; ns, no statistical difference.

Article Snippet: Quantitative PCR for human NLRP1 (Hs00248187_m1), NLRP3 (Hs00918082_m1), NLRP4 (Hs00370499_m1), NLRP6 (Hs00373246_m1), NLRP12 (Hs00536435_m1), NLRC3 (Hs01054716_m1), NLRC4 (Hs00368367_m1), NLRC5 (Hs00260008_m1), NLRX1 (Hs00226360_m1), NOD1 (Hs00196075_m1), NOD2 (Hs00223394_m1) and AIM2 (Hs00223394_m1) was performed by using TaqMan primer/probe sets and master mix (Applied Biosystems).

Techniques: Control, Expressing, MANN-WHITNEY

Journal: Oncotarget

Article Title: Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

doi:

Figure Lengend Snippet: Statistical analysis of NLRs and AIM2 expression in CRCs from different anatomical localizations

Article Snippet: Quantitative PCR for human NLRP1 (Hs00248187_m1), NLRP3 (Hs00918082_m1), NLRP4 (Hs00370499_m1), NLRP6 (Hs00373246_m1), NLRP12 (Hs00536435_m1), NLRC3 (Hs01054716_m1), NLRC4 (Hs00368367_m1), NLRC5 (Hs00260008_m1), NLRX1 (Hs00226360_m1), NOD1 (Hs00196075_m1), NOD2 (Hs00223394_m1) and AIM2 (Hs00223394_m1) was performed by using TaqMan primer/probe sets and master mix (Applied Biosystems).

Techniques: Expressing

Journal: Oncotarget

Article Title: Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

doi:

Figure Lengend Snippet: TNM stage information was exported from the TCGA colon database. A. NLRC3 and AIM2 showed statistically different levels of expression between healthy control and various CRC stages of cancer progression, where stage IV exhibited significantly lower expression of these two genes than stages 1-3. B. NLRP1, NLRP3, NLRC4, NOD1 and NOD2 exhibited statistically different levels of expression between healthy control and all four stages of CRC. C . Levels of NLRC5, NLRP12 and NLRP6 expression among control and cancer stages 1-4 were not significantly different. Data were presented as means ± SEM. * P < 0.05; ** P < 0.01; ** P < 0.001**** P < 0.0001.

Article Snippet: Quantitative PCR for human NLRP1 (Hs00248187_m1), NLRP3 (Hs00918082_m1), NLRP4 (Hs00370499_m1), NLRP6 (Hs00373246_m1), NLRP12 (Hs00536435_m1), NLRC3 (Hs01054716_m1), NLRC4 (Hs00368367_m1), NLRC5 (Hs00260008_m1), NLRX1 (Hs00226360_m1), NOD1 (Hs00196075_m1), NOD2 (Hs00223394_m1) and AIM2 (Hs00223394_m1) was performed by using TaqMan primer/probe sets and master mix (Applied Biosystems).

Techniques: Expressing, Control

Journal: Oncotarget

Article Title: Expression profile of innate immune receptors, NLRs and AIM2, in human colorectal cancer: correlation with cancer stages and inflammasome components

doi:

Figure Lengend Snippet: The quantitative RT-PCR data were obtained by the comparative C T method (2 −ΔΔCT ). A. The expression of NLRC3 and NLRP3 was significantly reduced in CRC. B. NLRP6, NLRP12 and NLRC5 expression was not significantly different between CRC and healthy controls. Results shown in A. and B. are congruent with the TCGA database presented in Figure . C. NLRC4, AIM2, NOD1, NOD2 and NLRP1 expression in FFPE CRC and adjacent normal controls were not significantly different. This data does not correlate with the TCGA database. The RT-PCR results are converted to a log2 scale. Data are expressed as mean ± SEM. Wilcoxon matched-pairs signed rank test was used to evaluate the significance of differential mRNA expression levels of candidate genes. * P < 0.05; ** P < 0.01; ** P < 0.001 **** P < 0.0001, and ns indicates the lack of a statistical difference. NLRP1 was only detected in 27 samples. Mann-Whitney unpaired test was used to perform statistical analysis on the GraphPad software.

Article Snippet: Quantitative PCR for human NLRP1 (Hs00248187_m1), NLRP3 (Hs00918082_m1), NLRP4 (Hs00370499_m1), NLRP6 (Hs00373246_m1), NLRP12 (Hs00536435_m1), NLRC3 (Hs01054716_m1), NLRC4 (Hs00368367_m1), NLRC5 (Hs00260008_m1), NLRX1 (Hs00226360_m1), NOD1 (Hs00196075_m1), NOD2 (Hs00223394_m1) and AIM2 (Hs00223394_m1) was performed by using TaqMan primer/probe sets and master mix (Applied Biosystems).

Techniques: Quantitative RT-PCR, Expressing, Reverse Transcription Polymerase Chain Reaction, MANN-WHITNEY, Software