Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Differential expression of Arabidopsis thaliana pri-miRNAs (white background) and mature miRNAs (gray background) under different abiotic stress conditions . Blue, red, and green colors indicate pri-miRNAs and mature miRNAs unchanged, upregulated and downregulated, respectively. (A) Upper panel depicts 30%SWC and 20%SWC drought stress (D30 and D20, respectively), middle panel shows half an hour and 6 h heat stress (H 0.5; H 6), lower panel shows 250 mM salinity (NaCl+), (B) upper panel presents copper deficiency (Cu−) and 10 μM copper excess (Cu+), lower panel depicts 10 μM cadmium excess (Cd+) and sulfur deficiency (S−). The fold change-based numbers of up- and downregulated pri-miRNAs and mature miRNAs shown in Venn diagrams were suggested by a two-tailed Student t test ( p ≤ 0.05) for pri-miRNA RT-qPCR analyses (modified mirEX high throughput real-time PCR platform, Bielewicz et al., , http://comgen.pl/mirex2/ ) and a One-Way Anova test ( p ≤ 0.05) for mature miRNA analyses by small RNA NGS, respectively.

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques: Quantitative Proteomics, Two Tailed Test, miRNA RT, Modification, High Throughput Screening Assay, Real-time Polymerase Chain Reaction

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Arabidopsis thaliana miRNAs revealed by Northern hybridization as affected under 30%SWC mild drought, 20%SWC severe drought, 0.5 h heat, 6 h heat, and salinity stress conditions . Only new stress responsive miRNAs unknown until now are shown. The white lines separate signals from miRNAs and U6snRNA loading control probed on the same blots. H 0 and H 6 samples were run on the same gel but not next to each other as indicated by the white separating lines between all signals probed. The star marks NGS revealed miRNAs with no statistic significance and the maintained tendency of expression level change as seen in Northern hybridization. Symbols representing various stresses are marked as described in the Figure .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques: Northern Blot, Hybridization, Control, Expressing

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Arabidopsis thaliana miRNAs revealed by Northern hybridization as affected under copper deficiency, copper excess, cadmium excess, and sulfur deficiency stress conditions . Only new stress responsive miRNAs unknown until now are shown. The white lines separate signals from miRNAs and U6snRNA loading control probed on the same blots. Control (Ctrl) and stress (Cu−, Cu+, Cd+, S−) samples were run on the same gel but not next to each other as indicated by the white separating lines between all signals probed. The star marks NGS revealed miRNAs with no statistic significance and the maintained tendency of expression level change as seen in Northern hybridization. Symbols representing various stresses are marked as described in the Figure .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques: Northern Blot, Hybridization, Control, Expressing

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Individual pri-miRNA—miRNA relationships under different abiotic stresses .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: 30%SWC (D30) and 20%SWC (D20) drought stress affected Arabidopsis miRNAs revealed by high-throughput small RNA NGS .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: 0.5 h (H 0.5) and 6 h (H 6) heat stress affected Arabidopsis miRNAs revealed by high-throughput small RNA NGS .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Salinity (NaCl+) stress affected Arabidopsis miRNAs revealed by high-throughput small RNA NGS .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: Copper deficiency (Cu−), copper excess (Cu+), cadmium excess (Cd+), and sulfur deficiency (S−) stresses affected Arabidopsis miRNAs revealed by high-throughput small RNA NGS .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: General stress-responsive miRNAs . (A) General abiotic stress responsive Arabidopsis thaliana miRNAs 319a/b, 319b.2, and 400 revealed by Northern hybridization under different abiotic stresses. The white lines separate signals from miRNAs and U6snRNA loading control as well as both signals for the following stress samples: NaCl+ and Cu− (for miR319a/b), control (Ctrl) and Cu− (for miR319b.2), Cd+ and H 0 (for miR400), all probed on the same blots. (B) The structure of the TBL10 and the predicted as well as 5′RACE identified slicing sites within its mRNAs. (C) The agarose gels showing the miR319b.2 directed 3′-TBL10 mRNA cleavage products in wt plants and Δ miR319b mutant plants. Arrow points to the expected length of the 5′RACE product. (D) The RT-qPCR of the TBL10 expression in wt plants under different abiotic stress conditions. Values on the chart are shown as the mean ± SD relative expression level from three independent experiments. Symbols representing various stresses are marked as described in the Figure .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques: Northern Blot, Hybridization, Control, Mutagenesis, Quantitative RT-PCR, Expressing

Journal: Frontiers in Plant Science

Article Title: Arabidopsis microRNA expression regulation in a wide range of abiotic stress responses

doi: 10.3389/fpls.2015.00410

Figure Lengend Snippet: General stress-responsive Arabidopsis microRNAs revealed by high-throughput small RNA NGS .

Article Snippet: The abundance of the selected microRNAs was tested using Northern hybridization (for detailed data see Tables S2, S3 and Figures , ) as well as TaqMan® miRNA assays (ABI, Life Technologies, USA) (Figure S2).

Techniques:

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Micro-CT images of the distal metaphysis of the femur. (B-H) Micro-CT analysis of the trabecular bone volume/total volume (B), connectivity density (C), trabecular number (D), bone mineral density (E), trabecular thickness (F), trabecular spacing (G), and structure model index (H). (I) H&E staining of femoral sections from irradiated mice and controls. (J) TRAP staining of femoral sections from irradiated mice and controls. RT-qPCR analysis of RANKL and OPG mRNA expression in flushed whole bone marrow. (K) RANKL/OPG ratio of RT-qPCR results. (L) Western blotting analysis of Crif1 expression in flushed whole bone marrow. *P < 0.05 , ** P < 0.01. All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Micro-CT, Staining, Irradiation, Quantitative RT-PCR, Expressing, Western Blot

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Histochemical staining analysis of Crif1 expression in femoral bone marrow. (B) RT-qPCR analysis of RANKL and OPG mRNA expression in flushed whole bone marrow of Crif1 fl/fl mice and Lyz2Cre;Crif1 fl/fl mice after 5 Gy radiation. (C) RANKL/OPG ratio of RT-qPCR results. (D)Micro-CT images of the distal metaphysis of the femur from Lyz2Cre;Crif1 fl/fl mice and Crif1 fl/fl mice. (E-K) Micro-CT analysis of the trabecular bone volume/total volume (E), connectivity density (F), trabecular number (G), bone mineral density (H), trabecular thickness (I), trabecular spacing (J), and structure model index (K). (L) H&E staining of femoral sections from Crif1 fl/fl mice and Lyz2Cre;Crif1 fl/fl mice. (M) TRAP staining of femoral sections from Crif1 fl/fl mice and Lyz2Cre;Crif1 fl/fl mice. * P < 0.05, ** P < 0.01. All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Staining, Expressing, Quantitative RT-PCR, Micro-CT

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Western blotting analysis of Crif1 expression in mouse BM-MSCs. Mouse BM-MSCs were transfected with a Crif1 lentiviral overexpression vector. (B) RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and Crif1-overexpressing BM-MSCs (OV). BM-MSCs and OV were cocultured with RAW264.7. (C) RANKL/OPG ratio of RT-qPCR results. (D) ELISA analysis of RANKL protein levels in coculture supernatant medium. (E) ELISA analysis of OPG protein levels in coculture supernatant medium. (F)RANKL/OPG ratio in coculture supernatant medium. (G) TRAP staining of RAW264.7 after 7 days in coculture. (H) Average number of TRAP-positive cells/well (arrow) from RAW264.7 cells in coculture. *P < 0.05 , ** P < 0.01. All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Western Blot, Expressing, Transfection, Over Expression, Plasmid Preparation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay, Staining

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Western blotting analysis of Crif1 and RANK expression in RAW264.7 cells. Crif1 was knocked out from RAW264.7 cells (RAW264.7-KO). (B) TRAP staining of RAW264.7-KO and controls after 7 days in coculture with mouse BM-MSCs. (C) Average number of TRAP-positive cells/well (arrow) from RAW264.7-KO and controls after 7 days in coculture with mouse BM-MSCs. (D) Western blotting analysis of Crif1 expression in BM-MSCs. Crif1 was knocked out from mouse BM-MSCs (KO), and KO and controls were irradiated with 9 Gy Co-60. (E) RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and Crif1 knockout BM-MSCs (KO). BM-MSCs and KO were cocultured with RAW264.7 (F) RANKL/OPG ratio of RT-qPCR results. (G) ELISA analysis of RANKL protein levels in coculture supernatant medium. (H) ELISA analysis of OPG protein levels in coculture supernatant medium. (I) RANKL/OPG ratio in coculture supernatant medium. (J) TRAP staining of RAW264.7 after 7 days in coculture. (K) Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture. *P < 0.05 , ** P < 0.01. All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Western Blot, Expressing, Staining, Irradiation, Quantitative RT-PCR, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Oil red O staining analysis of mouse BM-MSCs after 21 days of adipogenic differentiation. Crif1 was knocked out from mouse BM-MSCs (KO), and KO and controls were irradiated with 9 Gy Co-60. KO and controls were treated with mouse mesenchymal stem cell adipogenic differentiation medium (Ad) to induce adipogenesis. (B) The dye from oil red O staining was extracted using isopropanol, and the optical density at 510 nm was measured using a Benchmark Plus. (C) Western blotting analysis of adipogenesis-related markers and transcription factors PPARγ and AP2 in mouse BM-MSCs after 21 days of adipogenic differentiation. (D) RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and Crif1 knockout BM-MSCs (KO). (E) RANKL/OPG ratio of RT-qPCR results. (F) ELISA analysis of RANKL protein levels in supernatant adipogenic differentiation medium. (G) ELISA analysis of OPG protein levels in supernatant adipogenic differentiation medium. (H) RANKL/OPG ratio in supernatant adipogenic differentiation medium. *P < 0.05 , ** P < 0.01..All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Staining, Irradiation, Western Blot, Quantitative RT-PCR, Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A)RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and Crif1 knockout BM-MSCs (KO). BM-MSCs and KO were cocultured with RAW264.7 with or without 25 μM forskolin respectively. (B) RANKL/OPG ratio of RT-qPCR results. (C) ELISA analysis of RANKL protein levels in coculture supernatant medium with or without 25 μM forskolin. (D) ELISA analysis of OPG protein levels in coculture supernatant medium with or without 25 μM forskolin. (E) RANKL/OPG ratio in coculture supernatant medium with or without 25 μM forskolin. (F) RT-qPCR analysis of RANKL and OPG mRNA expression in BM-MSCs and Crif1-overexpressing BM-MSCs (OV). BM-MSCs and OV were cocultured with RAW264.7 with or without 20 μM H89 respectively. (G) RANKL/OPG ratio of RT-qPCR results. (H) ELISA analysis of RANKL protein levels in coculture supernatant medium with or without 20 μM H89. (I) ELISA analysis of OPG protein levels in coculture supernatant medium with or without 20 μM H89. (J) RANKL/OPG ratio in coculture supernatant medium with or without 20 μM H89. (K) TRAP staining of RAW264.7 in coculture treated with or without 25 μM forskolin. (L) Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture treated with or without 25 μM forskolin. (M) TRAP staining of RAW264.7 in coculture treated with or without 20 μM H89. (N) Average number of TRAP-positive cells/well (arrow) from RAW264.7 in coculture treated with or without 20 μM H89. (O)Western blotting analysis of CREB phosphorylation levels in BM-MSCs in coculture treated with or without 25 μM forskolin. (P) Western blotting analysis of CREB phosphorylation levels in BM-MSCs in coculture treated with or without 20 μM H89. *P < 0.05 , ** P < 0.01..All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Quantitative RT-PCR, Expressing, Knock-Out, Enzyme-linked Immunosorbent Assay, Staining, Western Blot, Phospho-proteomics

Journal: bioRxiv

Article Title: Crif1 Promotes Osteoporosis in Mice after Radiation

doi: 10.1101/725408

Figure Lengend Snippet: (A) Crif1− PKAα interaction model showing Crif1 (colored in rose red) and PKAα (colored in cyan). Interface amino acids are shown as sticks and colored in rose red (for Crif1) and cyan (for PKAα) and indicated as a zoomed-in view in the inset figure. (B-F) Chemical structure of each inhibitor molecule and their docked pose on Crif1 (colored in rose red, surface view). Docked molecule (stick) and the amino acids involved in the hydrophobic interactions (light purple) are shown. F0382-0033 (B), F3408-0076 (C), F1430-0134 (D), F3408-0031 (E) and F1430-0130 (F). (G-K) A tetrazolium salt (WST-8) assay was carried out to study the toxity effect of compounds on the hBM-MSCs. F0382-0033 (G), F3408-0076 (H), F1430-0134 (I), F3408-0031 (J) and F1430-0130 (K). (L)ELISA analysis of RANKL protein levels in supernatant medium. hBM-MSCs were pretreated with 5 different compounds followed by treatment with forskolin, and supernatant medium was collected for ELISA after 3 days. (M) ELISA analysis of OPG protein levels in supernatant medium. (N) RANKL/OPG ratio in supernatant medium. (O)Western blotting analysis of CREB phosphorylation levels. hBM-MSCs were pretreated with 5 different compounds followed by treatment with forskolin and total protein lysates were extracted for CREB phosphorylation detection after 1 hour. *P < 0.05, **P < 0.01 . All experiments were performed in triplicate, and the bars represent the mean ± SD.

Article Snippet: The primary antibodies used for blotting were as follows: Crif1 (M-222) (sc-134882; Santa Cruz), RANK (H-7) (sc-374360; Santa Cruz), A-FABP (AP2, sc-18661; Santa Cruz), PPARγ (sc-7273; Santa Cruz), β-actin (sc-47778; Santa Cruz), phospho-CREB rabbit mAb (#9198; Cell Signaling Technology), and CREB rabbit mAb (#9197; Cell Signaling Technology).

Techniques: Enzyme-linked Immunosorbent Assay, Western Blot, Phospho-proteomics

Journal: Obesity (Silver Spring, Md.)

Article Title: Skeletal muscle interleukin‐6 regulates metabolic factors in i WAT during HFD and exercise training

doi: 10.1002/oby.21139

Figure Lengend Snippet: Regulation of AMPK and HSL in iWAT in Floxed and IL‐6 MKO mice after 16 weeks on Chow, HFD, or HFD combined with exercise training (HFD ExTr). (a) AMPK Thr172 phosphorylation (phos), (b) HSL Ser660 phos, (c) HSL Ser565 phos, (d) AMPKα1 protein content, (e) HSL protein content, and (f) perilipin protein content ( n = 9‐10). Values are mean ± SE. *Significantly different from Chow within given genotype ( P < 0.05). ¤ Significantly different from HFD within given genotype ( P < 0.05). # Significantly different from Floxed within given intervention ( P < 0.05).

Article Snippet: The membrane was then blocked for 1 h in 3% fish gel (FG) (Sigma Aldrich, Copenhagen, Denmark) and incubated overnight in primary antibody against AMPKα1 protein (Hardie G.), AMPK Thr172 phosphorylation (#2535s Cell Signaling Technologies (CST), Danvers, MA), HSL protein, and HSL Ser660 and Ser565 as well as perilipin (#8334S CST), GLUT4 protein (#PAI‐1065, ABR), PEPCK protein (#10004943, Cayman Chemical Co., Ann Arbor, MI), PDK4 protein (Hardie G.), PDH‐E1α protein (Hardie G.), PDH Ser300 phosphorylation (Hardie G.), and PDH Ser232 phosphorylation (#AP1063, Millipore).

Techniques: Phospho-proteomics

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: Elevated CHEK1 expression is associated with poor outcomes in MM patients and promotes MM cell proliferation in vitro. A CHEK1 mRNA levels were significantly increased in MM samples. The signal level of CHEK1 is shown on the y-axis. Patients were designated as being healthy donors with normal bone marrow plasma cells (NP, n = 22), monoclonal gammopathy of undetermined significance (MGUS, n = 44), or multiple myeloma (MM, n = 351), and are sorted on the x-axis. B Increased CHEK1 mRNA expression was associated with poor overall survival (OS) in MM patients from the TT2 patient cohort. C Increased CHEK1 mRNA expression was associated with poor OS in MM patients from the HOVON65 cohort. D Western blot analysis revealed that CHEK1 was endogenously expressed in the specified MM cell lines. E Validation of CHEK1 overexpression (OE) in CHEK1- OE ARP1 and H929 cells relative to vehicle-transfected control cells (WT). F Four-day cell growth curve, as detected by trypan blue staining and counting of WT, CHEK1- OE ARP1, and H929 cells. G Confirmation of CHEK1 protein knockdown (KD) in ARP1 and H929 cells after transfection with three independent CHEK1- targeting shRNAs. H Four-day cell growth curve in WT, CHEK1- KD ARP1, and H929 cells. I Images of representative soft agar plates, revealing increased clonogenic growth of CHEK1 -OE cells and decreased clonogenic growth in CHEK1 -KD cells relative to WT. J Cell cycle analysis revealed that the proportion of G2/M phase cells significantly increased in CHEK1 -OE cells relative to WT. K Cell cycle analysis revealed that the proportion of G2/M phase cells significantly decreased in CHEK1 -KD cells

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Expressing, In Vitro, Clinical Proteomics, Western Blot, Biomarker Discovery, Over Expression, Transfection, Control, Staining, Knockdown, Cell Cycle Assay

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: CHEK1 is a marker for high-risk MM and induces drug resistance. A Heatmap of RNA-seq data showing significantly differentiated genes before and after doxycycline-induced CHEK1 OE. B Pathway enrichment analysis of RNA-seq data revealed enrichment of two pathways, which were related to cell cycle regulation and osteoclast differentiation. C Box plot representing CHEK1 expression in eight MM risk subgroups from the TT2 patient cohort. D In paired patient MM samples collected at first diagnosis and relapse, CHEK1 mRNA expression was increased in the relapsed samples relative to the corresponding samples from first diagnosis. E–F Increased CHEK1 expression was correlated with decreased OS in relapsed patients from the (E) TT2 and (F) APEX cohorts. G Western blotting confirmed that CHEK1 protein levels were significantly increased in MM1.R (dexamethasone-resistant) and ANBL6 DR (Bortezomib-resistant) cells. H Effects of Bortezomib and Adriamycin on the cell viability of H929 and ARP1 cells with or without CHEK 1 OE. I Western blots demonstrated that CHEK1 OE induced resistance to Adriamycin and Bortezomib in ARP1 and H929 cells, as indicated by cleavage of the apoptotic regulators PARP and Caspase 3. J–K Pro-apoptotic effects of (J) CHEK1 shRNA silencing and the (K) CHEK1 selective inhibitor LY2603618 in H929 and ARP1 cells, as demonstrated by increased cleavage of PARP and Caspase 3

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Marker, RNA Sequencing, Expressing, Biomarker Discovery, Western Blot, shRNA

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: CHEK1 evokes chromosomal instability (CIN) in MM. A–B Giemsa staining revealed that CHEK1 OE increased the separation error rate and number of multi-nuclear cells in (A) ARP1 and (B) H929 cells. C–D Increased chromosomal plate width and decreased mitotic bipolar spindle length in CHEK1- OE ARP1 and H929 cells relative to WT, as demonstrated by immunofluorescent (IF) staining for α-tubulin and DAPI. E A comparative genomic hybridization (CGH) array revealed significant gains and losses of multiple chromosomal segments in CHEK1 -OE ARP1 and H929 cells relative to WT. F In WT and CHEK1 -OE cells treated with vehicle or Borbezomib, chromosomal plate width was highest and mitotic spindle length lowest in the Borbezomib-treated CHEK1- OE group

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Staining, Hybridization

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: CHEK1 promotes CIN through CEP170 activation in MM. A–B Centrosomal Protein 170 (CEP170) was selected among candidate genes of the CIN-related gene list and genes associated with poor outcome in the TT2 MM patient cohort. C Increased CEP170 expression was associated with decreased OS in the TT2 patient cohort. D A Co-IP assay revealed that CHEK1 directly interacted with CEP170 in CHEK1 -OE ARP1 and H929 cells. E–F CEP170 OE significantly increased chromosomal plate width and decreased mitotic bipolar spindle length in ARP1 and H929 cells. G A Co-IP assay confirmed that CHEK1 physically interacted with and phosphorylated CEP170 in CHEK1 -OE cells compared with WT cells, as detected by total anti-phospho-serine antibody. H Mass spectrometry (MS) was used to determine the CHEK1 phosphorylation site of CEP170, Ser1260. I A Myc-tagged CEP170 Ser1260Ala mutant, containing a defective CHEK1 phosphorylation site, exhibited dramatically decreased interaction with flag-tagged CHEK1, as demonstrated by Co-IP followed by western blotting. J–K OE of mutated CEP170 Ser1260Ala decreased chromosomal plate width and increased mitotic bipolar spindle length in (J) ARP1 and (K) H929 cells

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Activation Assay, Expressing, Co-Immunoprecipitation Assay, Mass Spectrometry, Phospho-proteomics, Mutagenesis, Western Blot

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: CHEK1 induces macrophage osteoclast by upregulating NFATc1 expression. A Magnetic resonance imaging (MRI) revealed that increased CHEK1 expression was positively correlated with bone lesion formation in TT2 cohort MM patients. B–C TRAP staining revealed that Chek1 OE promoted osteoclast differentiation in RAW 264.7 mouse macrophages co-treated with RANKL (50 ng/mL) and M-CSF (15 ng/mL) in a time-dependent manner. D–E TRAP staining confirmed that Chek1 OE prompted osteoclast differentiation in RAW 264.7 cells treated with varying doses of RANKL and M-CSF in a manner dependent on RANKL and M-CSF dosages. F–G TRAP staining revealed that human primary peripheral blood mononuclear cells (PBMCs) transfected with human CHEK1 cDNA developed significant more osteoclasts than non-transfected control cells. H–I Western blotting and TRAP staining confirmed that the CHEK1 inhibitor LY2603618 decreased NFATc1 expression and suppressed osteoclast differentiation in RAW 264.7 cells. J Co-IP revealed that CHEK1 interacted with NFATc1 in RAW 264.7 cells. K Western blotting confirmed that the expression of NFATc1 was increased in Chek1 -OE RAW264.7 cells relative to WT cells. L CHEK1 knockdown prevented myeloma-associated bone loss in 5TMM3VT model. Micro-CT analysis of 5TMM3VT-involved tibia bone performed at 4 weeks confirmed the presence of osteolytic lesions and demonstrated decreased trabecular bone volume (BV/TV) compared with CHEK1 gene knockdown

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Expressing, Magnetic Resonance Imaging, Staining, Transfection, Control, Western Blot, Co-Immunoprecipitation Assay, Knockdown, Micro-CT

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: MM cells secrete circCHEK1_246aa circular RNA to induce MM CIN and promote osteoclast differentiation in the bone marrow microenvironment. A The number of exons and exact circCHEK1 sequences produced from CHEK1 were validated by Sanger sequencing. The blue arrow represents the “head-to-tail” splicing sites of circCHEK1. B mRNA levels of circCHEK1 and linear CHEK1 ± RNase R were determined by RT-PCR and qRT-PCR. C After pull-down using a CHEK1 antibody, protein samples at the expected size were excised and subjected to mass spectrometry (MS) analysis, and specific peptides from circCHEK1_246aa were identified. D A Co-IP assay revealed that circCHEK1_246aa more robustly interacted with native CEP170 than mutated CEP170. E–F circCHEK1 OE increased chromosomal plate width and decreased mitotic bipolar spindle length in ARP1 and H929 cells. G TRAP staining revealed that circCHEK1 -OE human primary PBMCs developed into significantly more osteoclasts relative to vehicle-transfected control cells. H Graphic illustrating that CHEK1 and circCHEK1_246aa promote multiple myeloma malignancy by evoking CIN and bone lesion formation

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: Produced, Sequencing, Reverse Transcription Polymerase Chain Reaction, Quantitative RT-PCR, Mass Spectrometry, Co-Immunoprecipitation Assay, Staining, Transfection, Control

Journal: Molecular Cancer

Article Title: CHEK1 and circCHEK1_246aa evoke chromosomal instability and induce bone lesion formation in multiple myeloma

doi: 10.1186/s12943-021-01380-0

Figure Lengend Snippet: CHEK1 promotes MM growth in vivo and is a potential therapeutic target . A Photographic images of xenograft-bearing mice from each group were taken at day 28. B Time course of tumor growth in NOD-SCID mice treated with vehicle, BTZ, or ADR. C Photographic images of xenografts from NOD-SCID mice of the specified groups on day 28. D Mean tumor weights in the six experimental groups at day 28 after implantation of the specified MM cells. E Photographic images of xenograft-bearing mice from the KD and KD + DOX groups were collected at day 28. F Time course of tumor growth in the NOD-SCID mice of the specified groups. G Xenografts from the NOD-SCID mice of the specified groups were collected at day 28. H Mean tumor weights in the specified two experimental groups at day 28 after implantation of MM cells

Article Snippet: Antibodies used were as follows: CHEK1 (sc-8408; Santa Cruz Biotechnology, USA); rabbit IgG (a7016); mouse IgG (a7028; Beyotime Institute of Biotechnology, China); FLAG (F-4020; Merck KGaA, Germany); PARP (9542S), Caspase-3 (9662S), β-actin (4970S; Cell Signaling Technology, USA); MYC (16286–1-AP), CEP170 (18899–1-AP; ProteinTech Group, China); and α-Tubulin (ab7291; Abcam, UK).

Techniques: In Vivo

Journal: eLife

Article Title: Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration

doi: 10.7554/eLife.13378

Figure Lengend Snippet: ( A ) Relative ATP level of T8993G compared to healthy control (BJ, H9 hESC) in iPSCs, NPCs and neurons. The relative percentage of ATP levels in T8993G was calculated by comparing to the mean of control cells respectively. Bars are mean ± SD, n=3. *p<0.05. Calculated by two-tailed t-test. Immunoblot analysis of AMPK Thr172 and ACC Ser79 phosphorylation in cell lysates prepared from primary fibroblasts, iPSCs, NPCs and neurons. ( B ) Cellular ATP level and secreted lactate from H9 NPCs and neurons treated with DMSO and oligomycin for 6 hr. The relative percentage of ATP levels was calculated by comparing to the mean of DMSO-treated cells respectively. Bars are mean ± SD, n=3. ( C ) Immunoblot analysis of representative enzymes in glycolysis, TCA and mitochondrial respiratory complexes in BJ and T8993G NPCs and neurons. 20 µg protein lysate from each sample were loaded for SDS-PAGE. ( D ) Measurement of lactate secreted by NPCs and neurons derived from human BJ iPSCs at 3 weeks. NPC and differentiated neurons at 3 weeks were incubated in fresh medium for 12 hr, and lactate in the medium is quantified. Bars represent mean ± SD of the absolute concentration of lactate after normalized to protein content. n=3. All the experiments were repeated at least three times. (see associated ). DOI: http://dx.doi.org/10.7554/eLife.13378.018 10.7554/eLife.13378.019 Figure 3—source data 1. DOI: http://dx.doi.org/10.7554/eLife.13378.019

Article Snippet: The primary antibodies and dilutions were used as follow: glycolysis antibody sampler kits (#8337&12866, Cell Signalling) used at 1:1000; the OXPHOS human WB Antibody cocktail, anti-CS and IDH2 used at 1:1000 (Abcam); anti-HSP60 and SUCLA2 (Santa Cruz); anti phospho AMPK T172 and AMPK, anti phospho ribosomal S6 Ser235/236 and S6; anti phospho mTOR Ser2481 and mTOR used at 1:1000 (Cell Signaling).

Techniques: Control, Two Tailed Test, Western Blot, Phospho-proteomics, SDS Page, Derivative Assay, Incubation, Concentration Assay

Journal: eLife

Article Title: Alleviation of neuronal energy deficiency by mTOR inhibition as a treatment for mitochondria-related neurodegeneration

doi: 10.7554/eLife.13378

Figure Lengend Snippet: ( A ) Metabolites measured by gas chromatography mass spectrometry (GC-MS). The metabolites were extracted from 3-week T8993G and control including two BJ and one H9 neurons. Relative cellular amino acids were shown. Bar are mean ± SD, n=3. ( B ) Metabolites of glycolysis and TCA reactions. The relative amount of metabolites in T8993G neurons was presented as percentage compared to the mean of control. Bar are mean ± SD, n=3. ( C ) A simplified metabolic flow diagram of glycolysis and the TCA cycle. ( D, E ) 3-week BJ neurons were treated with oligomycin (40 nM) and rotenone and antimycin A (1 µM each) for 6 hr. Bar are mean ± SD, n=3. *p<0.05. **p<0.01, calculated by two-tailed t-test. (see associated ). DOI: http://dx.doi.org/10.7554/eLife.13378.027 10.7554/eLife.13378.028 Figure 6—source data 1. DOI: http://dx.doi.org/10.7554/eLife.13378.028

Article Snippet: The primary antibodies and dilutions were used as follow: glycolysis antibody sampler kits (#8337&12866, Cell Signalling) used at 1:1000; the OXPHOS human WB Antibody cocktail, anti-CS and IDH2 used at 1:1000 (Abcam); anti-HSP60 and SUCLA2 (Santa Cruz); anti phospho AMPK T172 and AMPK, anti phospho ribosomal S6 Ser235/236 and S6; anti phospho mTOR Ser2481 and mTOR used at 1:1000 (Cell Signaling).

Techniques: Gas Chromatography, Mass Spectrometry, Gas Chromatography-Mass Spectrometry, Control, Two Tailed Test

Journal: Stem cells (Dayton, Ohio)

Article Title: Hypoxia and HIF1alpha repress the differentiative effects of BMPs in high-grade glioma.

doi: 10.1634/stemcells.2008-0402

Figure Lengend Snippet: Figure 2. Increasing oxygen tension induces p53 activation, leading to glial differentiation of HGG precursors. HGG precursors (pediatric and adult) were initially expanded in 2% oxygen, followed by acute exposure to 20% oxygen. (A–C): Expression of p53 using a pan-p53 antibody (green) in 2% oxygen (A) or after 24–48 hours of acute exposure to 20% oxygen (B). (C): Bar graph comparing total p53 expression with activation of p53 via phosphorylation at serine residues 20, 37, or 392. (D–F): Expression of Ki67 (red) and activated caspase-3 (green) in 2% oxygen (D) or 48 hours after acute exposure to 20% oxygen (E). (F): Quantitation. (G–I): Expression of p21cip1 (red) in 2% oxygen (G) or 48 hours after acute exposure to 20% oxygen (H); inset shows higher magnification of p21/DAPI colocalization. (I): Quantitation. (J–L): Expression of nestin (green) and GFAP (red) in 2% oxygen (J) or 48 hours after acute exposure to 20% oxygen (K). (L): Quantitation. Bar graphs show mean SEM, n 3–7. *, p .05. Bar 8 m for (A, B) and inset in (H). Bar 50 m for (D, E, G, H, J, K). Abbreviations: DAPI, 4,6-diamidino-2-phenylindole; GFAP, glial fibrillary acidic protein; HGG, high-grade glioma; SEM, standard error of the mean.

Article Snippet: Primary antibody staining was performed for Ki67 (mouse, 1:100; Dako, Fort Collins, CO, http:// www.dako.com), nestin (mouse, 1:200; Millipore, Billerica, MA, http://www.millipore.com), activated caspase-3 (rabbit, 1:1,000; Cell Signaling Technology, Danvers, MA, http://www.cellsignal. com), glial fibrillary acidic protein (GFAP) (mouse, 1:1,000; Sigma), -III-tubulin (rabbit, 1:2,000; Covance, Princeton, NJ, http://www.covance.com) or Tuj-1 (mouse, 1:1,000, Covance), p21cip1 (mouse, 1:800; Lab Vision, Fremont, CA, http://www. labvision.com), phospho-p53 Sampler Kit (as directed, Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com), phospho-Smad1, Smad5, and Smad8 (rabbit, as directed, Cell Signaling Technology), or Id1 (rabbit, 1:50; Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com).

Techniques: Activation Assay, Expressing, Phospho-proteomics, Quantitation Assay

Journal: Molecular Medicine Reports

Article Title: Proteinase-activated receptor 2 deficiency is a protective factor against cardiomyocyte apoptosis during myocardial ischemia/reperfusion injury

doi: 10.3892/mmr.2019.10618

Figure Lengend Snippet: Effects of PAR2 knockdown on MAPK signaling pathways in H9c2 cells after H/R injury. (A) Representative western blotting images for phosphorylation levels of ERK1/2, JNK, and p38 MAPK. (B) Densitometric analyses of antibody-bound protein bands in the western blot analyses (**P<0.01). PAR2, proteinase-activated receptor 2; H/R, hypoxia/reoxygenation; ERK, extracellular signal regulated kinase; JNK, c-Jun NH2-terminal protein kinase.

Article Snippet: The antibodies used were as follows: phospho-MAPK, total-MAPK, (phospho cat. no. 9910; 1:1,000; total cat. no. 9926, 1:1,000), poly-ADP-ribose polymerase (PARP; cat. no. 9532; 1:1,000), B-cell lymphoma 2 (Bcl-2; cat. no. 3498; 1:1,000 ( ) (all from Cell Signaling Technology, Inc.); PAR2 (cat. no. 180953, 1:1,000; Abcam).

Techniques: Knockdown, Protein-Protein interactions, Western Blot, Phospho-proteomics