Journal: Stem Cell Reports

Article Title: Anteroposterior Wnt-RA Gradient Defines Adhesion and Migration Properties of Neural Progenitors in Developing Spinal Cord

doi: 10.1016/j.stemcr.2020.08.016

Figure Lengend Snippet: Wnt/β-catenin Signaling Determines AP Gradients of Cell-Cell Adhesions (A) Schematic diagram of E12 embryo, and diagram of cell-sorting assay where cells pre-treated with DMSO, Chiron, Dkk1, or RA were allowed to self-sort based on their adhesion affinity. The gradient of purple color indicates the activation rate of different signals upon chemical treatment. (B) Quantification of the percentage of sorting patterns. NPCs treated with chemicals were from E12 T or Br. Br versus T resulted in significantly (80%) higher enveloped phenotype than segregated 5% and checkerboard 15%. Br versus T-Dkk1 resulted in significantly (58%) higher checkerboard phenotype than segregated 14% and enveloped 28% phenotypes. Br versus T-RA resulted in significantly (70%) higher checkerboard than 12% segregated and 18% enveloped. Br-Chiron versus T resulted in significantly (47%) higher checkerboard phenotype than segregated 30% and segregated 23% phenotypes. Br-Chiron versus T-Chiron E12 resulted in significantly (52%) higher enveloped phenotype than segregated 3% and checkerboard 45%. Br versus Br-DMSO resulted in significantly (88%) higher checkerboard phenotype than enveloped 12% phenotype. Br versus Br-Chiron resulted in significantly (72%) higher enveloped phenotype than checkerboard 28%. Data are shown as percentage; number of independent experiments = 3; number of examined neurospheres = 1,638. (C) RT-PCR analyses present the mRNA fold change in β-catenin , Lef1 , Tcf4 , Axin2 , and Cyclin D1 expression levels upon activation of Wnt signaling with 2 μM Chiron. Data are shown as mean ± SD; number of independent experiments = 3. ∗ p < 0.001 via Student's t test. (D) qRT-PCR analysis shows the fold change in mRNA levels of Wnt -related genes pre-treated with chemicals. Data are shown as mean ± SD; number of independent experiments = 3; ∗ p < 0.001 via one-way ANOVA. (E) qRT-PCR of Wnt -related genes in tail NPCs pre-treated with DMSO or 1 μM RA. Data are shown as mean ± SD; number of independent experiments = 3; ∗ p < 0.001 via Student's t test. Orange shading indicates the downstream genes of RA signaling. (F and I) Box plots for the directionality of brachial (F) and tail (I) NPCs pre-treated with chemicals during the scratch assay. Data are shown as median ± SD; number of independent experiments = 4; number of examined cells = 401; ∗ p < 0.001 via one-way ANOVA on ranks. (G and J) Top shows cell trajectory of migrating brachial (G) and tail (J) NPCs, and the bottom shows the angle histograms (polar graph) representing the direction of migrating cells at the leading and following domains. Different lengths of the angle bar represent different grouped cells with a particular angle. Front (angle 0°–180°) and rear (angle 180°–360°) cells were grouped based on the position of the centrosome. Number of independent experiments = 3; number of examined cells = 395. (H and K) Measurement of front-to-rear ratio of brachial (H) and tail (K) NPCs that were determined in polar graphs following chemical treatment. Data are shown as mean ± SD; number of independent experiments = 3; number of examined cells = 395; ∗ p < 0.001 via one-way ANOVA.

Article Snippet: For pre-treatment, NPCs at passage 0 were pre-treated with DMSO, 3 μM Chiron, 200 ng/mL DKK-1, 1 μM XAC-939, 1 μM Wnt-C59, or 1 μM RA for 4 consecutive days.

Techniques: FACS, Activation Assay, Reverse Transcription Polymerase Chain Reaction, Expressing, Quantitative RT-PCR, Wound Healing Assay

Journal: Cell metabolism

Article Title: Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia

doi: 10.1016/j.cmet.2023.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: Progression Free Survival Mice were considered to have reached the PFS endpoint when the volume of their tumors exceeded 2000 mm 3 , as measured by handheld calipers. . Dexamethasone treatment Dexamethasone 21-phosphate disodium salt (#D1159; Sigma-Aldrich) was dissolved in dH 2 O and administered intraperitoneally (i.p.) at 1mg/kg daily. . N-acetyl cysteine (NAC) treatment N-Acetyl-L-cysteine (#A9165; Sigma-Aldrich) was dissolved in 0.9% NaCl sterile saline solution (#Z1377; Thermo Fisher) and administered intraperitoneally (i.p.) at 150mg/kg daily. . Liproxstatin-1 treatment Liproxstatin-1 (#S7699; Selleck Chemicals) was dissolved in 2% Dimethyl Sulfoxide (DMSO) (#12611S; Cell Signaling), 40% Polyethylene glycol 300 (PEG300) (#S6704; Selleck Chemicals), 2% Tween-80 and 56% double-distilled water (ddH 2 O), individually and in this specific order, and administered intraperitoneally (i.p.) at 10mg/kg daily. . RSL3 treatment RSL3 ((1S,3R)-RSL3) (#HY-100218A; MedChemExpress) was dissolved in 10% DMSO (#12611S; Cell Signaling), 40% PEG300 (#S6704; Selleck Chemicals), 5% Tween-80 and 45% NaCl 0.9% sterile saline solution (#Z1377; Thermo Fisher) and administered intraperitoneally (i.p.) at 5mg/kg daily. . ACTH stimulation test (synacthen test) ACTH (#HOR-279, ProSpec) was reconstituted in dH 2 0 and injected intraperitoneally at a dose of 1 ug/g body weight.

Techniques: Recombinant, Sterility, Saline, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Iron Assay, Competitive ELISA, Software

Journal: Cell metabolism

Article Title: Ketogenic diet promotes tumor ferroptosis but induces relative corticosterone deficiency that accelerates cachexia

doi: 10.1016/j.cmet.2023.05.008

Figure Lengend Snippet: Key resources table

Article Snippet: Progression Free Survival Mice were considered to have reached the PFS endpoint when the volume of their tumors exceeded 2000 mm 3 , as measured by handheld calipers. . Dexamethasone treatment Dexamethasone 21-phosphate disodium salt (#D1159; Sigma-Aldrich) was dissolved in dH 2 O and administered intraperitoneally (i.p.) at 1mg/kg daily. . N-acetyl cysteine (NAC) treatment N-Acetyl-L-cysteine (#A9165; Sigma-Aldrich) was dissolved in 0.9% NaCl sterile saline solution (#Z1377; Thermo Fisher) and administered intraperitoneally (i.p.) at 150mg/kg daily. . Liproxstatin-1 treatment Liproxstatin-1 (#S7699; Selleck Chemicals) was dissolved in 2% Dimethyl Sulfoxide (DMSO) (#12611S; Cell Signaling), 40% Polyethylene glycol 300 (PEG300) (#S6704; Selleck Chemicals), 2% Tween-80 and 56% double-distilled water (ddH 2 O), individually and in this specific order, and administered intraperitoneally (i.p.) at 10mg/kg daily. . RSL3 treatment RSL3 ((1S,3R)-RSL3) (#HY-100218A; MedChemExpress) was dissolved in 10% DMSO (#12611S; Cell Signaling), 40% PEG300 (#S6704; Selleck Chemicals), 5% Tween-80 and 45% NaCl 0.9% sterile saline solution (#Z1377; Thermo Fisher) and administered intraperitoneally (i.p.) at 5mg/kg daily. . ACTH stimulation test (synacthen test) ACTH (#HOR-279, ProSpec) was reconstituted in dH 2 0 and injected intraperitoneally at a dose of 1 ug/g body weight.

Techniques: Recombinant, Sterility, Saline, Lysis, Protease Inhibitor, Red Blood Cell Lysis, Enzyme-linked Immunosorbent Assay, Iron Assay, Competitive ELISA, Software

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

doi: 10.1074/jbc.m008793200

Figure Lengend Snippet: FIG. 6. The peptide aldehyde, MG132, inhibits b-secretase cleavage of APPSw in stably transfected CHO cells. A, CHOAPPSw cells were incubated for 6 h with Me2SO vehicle or 20–100 mM MG132. Conditioned medium was collected and analyzed for se- creted APPSwb by ELISA. To control for any interference with the ELISA, MG132 was added to a final concentration of 100 mM after CHOAPPSw cells had conditioned medium for 6 h (white bar labeled 1001). B, an ELISA was also conducted on 10-mg aliquots of CHOAPPSw cell lysates to determine whether MG132 treatment al- tered levels of APPSw in the cells. C, MG132 inhibits secretion of APPSwb in a time-dependent manner. CHOAPPSw cells were incu- bated with either Me2SO vehicle (solid line) or 80 mM MG132 (dashed line) for 0, 0.5, 1, 2, 4, 6, or 8 h. Conditioned medium was collected and the levels of secreted APPSwb determined by ELISA.

Article Snippet: The peptide aldehyde, MG132, was dissolved in dimethyl sulfoxide (Me2SO) at a concentration of 10 mM (Peptides International).

Techniques: Stable Transfection, Transfection, Incubation, Enzyme-linked Immunosorbent Assay, Control, Concentration Assay, Labeling

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

doi: 10.1074/jbc.m008793200

Figure Lengend Snippet: FIG. 7. A comparison of AEBSF and MG132 incubation on in- tracellular production of APPSwb. CHOAPPSw cells were pulsed for 12 min with Tran35S-label. The cells were washed and then chased in complete DMEM containing Me2SO, 80 mM MG132, or 1 mM AEBSF for either 45 or 90 min as described under “Experimental Procedures.” Intracellular APPSwb indicated by an arrow (panel B) and full-length APPSw (panel A) were isolated from cell lysates by sequential immu- noprecipitation with 931 and 945 antisera, respectively. Conditioned medium was isolated and analyzed for secreted APPSwb (panel C) and APPSwa (panel D) by immunoprecipitation using the 931 and 6E10 antibodies, respectively.

Article Snippet: The peptide aldehyde, MG132, was dissolved in dimethyl sulfoxide (Me2SO) at a concentration of 10 mM (Peptides International).

Techniques: Comparison, Incubation, Isolation, Immunoprecipitation

Journal: Journal of Biological Chemistry

Article Title: The Protease Inhibitor, MG132, Blocks Maturation of the Amyloid Precursor Protein Swedish Mutant Preventing Cleavage by β-Secretase

doi: 10.1074/jbc.m008793200

Figure Lengend Snippet: FIG. 8. A 2-h preincubation with MG132 blocks intracellular production of APPSwb in CHOAPPSw cells. CHOAPPSw cells were preincubated for 2 h with either Me2SO control or 80 mM MG132. The cells were subsequently starved of methionine and cysteine, pulsed for 12 min with Tran35S-label and chased all in the presence of either Me2SO or 80 mM MG132 for either 45 or 90 min as described under “Experimental Procedures.” Intracellular APPSwb indicated by an ar- row (panel B) and full-length APPSw (panel A) were isolated from cell lysates by sequential immunoprecipitation with 931 and 945 antisera, respectively. Conditioned medium was isolated and analyzed for se- creted APPSwb (panel C) and APPSwa (panel D) by immunoprecipita- tion using the 931 and 6E10 antibodies, respectively.

Article Snippet: The peptide aldehyde, MG132, was dissolved in dimethyl sulfoxide (Me2SO) at a concentration of 10 mM (Peptides International).

Techniques: Control, Isolation, Immunoprecipitation