retinoid 9cra cat. no. r245015  (Toronto Research Chemicals)

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Comparison of RA resolution and isomerization during extraction. A , structures of RA isomers. Standards of RA isomers (100 fmol each) were resolved by either ( B ), original LC conditions or ( C ), revised LC conditions. D , atRA and ( F ), 9cRA standards in saline/ethanol were extracted and resolved by the original LC conditions. E , atRA and ( G ), 9cRA standards in methanol were extracted and resolved by the revised LC conditions. Analyses were done by LC/MS/MS using multiple reaction monitoring with a triple quadrupole mass spectrometer, which first isolated the molecular ion at m/z 301 [M + H] + in Q1 and then quantified its product ion at m/z 205 in Q3. 9cRA, 9- cis -retinoic acid; atRA, all- trans -retinoic acid; LC, liquid chromatography; LC/MS/MS, LC-tandem mass spectrometry; RA, retinoic acid.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Comparison, Extraction, Saline, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Isolation, Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Recovery of 9cRA from pancreas without isomerization. A , one microliter of a 100 nM solution of 9cRA in methanol (100 fmol) was injected into LC/MS/MS. B , a pancreas from a male mouse was homogenized in 1.5 ml methanol. Half of the homogenate was analyzed using the revised protocol. C , ten microliters of a 100 nM solution of 9cRA was added to the remaining half of the pancreas homogenate and analyzed using the revised protocol. Recovery was 96%. 9cRA, 9- cis -retinoic acid; LC/MS/MS, LC-tandem mass spectrometry.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Injection, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Mass spectrum of endogenous 9cRA. Pancreata from 46 mice were pooled, homogenized in methanol, and applied to LC. The 11.5 to 12.5 min fractions were collected, concentrated, and reinjected for a Q3 product ion scan. A , total ion chromatogram of the reinjected sample. B , Q3 mass scan of endogenous 9cRA. 9cRA, 9- cis -retinoic acid; LC, liquid chromatography.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Liquid Chromatography

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Derivatization of endogenous 9cRA with 3-NPH. A , reaction of 9cRA with 3-NPH. B , chromatogram of 9,13dcRA-3-NPH, 9cRA-3-NPH and atRA-3-NPH standards (300 fmol each). C , livers from six mice were homogenized with methanol and analyzed by LC/MS/MS for comparison to authentic 3-NPH derivatives. The Y -axes shows intensity of the product ion in Q3 (m/z 283.3). 3-NPH, 3-nitropenylhydrazine; 9cRA, 9- cis -retinoic acid; LatRA, all- trans -retinoic acid; C/MS/MS, LC-tandem mass spectrometry.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Liquid Chromatography with Mass Spectroscopy, Comparison, Tandem Mass Spectroscopy, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Variations of 9cRA and atRA with fasting versus refeeding

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques:

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: 9cRA biosynthesis in a β-cell line. 832/13 cells were FBS starved 16 h in medium with 11.1 mM glucose and then incubated 4 h with 250 nM all- trans -retinol or 9- cis -retinol and 3 mM or 15 mM glucose with continuing FBS starvation. Retinoids were quantified by LC/MS/MS. A , retinol isomers recovered. B , retinal isomers generated. C , atRA and 9cRA generated: ∗ p < 0.05, ∗∗ p < 0.02, # p < 0.001 versus 3 mM glucose. 9cRA, 9- cis -retinoic acid; atRA, all- trans -retinoic acid; FBS, fetal bovine serum; LC/MS/MS, LC-tandem mass spectrometry.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Incubation, Liquid Chromatography with Mass Spectroscopy, Generated, Mass Spectrometry

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Mechanism of glucose action. β-cells (832/13) were incubated 6 h with serum-free medium containing 3 mM or 15 mM glucose plus the additions noted. A , insulin effects on Rdh5 mRNA. Cells were incubated in the presence or absence of insulin (100 ng, n = 6 plates/group, ∗∗∗ p < 0.005 versus 3 mM glucose. B , IBMX (10 μM) and exendin-4 (10 nM) effects on Rdh5 mRNA (n = 6, ## p < 0.0001 versus 3 mM glucose; x p < 0.0001 versus 3 mM glucose and IBMX. C , IBMX (10 μM) and exendin-4 (10 nM) effects on Crem mRNA (## p < 0.0001 versus 3 mM glucose; ∗∗∗ p < 0.005 versus 3 mM glucose with IBMX; x p <0.0001 versus 15 mM glucose. D , riociguat (Rio) effects on Rdh5 mRNA (∗ p < 0.05 versus 3 mM glucose). E , effects of FoxO1 (100 nM AS1842856), CaMK (1 μM CK59), or Akt (1 μM Triciribine hydrate) inhibitors (## p < 0.0001 versus 3 mM glucose, ∗∗∗ p < 0.005 versus 15 mM glucose. F , effect of 9cRA on Atg7 mRNA (## p < 0.0001 versus 3 mM glucose). 9cRA (1 nM) increases Atg7 mRNA in 832/13 cells in the presence of 15 mM glucose (## p < 0.0001 versus 3 mM glucose). G , effects of inhibitors of mTOR (10 nM rapamycin), PKA (400 nM 14–22 amide) or GSK3β (100 nM XXV) (# p < 0.001 versus 3 mM glucose). 9cRA, 9- cis -retinoic acid; IBMX, 3-isobutyl-l-methylxanthine.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Incubation

Journal: The Journal of Biological Chemistry

Article Title: Energy status regulates levels of the RAR/RXR ligand 9- cis -retinoic acid in mammalian tissues: Glucose reduces its synthesis in β-cells

doi: 10.1016/j.jbc.2023.105255

Figure Lengend Snippet: Glucose and 9cRA exert opposing actions on β-cells. The glucose transporter Glut2 enables glucose uptake by β-cells. Glucokinase (Gck) catalyzes phosphorylation of intracellular glucose, which undergoes glycolysis to generate pyruvate. Acetyl-CoA formed from pyruvate enters the mitochondria to produce ATP. The increase in ATP induces depolarization of the K + channel, which prompts insulin release (). Increased ATP also stimulates cAMP biosynthesis via adenylate cyclase (Adcy), which induces calmodulin-dependent protein kinase (CaMK). CamK activates Akt, which phosphorylates the transcription factor FoxO1, resulting in its expulsion from the nucleus. Glucose metabolism also results in the repression of the vital autophagy regulator Atg7 . FoxO1 induces expression of Rdh5 , which catalyzes the first and rate-limiting reaction in 9cRA biosynthesis. The current data and previous reports show that 9cRA exerts diverse actions on β-cells ( , ). 9cRA rapidly reduces activities of Glut2 and Gck through nongenomic mechanisms. It represses transcription of Pdx1 and HNF4α . The latter two transcription factors induce transcription of Glut2 and Gck , decreasing glucose metabolism and thereby tempering GSIS. 9cRA also induces Atg7 , maintaining β-cell autophagy and vigor. Even though Adcy, CaMK, and Akt affect the activities of PKA, mTOR, and GSK3β, the latter do not affect Rdh5 mRNA. 9cRA, 9- cis -retinoic acid; GSIS, glucose-stimulated insulin secretion.

Article Snippet: Standards of atRA, 9cRA, 13cRA, and 3-nitrophenylhydrazine hydrochloride (3-NPH·HCl) and N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDC·HCl) were purchased from Sigma-Aldrich.

Techniques: Expressing

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Relative gene expression upon exposure to retinoids for 9 d (DIV13, a) or 18 d (DIV22, b). Data are normalized to the time-matched solvent control (i.e., DIV13 or DIV22); median log 2 -fold gene expression changes are displayed. Fold-change ratios are scalar color-coded between ± 3-fold change, changes > 3 (or, in the case of thyroid hormone signalling on DIV22 < −3) are shown in dark red. Detailed bar-chart representation of gene expression changes is provided in ; gene symbol abbreviations are explained in , . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, TH: thyroid hormone. n ≥ 3. * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Gene Expression, Solvent, Control

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Relative gene expression of selected markers upon exposure to retinoids for 18 d (DIV22). a-c: Markers of retinoic acid signaling, d: neural stem cell marker, e: marker of (mature) neurons, f: early glial marker. Data are normalized to the time-matched solvent control (i.e., DIV22); log 2 fold gene expression changes are displayed. Further studied genes and their relative expression, as well as the expression of selected genes on DIV13, are listed in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, SC: solvent control (0.004% methanol). Mean ± SEM; n ≥ 3. * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Gene Expression, Marker, Solvent, Control, Expressing

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Western blot detection and relative quantification of neurodifferentiation biomarkers upon exposure to retinoids for 18 d (DIV22). Data represent log 2 fold-change values relative to time-matched solvent control (SC). Housekeeping protein β actin was used for normalization (not shown). a) marker for NSCs, b) early neural marker, c) marker of dopaminergic neurons (TH = tyrosine hydroxylase), d) early astroglial marker, e) marker of astrocytes. Further studied proteins, their relative intensity, as well as the detection of selected markers on DIV13, are listed in . Further information on the studied markers and the respective antibodies and dilutions used are listed in . DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, TH: tyrosine hydroxylase. * p < 0.05, * * p < 0.01, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Western Blot, Quantitative Proteomics, Solvent, Control, Marker

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Proteomics analysis of neural protein markers determined upon exposure to retinoids for 18 d (DIV22). Protein abundance was normalized to the abundance of the housekeeping protein β actin. Further studied proteins, their relative abundance, and their detection on DIV13 are listed in ; protein quantities are given in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, SC: solvent control (0.004% methanol). n ≥ 3, * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Quantitative Proteomics, Solvent, Control

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Immunofluorescence staining of differentiating H9 NSCs at DIV22. Composite overview of RA concentration response effects and controls on network patterning visualized by IFL; representative image. Red: synaptophysin (SYP; synapses), green: βIII tubulin (TUJ1; neurons), blue: nuclei. The scale bar in subfigure a corresponds to 1000 µm. The edge length of inset detailed image sections corresponds to 400 µm. One representative image of 3 independent experiments and staining in duplicates is shown. Exemplary images of other biomarkers detected by IFL are listed in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Immunofluorescence, Staining, Concentration Assay

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Relative gene expression upon exposure to retinoids for 9 d (DIV13, a) or 18 d (DIV22, b). Data are normalized to the time-matched solvent control (i.e., DIV13 or DIV22); median log 2 -fold gene expression changes are displayed. Fold-change ratios are scalar color-coded between ± 3-fold change, changes > 3 (or, in the case of thyroid hormone signalling on DIV22 < −3) are shown in dark red. Detailed bar-chart representation of gene expression changes is provided in ; gene symbol abbreviations are explained in , . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, TH: thyroid hormone. n ≥ 3. * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Expressing, Solvent, Control

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Relative gene expression of selected markers upon exposure to retinoids for 18 d (DIV22). a-c: Markers of retinoic acid signaling, d: neural stem cell marker, e: marker of (mature) neurons, f: early glial marker. Data are normalized to the time-matched solvent control (i.e., DIV22); log 2 fold gene expression changes are displayed. Further studied genes and their relative expression, as well as the expression of selected genes on DIV13, are listed in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, SC: solvent control (0.004% methanol). Mean ± SEM; n ≥ 3. * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Expressing, Marker, Solvent, Control

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Western blot detection and relative quantification of neurodifferentiation biomarkers upon exposure to retinoids for 18 d (DIV22). Data represent log 2 fold-change values relative to time-matched solvent control (SC). Housekeeping protein β actin was used for normalization (not shown). a) marker for NSCs, b) early neural marker, c) marker of dopaminergic neurons (TH = tyrosine hydroxylase), d) early astroglial marker, e) marker of astrocytes. Further studied proteins, their relative intensity, as well as the detection of selected markers on DIV13, are listed in . Further information on the studied markers and the respective antibodies and dilutions used are listed in . DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, TH: tyrosine hydroxylase. * p < 0.05, * * p < 0.01, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Western Blot, Solvent, Control, Marker

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Proteomics analysis of neural protein markers determined upon exposure to retinoids for 18 d (DIV22). Protein abundance was normalized to the abundance of the housekeeping protein β actin. Further studied proteins, their relative abundance, and their detection on DIV13 are listed in ; protein quantities are given in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid, DIV4: non-differentiated sample collected on DIV4 before exposure commenced, diff: time-matched sample differentiated by growth factors BDNF and GDNF, SC: solvent control (0.004% methanol). n ≥ 3, * p < 0.05, * * p < 0.01, * ** p < 0.001, # p < 0.0001.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Solvent, Control

Journal: Toxicology

Article Title: Effects of all- trans and 9- cis retinoic acid on differentiating human neural stem cells in vitro

doi: 10.1016/j.tox.2023.153461

Figure Lengend Snippet: Immunofluorescence staining of differentiating H9 NSCs at DIV22. Composite overview of RA concentration response effects and controls on network patterning visualized by IFL; representative image. Red: synaptophysin (SYP; synapses), green: βIII tubulin (TUJ1; neurons), blue: nuclei. The scale bar in subfigure a corresponds to 1000 µm. The edge length of inset detailed image sections corresponds to 400 µm. One representative image of 3 independent experiments and staining in duplicates is shown. Exemplary images of other biomarkers detected by IFL are listed in . atRA: all-trans retinoic acid, 9cRA: 9-cis retinoic acid.

Article Snippet: Non-cytotoxic concentrations (8, 40, 200, 1000 nM) of two retinoids, atRA (cat. no. R250200, Toronto Research Chemicals, Toronto, ON, Canada), and 9cRA (cat. no. R245015, Toronto Research Chemicals, Toronto, ON, Canada) were used alongside two negative controls (“non-differentiated” cells kept in NSC maintenance medium containing bFGF and “naïve”/non-treated cells cultured in naïve medium) and a growth factor-induced differentiation-positive control (“differentiated”; cultured in naïve medium supplemented with BDNF and GDNF; see also , ).

Techniques: Immunofluorescence, Staining, Concentration Assay

Journal: PLoS ONE

Article Title: Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D 3

doi: 10.1371/journal.pone.0113722

Figure Lengend Snippet: (A) NBT-reducing activities. Cells were treated with vehicle control (Cont), 100 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH) 2 D 3 (D3) for 5 days. *, p <0.05; **, p <0.01; ***, p <0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). (B) Morphological changes of THP-1 and HL60 cells treated with 9cRA and/or 1,25(OH) 2 D 3 . Cells were treated with vehicle control (Cont), 100 nM 9cRA and/or 100 nM 1,25(OH) 2 D 3 for 5 days and the cell smears were stained with May-Grünwald-Giemsa. (C) Cell proliferations. Cells (1×10 5 /ml) were cultured with vehicle control (Cont), 100 nM 9cRA and/or 100 nm 1,25(OH) 2 D 3 (D3), and cell numbers were counted at indicated days. *, p <0.05; **, p <0.01; ***, p <0.001 vs Cont; ###, p <0.001 vs 9cRA; +++, p <0.001 vs D3 (one-way ANOVA followed by Tukey’s multiple comparisons). †††, p <0.001 (two-way ANOVA).

Article Snippet: 1,25(OH) 2 D 3 , ATRA and 9cRA were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Staining, Cell Culture

Journal: PLoS ONE

Article Title: Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D 3

doi: 10.1371/journal.pone.0113722

Figure Lengend Snippet: Representative histograms of CD14 expression (A) and CD11b expression (B) in THP-1 cells. Cells were treated with vehicle control (Cont), 100 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH) 2 D 3 (D3) for 96 hours. Filled curves, vehicle control. Similar results were obtained from repeated experiments. (C) CD14 mRNA levels in THP-1 and HL60 cells. Cells were treated with vehicle control (Cont), 30 nM 9cRA, ATRA, Am80 or HX630 in the absence or presence of 100 nM 1,25(OH) 2 D 3 (D3) for 72 hours. **, p <0.01; ***, p <0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

Article Snippet: 1,25(OH) 2 D 3 , ATRA and 9cRA were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Expressing

Journal: PLoS ONE

Article Title: Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D 3

doi: 10.1371/journal.pone.0113722

Figure Lengend Snippet: mRNA levels of the M2 markers CD163 (A), ARG1 (B), IL10 (C), and the M1 marker IL12B (D) in THP-1 and HL60 cells. (E) mRNA levels of the M2 marker TGFB1 , the M1 markers TNF , IL6 , and NOS2 in THP-1 cells. Cells were treated with vehicle control (Cont), 30 nM 9cRA, or ATRA in the absence or presence of 100 nM 1,25(OH) 2 D 3 (D3) for 72 hours. *, p <0.05; **, p <0.01; ***, p <0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). n.d., not detected.

Article Snippet: 1,25(OH) 2 D 3 , ATRA and 9cRA were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Marker

Journal: PLoS ONE

Article Title: Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D 3

doi: 10.1371/journal.pone.0113722

Figure Lengend Snippet: (A) Representative histograms of CD14 and CD163 expression. (B) Quantification of mean fluorescence intensity of CD163 and CD14 expression. (C) Quantification of percentages of CD163+/CD14+ cells and CD163−/CD14+ cells. Cells were treated with vehicle control (Cont), 100 nM 9cRA and/or 100 nM 1,25(OH) 2 D 3 (D3) for 96 hours. **, p <0.01; ***, p <0.001 (one-way ANOVA followed by Tukey’s multiple comparisons).

Article Snippet: 1,25(OH) 2 D 3 , ATRA and 9cRA were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: Expressing, Fluorescence

Journal: PLoS ONE

Article Title: Induced Differentiation of Human Myeloid Leukemia Cells into M2 Macrophages by Combined Treatment with Retinoic Acid and 1α,25-Dihydroxyvitamin D 3

doi: 10.1371/journal.pone.0113722

Figure Lengend Snippet: Cells were treated with vehicle control (Cont), 30 nM 9cRA and/or 100 nM 1,25(OH) 2 D 3 (D3) for 72 hours and secreted IL-10 levels in media were measured. **, p <0.01; ***, p <0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). n.d., not detected.

Article Snippet: 1,25(OH) 2 D 3 , ATRA and 9cRA were purchased from Wako Pure Chemical Industries (Osaka, Japan).

Techniques: