DNA microarrays (60-mer, Agilent Technologies, USA) based on the full P. falciparum genome as previously described [96 (link)] were used to assess global transcriptomic changes in gametocytes treated with JIB-04. Day 2 and day 3 gametocyte cultures (1–3% gametocytaemia, 4% haematocrit) were treated with 5 µM JIB-04 (Cayman Chemicals) for 24 h followed by isolation of gametocytes using 0.01% (w/v) saponin. Total RNA was isolated with a combination of TRIzol (Sigma-Aldrich, USA) and phenol–chloroform extraction and subsequently used to synthesise cDNA as previously described [96 (link)] for the untreated and JIB-04 treated day 2 and day 3 gametocyte samples. Sample cDNA was labelled with Cy5 dye (GE Healthcare, USA) prior to hybridisation to arrays with an equal amount (350–500 ng) of Cy3-labelled (GE Healthcare, USA) reference pool containing equal amounts of cDNA from each gametocyte sample and mixed stage 3D7 asexual parasites. After hybridisation, the slides were scanned on a G2600D (Agilent Technologies, USA) scanner and normalised signal intensities for each oligo were extracted using the GE2_1100_Jul11_no_spikein protocol and Agilent Feature Extractor Software (v 11.5.1.1) as described before [96 (link)].
Jib 04
Manufactured by Merck Group
Sourced in United States
The JIB-04 is a laboratory equipment product manufactured by Merck Group. It is designed for general laboratory use, with a core function of providing a controlled environment for various scientific experiments and analysis procedures. The JIB-04 ensures a stable and consistent environment for the samples or materials being studied.
Automatically generated - may contain errors
Lab products found in correlation
Apicidin, by Merck Group (2 mentions)
Sirtinol, by Merck Group (2 mentions)
Unc0638, by Merck Group (2 mentions)
Gsk lsd1, by Merck Group (2 mentions)
Mithramycin a, by Merck Group (2 mentions)
Gsk 126, by Thermo Fisher Scientific (2 mentions)
Gsk j4, by Abcam (2 mentions)
Ly294002, by Merck Group (2 mentions)
Shikonin, by Merck Group (2 mentions)
Avastin, by Roche (2 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Jib 04, by Cayman Chemical (1 mentions)
Cy5 dye, by GE Healthcare (1 mentions)
Cyanine3 (cy3), by GE Healthcare (1 mentions)
Trizol, by Merck Group (1 mentions)
Tropicamide, by Alcon (1 mentions)
Proparacaine, by Alcon (1 mentions)
Phenylephrine, by Akorn (1 mentions)
Glucose free dmem, by Thermo Fisher Scientific (1 mentions)
Dithiothreitol (dtt), by Merck Group (1 mentions)
9 protocols using jib 04
1
Transcriptomic Analysis of Malaria Gametocytes
2
Small Molecule Inhibition Screening
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For all small molecule inhibition studies, the drug was added on the day of encapsulation and replaced with each subsequent media change. The inhibitors used were GSK 126 (100 nM, Fisher Scientific), UNC 0638 (250 nM, Sigma), JIB-04 (1 μM, Sigma), GSK-J4 (10 μM, Abcam), GSK-LSD1 (100 nM, Sigma), Sirtinol (50 μM, Sigma), apicidin (1 μM, Sigma), mithramycin A (50 nM, Sigma), LY294002 (20 μM, Sigma) and SAHA (1 μM, Sigma). All drugs were dissolved in DMSO and diluted in basal medium before adding to the culture medium. DMSO alone was added to the culture medium as a vehicle control.
3
Optimizing Compound Dissolution and Usage
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DNL was purchased from the Ko Da Company (Taipei, Taiwan) and dissolved in ddH2O. Moscatilin was purchased from EMMX Biotechnology (EN10271, CA, USA) and dissolved in dimethyl sulfoxide (DMSO; vehicle). Various inhibitors/antibodies were purchased from various companies, namely JIB-04 (Sigma-Aldrich), Shikonin (S7576; Sigma-Aldrich), Avastin (Hoffmann-La Roche), or Eylea (Regeneron Pharmaceuticals Inc.).
4
Neuroprotective Effect of XFZYD in Retinal Ischemia
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An intake of 1.35 or 2.7 g/kg/day XFZYD (Sun Ten Pharmaceutical CO, Taichung, Taiwan) was administered for seven continuous days before or after HIOP induced retinal ischemia and the rats were then sacrificed. The test rat’s eye that received ischemia was provided with a constant quantity (4 ml) of XFZYD or the “same” volume of vehicle.
After anesthesia as described, the rats’ pupils were dilated with 1% tropicamide (Alcon, ZG, Switzerland) and 2.5% phenylephrine (Akorn, Inc., IL, USA); in addition there was anesthesia of the ocular surface with 0.5% proparacaine (Alcon, ZG, Switzerland); then, a 30-gauge needle attached to a 25 μl syringe was used to perform the intravitreal injections. In certain instances, intravitreal injections (5 μl) of 4 μM Shikonin (Sigma-Aldrich, MO, USA), of 10 μM JIB-04 (Sigma-Aldrich, MO, USA), of 100 mg/4 ml Avastin (Hoffmann-La Roche, Basel, Switzerland) or of vehicle (an equal volume of dimethyl sulfoxide; J.T.Baker, NJ, USA) were given to ischemic eyes for fifteen minutes and retinal ischemia was induced by HIOP later.
After anesthesia as described, the rats’ pupils were dilated with 1% tropicamide (Alcon, ZG, Switzerland) and 2.5% phenylephrine (Akorn, Inc., IL, USA); in addition there was anesthesia of the ocular surface with 0.5% proparacaine (Alcon, ZG, Switzerland); then, a 30-gauge needle attached to a 25 μl syringe was used to perform the intravitreal injections. In certain instances, intravitreal injections (5 μl) of 4 μM Shikonin (Sigma-Aldrich, MO, USA), of 10 μM JIB-04 (Sigma-Aldrich, MO, USA), of 100 mg/4 ml Avastin (Hoffmann-La Roche, Basel, Switzerland) or of vehicle (an equal volume of dimethyl sulfoxide; J.T.Baker, NJ, USA) were given to ischemic eyes for fifteen minutes and retinal ischemia was induced by HIOP later.
5
Cellular Response to Chemical Stresses
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Cells were treated with the following chemical and metabolic stresses for 24 h at doses used previously: 2 µg/mL TU (Abcam), 60 µM H2O2 (Thermo Fisher Scientific), reduced-serum DMEM (0.1% FBS), glucose-free DMEM (Gibco), 2 mM DTT (Sigma), 5 mM NAC (Sigma), and 1 µM DMNQ (Sigma). For heat-shock treatment, cells were incubated for 30 min at 43°C and returned for 24 h prior to collection to 37°C .
For G1/S synchronization, cells were treated with 2 mM HU (Sigma) for 20 h. To release, cells were washed twice with culture medium preconditioned in normoxia or hypoxia and supplied with fresh preconditioned medium. For JIB-04 treatment, normoxic cells were pretreated with 62.5 nM JIB-04 (Xcessbio) for 24 h and then treated again with JIB-04 and either transferred to 1% O2 or maintained in normoxia for an additional 24 h. Succinate (Sigma, S9637) was administered at a final concentration of 2 mM, and cells were either maintained in normoxia for 72 h or maintained in normoxia for 48 h prior to being transferred to 1% O2 for 24 h.
For G1/S synchronization, cells were treated with 2 mM HU (Sigma) for 20 h. To release, cells were washed twice with culture medium preconditioned in normoxia or hypoxia and supplied with fresh preconditioned medium. For JIB-04 treatment, normoxic cells were pretreated with 62.5 nM JIB-04 (Xcessbio) for 24 h and then treated again with JIB-04 and either transferred to 1% O2 or maintained in normoxia for an additional 24 h. Succinate (Sigma, S9637) was administered at a final concentration of 2 mM, and cells were either maintained in normoxia for 72 h or maintained in normoxia for 48 h prior to being transferred to 1% O2 for 24 h.
6
Small Molecule Inhibition Screening
7
Dose-dependent Cellular Growth Inhibition Assay
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Cells for dose-response curves using were plated in 96-well plates and JIB-04 (Sigma-Aldrich, Gillingham, Dorset, UK) was added at increasing dose increments from 0 (DMSO (Sigma-Aldrich) only) to 10 µM and growth measured using using CellTiter 96® AQueous One Solution Cell Proliferation Assay (MTS) kit (Promega) at 96 h. GI50s were calculated in GraphPad Prism 7 (GraphPad Software Inc., San Diego, CA, USA) using the mean of six replicates.
8
Oxidative Stress and KDM4C Regulation in HEK293 Cells
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HEK293 cells were cultured under conditions suggested by ATCC. H2O2 treatment (at a concentration of 100 mM) was used to induce oxidative stress in vitro. KDM4C knockdown was carried out using KDM4C human siRNA oligo duplex (Locus ID 23081) (Origene, Rockville, MD, USA). JIB04 (Merck & Co., Inc., Kenilworth, NJ, USA) was used to inhibit KDM4C activity. For siRNA plasmid transfection, HEK293 cells were seeded in a 6-well plate (1 × 105 cells/well) and cultured for 24 h at 37 °C. The cells were then co-incubated with 10 µL Lipofectamine® 2000 (Thermo Fisher Scientific, Inc., Waltham, MA, USA) and 4 µg of plasmids at 37 °C for 6 h. Subsequently, the cells were cultured with fresh medium for an additional 24 h. For the autophagy study, serum starvation was performed with serum-free culture.
SeeSupplementary Figure S1B,C for Further Details of the Study Design.
See
9
Macrophage differentiation and oxidized LDL protocol
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Murine macrophage cell line RAW264.7 [23 (link)] and human monocytic cell line THP-1 were obtained from American Type Culture Collection (ATCC; Rockville, MD). RAW264.7 cells were maintained in DMEM medium (Corning, NY, USA) containing 10% heat inactivated fetal bovine serum (FBS; Gibco, Thermo Fisher Scientific, Frederick, MD) and supplemented with 2 mM L-glutamine. THP-1 cells were cultured in RPMI-1640 medium (Corning) containing 10% heat inactivated FBS. To prepare human macrophages (M0), THP-1 cells were incubated with 30 ng/ml phorbol 12-myristate 13-acetate (PMA; Sigma, St. Louis, MO) for 48 hrs, followed by incubation with RPMI medium without PMA for additional 24 hrs [28 (link)].
Human oxidized LDL was obtained from Peking Union-Biology Co. Ltd (Beijing, China). The IκB kinase (IKK) inhibitor parthenolide [36 (link)] and the pan-selective Jumonji histone demethylase inhibitor JIB-04 [35 (link)] were purchased from Merck Millipore (Billerica, MA).
Human oxidized LDL was obtained from Peking Union-Biology Co. Ltd (Beijing, China). The IκB kinase (IKK) inhibitor parthenolide [36 (link)] and the pan-selective Jumonji histone demethylase inhibitor JIB-04 [35 (link)] were purchased from Merck Millipore (Billerica, MA).
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