Cells were seeded so that they would reach at ~90% confluency on the following day. After one wash with PBS, cells were treated with the medium supplemented with the vehicle solvent, sodium propionate (Sigma-Aldrich, P5436), sodium valproate (Sigma-Aldrich, P4543), SAHA (Sigma-Aldrich, SML0061), β-hydroxybutyrate (Sigma-Aldrich, H6501), TSA (Sigma-Aldrich, T8552), sodium butyrate (Sigma-Aldrich, B5887), or HDAC3 inhibitor RGFP966 (Selleckchem, S7229) for 24 hours. Cells were washed with PBS and lysed in the RIPA buffer (Thermo Fisher Scientific, 89900) to prepare soluble protein extracts or in the Triton extraction buffer (PBS containing 0.5% Triton X-100, 2 mM phenylmethylsulfonyl fluoride, 0.02% NaN3, and protease inhibitors) to prepare protein extracts. Extracts were prepared in buffer K and further analyzed by immunoblotting with various antibodies as described above.
P5436
Manufactured by Merck Group
Sourced in United States
P5436 is a laboratory centrifuge designed for general-purpose applications. It has a maximum speed of 6,000 RPM and can accommodate a variety of sample sizes and types. The centrifuge provides reliable and consistent performance to support various laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Ripa buffer, by Thermo Fisher Scientific (2 mentions)
Rnaimax, by Thermo Fisher Scientific (2 mentions)
Opti mem, by Thermo Fisher Scientific (2 mentions)
Rgfp966, by Selleck Chemicals (1 mentions)
T8552, by Merck Group (1 mentions)
S2889, by Merck Group (1 mentions)
Hy 112675, by MedChemExpress (1 mentions)
I29204, by Merck Group (1 mentions)
Penicillin g, by Sangon (1 mentions)
L1750, by Merck Group (1 mentions)
Fetal bovine serum (fbs), by Thermo Fisher Scientific (1 mentions)
Streptomycin, by Sangon (1 mentions)
P8765, by Merck Group (1 mentions)
Multiscan fc, by Thermo Fisher Scientific (1 mentions)
Pf 429242, by Merck Group (1 mentions)
6 protocols using p5436
1
Protein Extraction and Analysis of HDAC Inhibitors
2
Short-chain fatty acid treatment
3
Culturing Mycobacterium Strains in Minimal Media
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The NTM strains used in this study are listed in Table 1 . All clinical samples are from CF patients while lettered samples are mixed morphology cultures from the same date and same patient. Bacteria were cultured at 37° C in 7H9 broth with OADC (Remel, R450605). For minimal media experiments, a modified 7H9 broth was used based on the methods described by Muñoz-Elías and McKinney [58 (link)], specifically, NTM were grown in 7H9 + 0.5% albumin, 0.085% NaCl, 0.05% Tween-80, and carbon substrate (10 mM short chain fatty acid) for the indicated number of days. For the pH adjusted minimal media, the media with all the above listed components was pH adjusted and then filter sterilized prior to SCFA addition and use. IA (Sigma-Aldrich, I29204) was resuspended in sterile ddH2O, pH adjusted (for the pH = 7 IA), filter sterilized, and added to the 7H9 media to the desired concentration. For pH-neutral IA experiments, IA received NaOH to bring the pH to a neutral range before being brought to the desired final volume and filter sterilized for addition to the 7H9 media. 4-Octyl itaconate (MedChem Express, HY-112675) was resuspended in DMSO and added to THP-1 cells at the desired concentrations. The short chain fatty acids (SCFA) acetate, propionate, and butyrate (Sigma-Aldrich, S2889, P5436, & 303410, respectively) were resuspended in sterile ddH20 and filter sterilized prior to use.
4
SW480 Cell Line Culture and Lactic/Propionic Acid Treatment
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SW480 cell line was obtained from ATCC and cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) (Invitrogen, Waltham, MA, United States), 100 mg/mL streptomycin (Sangon, Shanghai, China) and 100 U/mL penicillin G (Sangon, Shanghai, China) at 37°C in an incubator containing 5% CO2. SW480 was cultured in a petri dish. Next, the culture medium of SW480 cells that had grown adherently was discarded and washed with PBS three times, and then fresh culture medium and lactic acid (L1750, Sigma-Aldrich, United States) or propionic acid (P5436, Sigma-Aldrich, United States) were added to culture for 48 h for subsequent experiments. The experiment was repeated thrice.
5
Cell Viability Assay with LPS Stimulation
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To perform the cell toxicity assay, using counting kit-8 (CCK8; CA1210, Solarbio, Beijing, China), according to the manufacturer‘s direction, the cell was adjusted to 1 × 105 cells/mL and seeded in 96-well plates. Cell medium was added 15, 25, 35 mM SP (P5436, Sigma-Aldrich, St Louis, MS, USA) and 10 μM pyrrolidine dithiocarbamate (PDTC; P8765, Sigma-Aldrich, St Louis, MS, USA), then incubated for 2 h. The cells were stimulated by 1, 5 and 10 μg/mL LPS (L4391, from the E. coli 0111:B4, Sigma-Aldrich, St Louis, MS, USA) for 6 h. After this, 10 μL CCK8 solution was added to each well and then the cells were incubated for another 3 h. The OD value was measured at 450 nm by a microplate reader (Multiscan FC, Thermo Fisher Scientific, Waltham, MA, USA).
6
Propionic and Methylmalonic Acid Effects on Lipid Metabolism
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For propionic acid studies, Huh7 cells were cultured in DMEM supplemented with 10% (v/v) LPDS for 24 h and incubated in culture media containing 5 μM statin, the indicated doses of propionic acid sodium salt (Sigma #P5436) or vehicle control (ethanol) for an additional 24 h. For methylmalonic acid studies, Huh7 cells were cultured in Opti-MEM™ with RNAiMAX (ThermoFisher) for 24 h to enhance uptake of methymalonic acid. After this pretreatment, cells were incubated in DMEM containing 10% (v/v) LPDS containing the indicated doses of methylmalonic acid (Sigma #M54058), 5 μM statin or vehicle control (ethanol) for an additional 24 h. Following treatment, gene expression, LDLR activity, total sterol content, and/or 14C-acetate incorporation into cholesterol were assessed as described. In another set of experiments, cells were pretreated with 10 μM S1P inhibitor (PF-429242; Sigma #SML0667) for 2 h prior to addition of statin or propionate; gene expression was assessed as described below.
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