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DDIT3 is a gene that encodes for the CCAAT/enhancer-binding protein (C/EBP) homologous protein (CHOP), a transcription factor involved in the cellular stress response. The DDIT3 product plays a role in the regulation of gene expression in response to various cellular stressors.

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Lab products found in correlation

Rneasy mini kit, by Qiagen (3 mentions) Hmox1, by Thermo Fisher Scientific (2 mentions) Rneasy kit, by Qiagen (1 mentions) Taqman reverse transcription reagent, by Thermo Fisher Scientific (1 mentions) Icam 1, by Thermo Fisher Scientific (1 mentions) Ch25h, by Thermo Fisher Scientific (1 mentions) Vcam 1 mm01320970 m1, by Thermo Fisher Scientific (1 mentions) Ht7900 abi sequence detection system, by Thermo Fisher Scientific (1 mentions) Taqman gene expression master mix, by Thermo Fisher Scientific (1 mentions) Taqman assay, by Thermo Fisher Scientific (1 mentions) Reliaprep rna tissue miniprep system, by Promega (1 mentions) Abi prism 7900, by Thermo Fisher Scientific (1 mentions) High capacity cdna reverse transcription kit, by Thermo Fisher Scientific (1 mentions) Power sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions) Mm00517691 m1, by Thermo Fisher Scientific (1 mentions) Dnase, by Promega (1 mentions) Abi 7500 real time pcr system, by Thermo Fisher Scientific (1 mentions) Hspa1a, by Thermo Fisher Scientific (1 mentions) Viia7, by Thermo Fisher Scientific (1 mentions) Fam dye labeled taqman probes, by Thermo Fisher Scientific (1 mentions)

6 protocols using ddit3

1

Quantitative Gene Expression Analysis

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RNA was isolated by RNeasy Kit (Qiagen). Complementary DNAs were generated from 1.5 μg of purified RNA using TaqMan reverse transcription reagents from Applied Biosystems. Real-time PCR was performed in triplicate with TaqMan PCR Mix (Applied Biosystems) in the HT7900 ABI sequence Detection System (Applied Biosystems). Predesigned primers were purchased from Applied Biosystems (Vcam1 [Mm01320970_m1], Atf4 [Mm00515325_g1], Ddit3 [Mm01135937_g1], Ch25h [Mm00515486_s1], and Icam1 [Mm00516023_m1]), using Gusb (Mm00446953_s1) or Gapdh (Mm99999915_g1) as a housekeeping gene for normalization.
Madenspacher J.H., Morrell E.D., McDonald J.G., Thompson B.M., Li Y., Birukov K.G., Birukova A.A., Stapleton R.D., Alejo A., Karmaus P.W., Meacham J.M., Rai P., Mikacenic C., Wurfel M.M, & Fessler M.B. (2023). 25-Hydroxycholesterol exacerbates vascular leak during acute lung injury. JCI Insight, 8(7), e155448.
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2

Quantitative PCR Analysis of Mouse and Human Samples

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RNeasy Mini Kit (QIAGEN) was used to prepare total RNA from mouse tissues and cultured cells, and ReliaPrep RNA Tissue Miniprep System (Promega) from human samples. Reverse-transcription reaction was carried out with a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems), after treatment with DNAse (Promega). Quantitative PCR analyses were performed using ABI Prism 7900, with TaqMan Gene Expression Master Mix or Power SYBR Green PCR Master Mix (Applied Biosystems). The relative expression levels were normalized by the expression level of Ppia (encoding Cyclophilin A) in samples of mouse tissues and cultured cells, control IgG and control upstream region in ChIP assays, and GAPDH in human samples, respectively. TaqMan Assays were purchased from Applied Biosystems: Hspa5 (BiP), Mm00517691_m1; Ddit3 (CHOP), Mm00492097_m1; Sdf2l1, Mm00452079_m1; Ero1lb, Mm00470754_m1; Pdia3, Mm00433130_m1; Sdf2, Mm00485985_m1; Pparg, Mm00440945_m1; Pten, Mm00477208_m1; Sod1, Mm01344233_g1; Sod2, Mm01313000_m1; Tnf, Mm99999068_m1; Ppara: Mm00627559_m1; Fasn, Mm00662319_m1; Acacb, Mm01204671_m1; Pck1: Mm00440636_m1.
Sasako T., Ohsugi M., Kubota N., Itoh S., Okazaki Y., Terai A., Kubota T., Yamashita S., Nakatsukasa K., Kamura T., Iwayama K., Tokuyama K., Kiyonari H., Furuta Y., Shibahara J., Fukayama M., Enooku K., Okushin K., Tsutsumi T., Tateishi R., Tobe K., Asahara H., Koike K., Kadowaki T, & Ueki K. (2019). Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism. Nature Communications, 10, 947.
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3

Quantifying Stress Response Genes

Cited 1 time
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Total cellular mRNA was isolated using the Qiagen RNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol. Human primer probes [DDIT3 (Hs_00358796_g1), HSPA6 (Hs_00275682_s1), HSPA1A (Hs_00359163_s1), EGR1 (Hs_00152928_m1), HMOX1 (Hs_00157965_m1), SOD2 (Hs_00167309_m1), RSP18 (housekeeping gene; Hs_01375212_g1)], were obtained from Thermo Fisher Scientific. After cDNA synthesis, quantitative PCR reactions were performed as follows: 10 min (95 °C) followed by 15 s (95 °C), 1 min (60 °C), 40 cycles, using the ABI7500 Real-Time PCR System (Applied Biosystems). Amplification plots were generated, and Ct values were recorded as published before.34 (link)
Jandova J., Park S.L., Corenblum M.J., Madhavan L., Snell J.A., Rounds L, & Wondrak G.T. (2022). Mefloquine induces ER stress and apoptosis in BRAFi-resistant A375-BRAFV600E/NRASQ61K malignant melanoma cells targeting intracranial tumors in a bioluminescent murine model. Molecular carcinogenesis, 61(6), 603-614.
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4

Quantitative RNA Expression Analysis

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Total RNA content from 1 × 105 cells were extracted using PureLink® RNA Mini Kit (Applied Biosystems) per manufacturer’s protocol and cDNA conversion was done using High-Capacity cDNA RT Kit (ThermoFisher). Quantitative PCR reactions and analysis were performed using ViiA 7 (ThermoFisher). For BM APC experiments, cells were enriched using CD45 magnetic beads. TaqMan®FAM™ dye-labeled probes including GAPDH (Hs02786624_g1, Mm99999915_g1), GADD45A (Hs00169255_m1, Mm00432802_m1), DDIT3 (Hs00358796_g1, Mm01135937_g1), HERPUD1 (Hs01124269_m1, Mm00445600_m1), IFNA1 (Mm03030145_gH), and IFNB1 (Mm00439552_s1) (ThermoFisher). GAPDH was used to normalize signal expression. Fold change comparison were performed between control and treated samples using the ΔΔCT method as described31 (link).
von Roemeling C.A., Wang Y., Qie Y., Yuan H., Zhao H., Liu X., Yang Z., Yang M., Deng W., Bruno K.A., Chan C.K., Lee A.S., Rosenfeld S.S., Yun K., Johnson A.J., Mitchell D.A., Jiang W, & Kim B.Y. (2020). Therapeutic modulation of phagocytosis in glioblastoma can activate both innate and adaptive antitumour immunity. Nature Communications, 11, 1508.
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5

Gene Expression Analysis in Cultured Cells

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Total RNA was isolated from cultured cells using RNeasy mini kit, Qiagen (Venlo, Netherlands) and cDNA was produced using Iscript cDNA synethesis kit, Bio-Rad (Hercules, CA; #1708890). qRT-PCR was performed using TaqMan master mix, ThermoFisher (Waltham, MA; #4444557). Primers: DDIT3 (HS00358796_g1), BOK (Hs011006404_m1), NOXA1 (Hs00611456_g1), HMOX1 (#Hs00157965_m1), NQO1 (#Hs01045993_g1), GCLM (#Hs00978072_m1), NFE2L2 (Hs00975961_g1), and POLR2A (Hs00172187_m1) were purchased from ThermoFisher. POLR2A was used as the endogenous control. The relative fold change was calculated based on the formula R = 2−(ΔCt sample − ΔCt control).
Parkhurst A., Wang S.Z., Findlay T.R., Malebranche K.J., Odabas A., Alt J., Maxwell M.J., Kaur H., Peer C.J., Figg W.D., Warren K.E., Slusher B.S., Eberhart C.G., Raabe E.H, & Rubens J.A. (2022). Dual mTORC1/2 inhibition compromises cell defenses against exogenous stress potentiating Obatoclax-induced cytotoxicity in atypical teratoid/rhabdoid tumors. Cell Death & Disease, 13(4), 410.
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6

Knockdown of BIP, CHOP, and SIRT3 in 661W Cells

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To knock down BIP or CHOP in 661 W cells, we introduced either control siRNA (Negative Control Lo GC, Thermo Fisher Scientific), or Bip siRNA (Hspa5 MSS204938, Thermo Fisher Scientific) or Chop siRNA (Ddit3 MSS273951, Thermo Fisher Scientific), using Lipofectamine RNAiMAX Reagent (Thermo Fisher Scientific), according to the manufacturer’s protocol, and incubated for 24 h before collecting the cells for experiments. To knock down SIRT3 in 661 W cells, we used Sirt3 shRNA (#NM_022433; Qiagen, Hilden, Germany) with Attractene Transfection Reagent (Qiagen) according to the manufacturer’s protocol. In brief, 661 W cell suspension and transfection complexes were mixed and seeded to 24-well plate, and then transfected cells are incubated for 48 h before the experiments.
Ban N., Ozawa Y., Osada H., Lin J.B., Toda E., Watanabe M., Yuki K., Kubota S., Apte R.S, & Tsubota K. (2017). Neuroprotective role of retinal SIRT3 against acute photo-stress. NPJ Aging and Mechanisms of Disease, 3, 19.
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