Journal: Infection and Immunity

Article Title: ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress

doi: 10.1128/iai.00300-24

Figure Lengend Snippet: Pertussis toxin induces canonical markers of ER stress independent of catalytic activity, and ER stress contributes to the effect of PTx on STING. ( A ) BMDMs were treated overnight with media alone, 250 ng/mL of WT, or 250 ng/mL R9K;E129A mutant purified pertussis toxin. The following morning, media containing toxin were removed and replaced with fresh culture media for 4 hrs. Total RNA was then harvested, and the expression of Bip/GRP78 was quantified by qRT-PCR. ( B ) BMDMs were treated as in ( A ), and whole-cell lysates were harvested and analyzed by immunoblot for the indicated genes. Representative of n = 4 ( C ) BMDMs was cultured overnight with media alone or 250 ng/mL of WT PTx in the presence or absence of 100 µM TUDC. The following morning, media containing toxin was removed and replaced with fresh culture media. BMDMs were stimulated by the addition of 10 µg/mL cGAMP to culture media for 4 hrs. Total RNA was harvested, and the expression of indicated genes was quantified by qRT-PCR. * P < 0.01.

Article Snippet: Sodium tauroursodeoxycholate (TUDC) was purchased from Selleckchem (cat no. S7896).

Techniques: Activity Assay, Mutagenesis, Purification, Expressing, Quantitative RT-PCR, Western Blot, Cell Culture

Journal: Infection and Immunity

Article Title: ER-transiting bacterial toxins amplify STING innate immune responses and elicit ER stress

doi: 10.1128/iai.00300-24

Figure Lengend Snippet: The catalytically inactive cholera toxin B subunit induces ER stress and primes STING responses. (A and B) BMDMs were cultured in media alone or intoxicated for 3 hrs with 10 µg of purified cholera toxin. BMDMs were subsequently stimulated by the addition of 10 µg/mL cGAMP to culture media for 4 hrs. Total RNA was harvested, and the expression of indicated genes was quantified by qRT-PCR. * P ≤ 0.05. (C) BMDMs were pre-treated with Forskolin for 3 hrs prior to stimulation with cGAMP for 4 hrs. Representative of n = 3. * P ≤ 0.05. (D) BMDMs were pre-treated with purified CTx B overnight prior to stimulation with 10 µg of cGAMP for 3 hrs. * P = 0.003. (E) BMDMs were treated with CTx B overnight as in (D). Whole-cell lysates were harvested and analyzed by immunoblot using antibodies directed against the indicated protein targets. Representative of n = 3. (F) BMDMs were cultured overnight with media alone or 20 ng of CTx B in the presence or absence of 100 µM TUDC. The following morning, media containing toxin were removed and replaced with fresh culture media. BMDMs were stimulated by the addition of 10 µg/mL cGAMP to culture media for 4 hrs. Total RNA was harvested, and the expression of indicated genes was quantified by qRT-PCR. * P < 0.05. N = 3. (G) Immortalized murine intestinal epithelial cells were treated overnight with media alone, 20 ng/mL CTx B, or 200 ng/mL PTx followed by stimulation with DMXAA for 4 hrs.

Article Snippet: Sodium tauroursodeoxycholate (TUDC) was purchased from Selleckchem (cat no. S7896).

Techniques: Cell Culture, Purification, Expressing, Quantitative RT-PCR, Western Blot