Fig 1: The cGAS-STING pathway is required for HMGB1 release(A) Confocal microscopy images depicting HMGB1 (green) and DNA (DAPI, blue) in sgNTC, cGas-deficient, and Sting-deficient PyMT-B6 cells treated with DTX. Representative images of one of three independent experiments shown (left). Scale bars are 25 μM. Quantification reflects MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test, shown as ∗∗∗p < 0.001 (right).(B) Tumor volume in mice bearing orthotopic Cgas- or Sting-deficient PyMT-B6 tumors treated with PTX and either IgG2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm3 (day 0). One of two representative experiments shown with n = 5–10 mice per group as indicated. Data displayed as mean ± SEM, with significance determined by two-way ANOVA, shown as ∗∗p < 0.01.(C) Relative mRNA levels for Cxcl9, Ifnb1, and Ifna1 in PyMT-B6 cells treated with DTX or DMXAA, along with blockade of IFNAR1, as determined by RT-PCR.(D) Release of IFN-β measured by ELISA following treatment with DTX, PTX, or DMXAA. For (C and D), data reflect the mean ± SD of three biological replicates from one of two independent experiments, with significance determined by one-way ANOVA and Sidak’s test for multiple comparisons, shown as ∗p < 0.05, ∗∗p < 0.01, and ∗∗∗p < 0.001.(E) Western blot of phospho-IRF3 (pIRF3) and IRF3 protein present in PyMT-B6 cells exposed to PTX, Tunicamycin, or DMXAA or DTX ± TLR4 inhibitor as indicated. β-actin used as loading control. Molecular weights in kDa are shown to the left. Representative images from one of two independent experiments shown.(F) Confocal microscopy images (left) depicting the colocalization of the Golgi (red), STING (green), and DNA (DAPI, blue) in control, cGas-deficient, and Sting-deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the Golgi as determined by Mander’s overlap (top right) and the MFI of STING (bottom right).(G) Confocal microscopy images (left) depicting calreticulin (ER marker, red), STING (green), and DNA (DAPI, blue) in control, cGas-deficient, and Sting-deficient PyMT-B6 cells treated with DTX. Scale bars, 25 μM. Quantification reflects the colocalization of STING with the ER as determined by Mander’s overlap (top right) and the MFI of calreticulin (bottom right). For (F) and (G), data reflect the mean ± SD in individual cells from two to three images, with significance determined by t test and shown as ∗∗p < 0.01, ∗∗∗p < 0.001, and one of three independent experiments is shown.
Fig 2: STING-dependent ER stress and HMGB1 release in a model of TNBC(A) Confocal microscopy images (left) depicting HMGB1 (green) and DNA (DAPI, blue) in KP1 cells treated with PTX or DTX, with and without TLR4 inhibitor. Scale bars are 25 μM. Representative images of one of three independent experiments shown. Quantification reflects the MFI ± SD in individual cells from 2 to 3 images, with significance determined by t test and shown as ∗∗∗p < 0.001 (right).(B) Quantification of MFI ± SD in individual cells from two to three confocal microscopy images assaying cytosolic dsDNA post-PTX or -DTX treatment, with significance determined by t test and shown as ∗∗p < 0.01 and ∗∗∗p < 0.001.(C) Live cell imaging with ER Tracker Blue-White depicting ER expansion in KP1 cells pretreated with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the MFI ± SD of three biological replicates.(D) Extracellular flow cytometric detection of LAMP1 on KP1 cells 24 h post-pretreatment with NAC or TLR4 inhibitor for 1 h prior to treatment with DTX. Data reflect the percent positivity ± SD of three biological replicates. For (C and D), significance was determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗p < 0.05 and ∗∗∗p < 0.001. One of three independent experiments shown.(E) Tumor volume in mice bearing orthotopic sgNTC or sgSting KP1 tumors treated with PTX and either IgG2a isotype control or αTIM-3. Treatment was initiated when tumors reached ∼50–100 mm3 (day 0).(F) Tumor volume in mice bearing orthotopic sgNTC or sgSting KP1 tumors treated with PTX and either IgG2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm3 (day 0).(G) Flow cytometric analysis of CD103+ cDC1s and Ly6G+ neutrophils in the tumors from (F). Data from one of two independent experiments with n = 5 mice per group. Data shown as mean ± SEM, with significance determined by one-way ANOVA with Sidak’s test for multiple comparisons, shown as ∗p < 0.05 and ∗∗p < 0.01.(H) Tumor volume in mice bearing orthotopic sgNTC control or Ifnb1-deficient KP1 tumors treated with PTX and IgG2a isotype control or αPD-1. Treatment was initiated when tumors reached ∼50–100 mm3 (day 0). For (E), (F), and (H), data merged from two independent experiments, with n = 9–10 mice per group. Data shown as mean ± SEM with significance determined by two-way ANOVA displayed with ∗p < 0.05 and ∗∗p < 0.01.
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