Attenuation of cGAS/STING activity during mitosis

HaCaT cells were pretreated with vehicle, NOC, GCA, or BFA before a 90-min transfection with H₂O or pGL3. (A, B) Cells were fixed and stained for (A) STING and p230 or (B) IRF3 and TGN46 before DAPI staining. (A, B) Representative micrographs are shown in (A, B), white arrowheads indicate overlap. (C, D) Manders’ overlap coefficients from multiple micrographs were plotted for (C) STING:p230 and (D) IRF3:DAPI. *P < 0.01, ***P < 0.0001. Scale bars = 10 μm.

(A) Cells were treated with vehicle, nocodazole (NOC), golgicide A (GCA), or brefeldin A (BFA) for 1 h prior and during a 90-min transfection with HSV-60 oligonucleotide, calf-thymus DNA (CTD), or plasmid pGL3. (B, C, D) Transfection of vehicle- or GCA-treated cells with pGL3 or pIC, and (C, D) densitometric quantification of pIRF3 blots. **P < 0.001, n = 5 biological replicates. (E) Vehicle- or GCA-treated cells were treated with H₂O or 12.5 μg cGAMP for 90 min before SDS–PAGE and Western blot for cGAS/STING pathway components. (F) cGAMP production in vehicle- and GCA-treated cells upon pGL3 transfection. n = 3 biological replicates, with n = 2 technical replicates each.

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HaCaT cells were treated with GCA or vehicle for 1 h before a 90-min pGL3 transfection. (A, B, C, D, E, F, G, H) Transcript levels of (A) IFNB1, (B, C, D, E, F) ISGs, and (G, H) chemokine genes were measured via RT-qPCR and normalized to TATA-binding protein. *P < 0.01, **P < 0.001, ***P < 0.0001, for comparisons of 4 and 8 h DMSO-treated pGL3 groups to 4 and 8 h DMSO-treated H₂O controls and for 4 and 8 h DMSO-treated pGL3 groups to 4 and 8 h GCA-treated pGL3 groups, n = 3 technical replicates.

(A) Cell synchronization method. Cells were pre-synchronized in G₁ by contact inhibition and growth in low serum. Cells were then released by replating at subconfluence in 10% serum and synchronized in prometaphase with NOC. Cell cycle analysis by propidium iodide staining. Cells were transfected with pGL3 for 90 min at various times post-release from NOC. (B) pIRF3 activation in response to pGL3 was dampened when arrested cells were transfected, gradually returning as cells completed mitosis and returned to interphase. (C) Densitometric quantification of relative pIRF3 increase in response to pGL3 transfection across multiple blots. *P < 0.05, for comparison of 180 min to NR groups, n = 4 independent biological replicates. (D) Golgi localization of STING was impaired when arrested cells (NR) were transfected, but was restored upon return to interphase (180 min). (E) Manders’ overlap coefficients from multiple micrographs were plotted for STING:p230, **P < 0.001, scale bars = 10 μm.