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. 2021 Aug 17;2(8):100370. doi: 10.1016/j.xcrm.2021.100370

Figure 6.

Figure 6

mtDNA accumulation is responsible for inflammation and rhabdomyolysis that can be reversed by steroids or chloroquine (CLQ) treatment in vivo

(A) Real-time calcium flux in myoblasts from 4 controls and patients challenged with CpG-A, after pre-incubation with ODN151, a synthetic TLR9 antagonist. Each dot plot with lines depicts the mean ± SD of the intensity of the fluo-4 calcium probe as normalized to basal intensity of 1 single individual (≥10 cells/condition).

(B) Death of myoblasts was evaluated by staining cells from 4 control individuals transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein (+LPIN1) and 4 patients transduced with a lentivirus expressing a plasmid encoding for myc-DDK-tagged lipin1 protein or an empty vector, for the apoptosis marker caspase3/7 green and propidium iodide or 7-actinomycin D (7-AAD), a dye that stains both apoptotic and necrotic cells. Staurosporin (ST): positive control. Fluorescence-activated cell sorting (FACS) plots show the codistribution of the fluorescence intensity of caspase3/7 and of 7-AAD in 1 of 4 controls and patients.

(C) Dot plots (≥1,000 single cells/dot) depict the mean ± SD of the proportion of dead cells (ie, cells positive for capsase3/7 and/or 7-AAD) from (B) (mean effect of interaction F(8,45) = 5.296, p = 0.0001, of stimulus F(4,45) = 70.03, p < 0.0001, of subjects F(2,45) = 6.607, p = 0.003).

(D) As in (C), but using immortalized myoblast cell line transduced with Sh-Sc or Sh-LPIN1 shRNA (mean effect of interaction F(4,20) = 6.154, p = 0.0021, of stimulus F(4,20) = 48.93, p < 0.0001, of subjects F(1,20) = 15.62, p = 0.0008).

(E and F) As in (C), but myoblasts were pre-treated with Et. B. or vehicle (mean effect of interaction F(3,24) = 21.28, p < 0.0001, of stimulus F(3,24) = 114.8, p < 0.0001, of subjects F(1,24) = 25.79, p < 0.0001) for 5 days (E) or dexamethasone (DXM) or vehicle (mean effect of interaction F(3,24) = 9.490, p = 0.0003, of stimulus F(3,24) = 33.95, p < 0.0001, of subjects F(1,24) = 11.56, p = 0.0024) for 8 h (F), before challenging myoblasts with CpG-A in the presence of GM or EBSS.

(G) CK levels (means ± SDs of 3 individual values obtained from independent blood samples drawn the same day) were measured in 2 patients during a flare before and after the administration of steroids.

(H) As in (C) and Figure 5, but myoblasts from 4 controls and patients were pre-treated with CLQ or ODN151, or vehicle for 8 h followed by challenging with CpG-A together with GM or EBSS for another 16 h (mean effect of interaction F(5,36) = 19.53, p < 0.0001, of stimulus F(5,36) = 38.32, p < 0.0001, of subjects F(1,36) = 67.60, p < 0.0001). Dot plots (mean of 4–6 technical replicates/dot) show the mean ± SD of IL-6 concentration at the end of the experiment.

(I) As in (H), but evaluating the proportion of cell death (mean effect of interaction F(5,36) = 17.51, p < 0.0001, of stimulus F(5,36) = 73.41, p < 0.0001, of subjects F(1,36) = 40.06, p < 0.0001). Dot plots (≥1,000 single cells/dot) show the means ± SDs. IL-6 concentration is presented after background (which corresponds to values obtained from culture supernatants of cells exposed to vehicle) subtraction.

∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001: adjusted p values as determined by a between-subjects 2-way ANOVA (C–F, H, and I) and post hoc Sidak’s correction for multiple comparisons. Results are from 1 representative of 2 (D) or at least 3 (A–C, E–I) independent experiments.