Fig 2: Myospreader improves stability and localization of Cas9/dCas9 in myoblasts and myofibers. (A) Representative IF images of C2C12 myoblasts transfected with plasmids encoding 2xSV40 Cas9 or Myospreader Cas9. SaCas9 is shown in green, nucleolin in yellow, and DAPI in blue. (Scale bars: 10 µm.) (B) Schematic of the experiment to assess Myospreader Cas9 stability and editing efficiency in C2C12 myoblasts. (C) tdTomato mRNA expression as assessed by RT-qPCR in AI14 reporter C2C12s following transfection with plasmids encoding 2xSV40 Cas9 or Myospreader Cas9. Plotted relative to the mean of the 2xSV40 NLS Cas9 group. Significance by Student’s t test. (D) Cas9 mRNA expression as assessed by RT-qPCR in AI14 reporter C2C12s following transfection with plasmids encoding 2xSV40 Cas9 or Myospreader Cas9. Plotted relative to the mean of the 2xSV40 NLS Cas9 group. Significance by Student’s t test. (E) Western blot against Cas9 and Gapdh in Ai14 reporter C2C12s following transfection with plasmids encoding 2xSV40 Cas9 or Myospreader Cas9. Relative signal intensity determined by densitometry at the bottom. A.U.: arbitrary unit, normalized to Gapdh. (F) Schematic of dCas9 impeding transcription of toxic CUG RNA and experiment. Each treatment was packaged in a single AAV vector systemically delivered to 4-wk-old HSALR mice at a dose of 5E+13 vg/kg. (G) HCR-FISH to detect CUG RNA foci (magenta) and dCas9 mRNA (green) in myonuclei of representative TA myofibers isolated from treated HSALR mice. Myonuclei borders are indicated with dashed lines. Asterisks indicate dCas9-positive myonuclei. (Scale bar: 30 µm.) (H) Cumulative distribution functions of HCR-FISH CUG RNA signal in myonuclei proximal to dCas9-expressing myonuclei in myofibers isolated from treated HSALR mice. Significance by Kolmogorov–Smirnov test. (I) dCas9 mRNA expression as assessed by RT-qPCR in gastrocnemius muscle of treated HSALR mice. Plotted relative to the mean of the 2xSV40 NLS Cas9 nontargeting group. Significance by Tukey’s HSD. (J) Western blot detecting dCas9 and Gapdh in gastrocnemius muscle of treated mice. Relative signal intensity determined by densitometry at the bottom. A.U.: arbitrary unit, normalized to Gapdh (ns = not significant; *P < 0.05; **P < 0.01; ***P < 0.001) (error bars = 95% CI).

Fig 3: HDMECs restrict R. rickettsii Iowa, but not Sheila Smith, through IFN-β signaling. (A) Lysis of HDMECs was determined by LDH activity in the culture supernatants after 12, 18, and 24 h of infection. Select samples were also treated with isotype control or anti-IFNAR2 (mean ± S.D., N = 6). (B) Normalized bacterial burdens within HDMECs infected with R. rickettsii Iowa or Sheila Smith treated with isotype control or anti-IFNAR2 for 24 h (mean ± S.D., N = 6). Significance in A was assessed with two-way ANOVA and one-way ANOVA in B; P < 0.05, *; P < 0.0001, ****. (C) Western blotting with anti-p-STAT1, STAT1, p-STAT2, STAT2, and GAPDH of HDMECs infected with R. rickettsii strains treated with isotype control or anti-IFNAR2 antibodies (3 mg/mL) for 6 and 12 h (representative results are shown, N = 4). (D) Western blotting with anti-TRAIL, IDO, and GAPDH of HDMECs infected with R. rickettsii strains treated with isotype control or anti-IFNAR2 for 12, 18, and 24 h (representative results are shown, N = 3), ns = not significant.

Fig 4: SORL1 harboring the N1358S mutation exhibits increased seeding capacity in HEK293T cells. HEK293T Tau RD P301S FRET Biosensor cells were transfected with plasmid containing WT SORL1 or SORL1 harboring the G511R or N1358S mutations. A, expression level of different variants was measured by Western blot (GAPDH was used as a loading control). These cells were incubated with (B) HMW or LMW SEC fractions from human AD patient brain or vehicle control and tau seeding was quantified. In a separate experiment, transfected cells expressing equal levels of the SORL1 variants (C) were incubated with (D) 20 nM 125I-labeled tau in the presence or absence of 1 μM RAP for 2 h and then internalized tau was quantified. (Mean ± SEM; 2-way ANOVA, Tukey’s multiple comparison test). AD, Alzheimer’s disease; HMW, high molecular weight; LMW, low molecular weight; RAP, receptor associated protein; SORL1, sortilin-related receptor 1.

Fig 5: Virulent rickettsia eventually induce apoptosis, while non-pathogenic rickettsia do not. (A) HDMECs were infected with rickettsia or treated with 2 µM Staurosporine (Stauro), and Caspase-3/7 activity was measured after 4, 6, 12, and 24 h (mean ± S.D., N = 6–10). (B) Western blots with anti-PARP, cleaved PARP, GSDME, and GAPDH of HDMECs infected with rickettsia (MOI 2.5) for 6, 12, or 24 h (representative results are shown, N = 4).