Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Upper panel: Chemical Structure of 7-nitrobenz-2-oxa-1,3-diazole (NBD)-labeled PAPTP (PAPTP-NBD). Lower panel: Synthesis of PAPTP-NBD. Reagents and Conditions (i) TsCl, DMAP, pyridine, DCM, r.t., 18 h; (ii) NaN 3 , DMF, 90 °C, 3 h; (iii) Pd/C, H2, MeOH, r.t., 16 h; (iv) NBD-Cl, DIPEA, MeOH, r.t., 16 h; (v) TsCl, DMAP, pyridine, DCM, r.t., 5 h; (vi) NaI, acetone, 40 °C, 16 h; (vii) PAPTP-OH, K 2 CO 3 , DMF, r.t., 16 h. Synthesis of 2-(2-(2-hydroxyethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (1). To a solution of triethylene glycol (PEG3-OH, 77.28 g, 514.6 mmol, 8.0 equiv) in dichloromethane (DCM, 255 mL) at 0 °C were added 4-dimethylaminopyridine (DMAP, 15.7 g, 128.6 mmol, 2.0 equiv) and pyridine (10.18 g, 128.6 mmol, 2.0 equiv). After 10 min of stirring, p-toluenesulfonyl chloride (TsCl, 12.3 g, 64.3 mmol, 1.0 equiv), previously dissolved in DCM (165 mL), was added dropwise. The reaction mixture was stirred at room temperature for 18 h. The reaction was then quenched by dilution with 300 mL of 1.0 M HCl, the organic layer separated and the aqueous phase was extracted with DCM (2 × 250 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using a DCM/acetone mixture (8:2) to afford 1 as a pale yellow oil (13.2 g, 43.4 mmol, yield: 67%). 1H NMR (400 MHz, CDCl3) δ 7.80 (d, J = 8.4 Hz, 2H), 7.34 (d, J = 8.4 Hz, 2H), 4.18–4.15 (m, 2H), 3.72–3.69 (m, 4H), 3.61 (s, 4H), 3.58–3.56 (m, 2H), 2.44 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 145.01, 133.07, 129.98, 128.11, 72.59, 70.92, 70.43, 69.29, 68.85, 61.89, 21.78. ESI-MS (ion trap): m /z 305 [M + H]+. Synthesis of 2-(2-(2-azidoethoxy)ethoxy)ethan-1-ol (2). To a solution of compound 1 (13.2 g, 43.4 mmol, 1.0 equiv) in anhydrous N,N-dimethylformamide (DMF, 100 mL), sodium azide (NaN₃, 8.5 g, 130.2 mmol, 3.0 equiv) was added. The reaction mixture was stirred at 90 °C for 3 h until thin-layer chromatography (TLC) analysis (EtOAc/PE, 6:4) indicated complete consumption of the starting material. The reaction mixture was then diluted with ethyl acetate (EtOAc, 300 mL) and washed with brine/water 1:1 (5 × 100 mL). The organic layer was dried, and the solvent was removed under reduced pressure. The flask was left under high vacuum overnight to remove residual DMF, yielding compound 2 as a pale yellow oil (6.8 g, 38.7 mmol, yield: 89%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.71 (m, 2H), 3.68–3.64 (m, 6H), 3.61–3.59 (m, 2H), 3.38 (t, J = 5.0 Hz, 2H), 2.45 (s, 1H).13 C NMR (101 MHz, CDCl3) δ 72.59, 70.74, 70.48, 70.13, 61.84, 50.74. ESI-MS (ion trap): m / z 176 [M + H]+. Synthesis of 2-(2-(2-aminoethoxy)ethoxy)ethan-1-ol (3). Palladium on carbon (Pd/C, 10% w/w, 0.70 g) was suspended in methanol (30 mL) in a round-bottom flask under nitrogen atmosphere. Compound 2 (6.8 g, 38.7 mmol, 1.0 equiv), previously dissolved in methanol (10 mL), was added to the suspension. The reaction atmosphere was then replaced with hydrogen, and the mixture was stirred at room temperature for 16 h, until thin-layer chromatography (TLC) analysis (DCM/acetone, 8:2) confirmed complete consumption of the starting material. Hydrogen was removed by nitrogen stream and the reaction mixture was filtered through a celite pad to remove the catalyst. The solvent was evaporated under reduced pressure and the crude product was purified by flash column chromatography on silica gel using a DCM/MeOH/NH3(aq) (87:12:1) as the eluent, affording compound 3 as a colorless oil (2.6 g, 17.4 mmol, yield: 45%). 1H NMR (400 MHz, CDCl3) δ 3.73–3.70 (m, 2H), 3.68–3.62 (m, 4H), 3.61–3.58 (m, 2H), 3.56–3.53 (m, 2H), 2.88 (t, J = 5.1 Hz, 2H), 2.55 (s, 3H).13 C NMR (101 MHz, CDCl3) δ 72.81, 72.79, 70.47, 70.24, 61.63, 41.53. ESI-MS (ion trap): m / z 150 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethan-1-ol (4). To a solution of NBD-Cl (1.0 g, 5.0 mmol, 1.0 equiv) in methanol (25 mL) at 0 °C were added N,N-diisopropylethylamine (DIPEA, 2.6 g, 20 mmol, 4.0 equiv) and compound 3 (0.82 g, 5.5 mmol, 1.1 equiv). The reaction mixture was allowed to warm to room temperature and stirred for 16 h. The mixture was then diluted with ethyl acetate (150 mL) and washed with saturated NH4Cl solution (3 × 50 mL). The organic layers were combined, dried, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using an EtOAc/MeOH mixture (99:1) as the eluent, affording compound 4 as a brown powder (1.0 g, 3.2 mmol, yield: 64%). 1H NMR (400 MHz, MeOD) δ 8.51 (d, J = 8.9 Hz, 1H), 6.43 (d, J = 8.9 Hz, 1H), 3.84–3.80 (m, 2H), 3.76 (s, 2H, broad signal), 3.71–3.66 (m, 2H), 3.66–3.62 (m, 4H), 3.56–3.53 (m, 2H).13 C NMR (101 MHz, DMSO) δ 154.82, 153.90, 153.60, 147.35, 130.29, 109.01, 81.83, 79.36, 79.22, 77.44, 69.66, 52.88. ESI-MS (ion trap): m / z 313 [M + H]+. Synthesis of 2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethyl 4-methylbenzenesulfonate (5). To a solution of compound 4 (100 mg, 0.32 mmol, 1.0 equiv) in dichloromethane (DCM, 1.5 mL) at 0 °C were added pyridine (76 mg, 0.96 mmol, 3.0 equiv), 4-dimethylaminopyridine (DMAP, 78 mg, 0.64 mmol, 2.0 equiv), and p-toluenesulfonyl chloride (TsCl, 122 mg, 0.64 mmol, 2.0 equiv). The reaction mixture was stirred at room temperature for 5 h. The mixture was then diluted with brine (50 mL) and extracted with DCM (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using DCM/MeOH (99:1) as the eluent, affording compound 5 as a brown solid (94 mg, 0.20 mmol, yield: 63%). 1H NMR (400 MHz, (CD3)2CO) δ 8.50 (d, J = 8.8 Hz, 1H), 7.77 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.49 (d, J = 8.8 Hz, 1H), 4.19–4.11 (m, 2H), 3.84–3.80 (m, 4H), 3.71–3.66 (m, 2H), 3.64 – 3.60 (m, 2H), 3.60–3.53 (m, 2H), 2.86 (s, 1H), 2.42 (s, 3H).13 C NMR (101 MHz, (CD3)2CO) δ 145.78, 145.45, 145.08, 137.77, 134.22, 130.77, 128.63, 123.50, 99.90, 71.24, 71.07, 70.61, 69.34, 44.68, 21.49. ESI-MS (ion trap): m/z 467 [M + H]+. Synthesis of N-(2-(2-(2-iodoethoxy)ethoxy)ethyl)-7-nitrobenzo[c][1,2,5]oxadiazol-4-amine (6). To a solution of compound 5 (30 mg, 0.064 mmol, 1.0 equiv) in acetone (0.8 mL) was added sodium iodide (NaI, 39 mg, 0.257 mmol, 4.0 equiv). The reaction mixture was stirred at 40 °C for 16 h in a sealed vial. After completion, the mixture was diluted with ethyl acetate (EtOAc, 40 mL) and washed with brine (3 × 10 mL). The organic layer was dried over anhydrous Na₂SO₄, and concentrated under reduced pressure. The crude product was purified by flash column chromatography on silica gel using EtOAc/petroleum ether (1:1) as the eluent, affording compound 6 as a brown solid (14.5 mg, 0.034 mmol, yield: 54%). 1H NMR (400 MHz, (CD3)2CO) δ 8.54 (d, J = 8.8 Hz, 1H), 8.13 (s, 1H, broad signal), 6.55 (d, J = 8.8 Hz, 1H), 3.93–3.79 (m, 4H), 3.73–3.58 (m, 6H), 3.29 (t, J = 6.5 Hz, 2H).13 C NMR (101 MHz, (CD3)2CO) δ 145.81, 145.41, 145.02, 137.67, 123.43, 99.98, 72.35, 71.08, 70.67, 69.32, 44.59, 4.26. ESI-MS (ion trap): m/z 423 [M + H]+ Synthesis of (4-(2-(2-(2-((7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino)ethoxy)ethoxy)ethoxy)phenyl) (3-(4-(4-((7-oxo-7H-furo[3,2-g]chromen-4-yl)oxy)butoxy)phenyl)propyl)diphenylphosphonium (PAPTP-NBD) A solution of compound 6 (14.5 mg, 0.034 mmol, 1.1 equiv) in DMF (0.5 mL) was cooled to 0 °C, and PAPTP-OH (25 mg, 0.031 mmol, 1.0 equiv, synthesized as previously reported, 10.3390/ph14020129) and potassium carbonate (K₂CO₃, 4 mg, 0.031 mmol, 1.0 equiv) were added. The reaction mixture was stirred at room temperature for 16 h. After completion, the reaction was diluted with EtOAc (30 mL) and washed with 0.5 M HCl (2 × 10 mL) followed by brine (1 × 10 mL). The combined organic layers were dried over anhydrous Na₂SO₄ and concentrated under reduced pressure. The crude product was purified by preparative HPLC and lyophilized to afford PAPTP-NBD as an orange solid (9.6 mg, 0.009 mmol, yield: 29%, purity UPLC > 95%). 1H NMR (400 MHz, (CD3)2CO) δ 8.42 (s, 1H, broad signal), 8.21 (dd, J = 9.8, 0.7 Hz, 1H), 7.93–7.68 (m, 13H), 7.26 (dd, J = 2.4, 1.0 Hz, 1H), 7.22 (dd, J = 9.0, 2.6 Hz, 2H), 7.16–7.08 (m, 3H), 6.82 (d, J = 8.6 Hz, 2H), 6.55 (s, 1H, broad signal), 6.19 (d, J = 9.8 Hz, 1H), 4.67 (t, J = 5.8 Hz, 2H), 4.24–4.21 (m, 2H), 4.09 (t, J = 5.8 Hz, 2H), 3.90–3.82 (m, 6H), 3.68 (s, 4H), 3.60–3.48 (m, 2H), 2.84 (t, J = 5.8 Hz, 2H), 2.12–1.98 (m, 6H). 13 C NMR (101 MHz, (CD3)2CO) δ 165.18, 159.24, 158.66, 153.86, 150.18, 146.37, 140.10, 136.81 (d, J = 11.5 Hz), 135.87 (d, J = 3.0 Hz), 134.61 (d, J = 10.0 Hz), 133.12, 131.25 (d, J = 12.6 Hz), 130.52, 120.83, 119.97, 117.48, 117.35, 115.48, 113.33, 109.39, 108.46, 107.37, 106.47, 94.02, 73.61, 71.46, 71.36, 70.02, 69.73, 69.23, 68.23, 49.07, 36.03 (d, J = 16.9 Hz), 27.59, 26.72, 25.49 (d, J = 3.7 Hz), 22.25 (d, J = 52.6 Hz). ESI-MS (ion trap): m / z 363 [M]+. ( B ) Confocal microscopy images showing the fluorescent signal in untreated cells and in those treated with fluorescent PAPTP (PAPTP-fluor) in mitochondria of CD4 + CD25 - Tconv cells isolated from healthy mice before and after treatment with 100 nM PAPTP-Fluor for 30 min. The scale bar is 10 μm. Control experiment for Fig. . The same cells shown in this representative image are also shown in Fig. at higher magnification. ( C ) Left panels: Upper panel: Downregulation of Kv1.3 in cells treated with CRIPSR/Cas9 and Kv1.3 staining in sorted cells. Yellow: CRISPR/Cas transfected, unstained. Blue: CRISPR/Cas transfected, cells positively sorted for Kv1.3. This is the fraction, which was positively sorted and then stained with FITC-anti-Kv1.3 antibodies.For sorting, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (BioLegend, #101302; 1:50 dilution) for 15 min at 4 °C, washed, and labeled with biotin-conjugated anti-Kv1.3 antibody (Alomone Labs, #APC-101B) for 30 min at 4 °C. Following a second wash, cells were incubated with streptavidin-conjugated microbeads (Miltenyi Biotec, #130-048-101) for 30 min at 4 °C. Kv1.3-positive and -negative populations were isolated using LS columns (Miltenyi Biotec, 130-042-401). For the flow cytometry, Fc-receptors were blocked with True stain (1:50 dilution, cells were collected, washed with H/S, incubated with Fc receptor blocking reagent (Clone S17011E, BioLegend, #156604) and then an aliquot of the samples was stained with a FITC-coupled anti-rabbit IgG (1:500, Jackson Immunoresearch 711-096-152) to detect the anti-Kv1.3, which was already bound to the cells. Red: CRISPR/Cas transfected, cells negatively sorted for Kv1.3. This is the fraction, which was negatively sorted and then stained exactly as the blue fraction. The staining was done on aliquots just before retransfection of the mito-Kv1.3 construct to confirm downregulation. Lower panel: Same as above, but with aliquots that were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again and then stained. This confirms downregulation of extra- and intracellular Kv1.3. Right upper panel: Flow cytometry of sorted Kv1.3-negative cells that were re-transfected. Blue: Control-transfected (empty vector). Aliquots were fixed for 10 min at room temperature in 1% buffered PFA, then washed and permeabilized for 8 min with 0.1% Triton X-100 at room temperature, washed again, Fc-receptors were blocked with True stain (1:50, Biolegend, #156604) and then stained with FITC-coupled anti-Kv1.3 (Alamone, #APC-101-F). Red: EYFP-Mito-Kv1.3 transfected cells, as above. Right lower panel: Representative dot plot showing the gating strategy for the identification of EYFP-mitoKv1.3 + Annexin + and EYFP-mitoKv1.3 - Annexin + cells after PAPTP treatment. Kv1.3 - cells were transfected with EYFP-mitoKv1.3 construct and subsequently treated with PAPTP for 48 h. Cells were then analyzed for apoptosis using flow cytometry. ( D ) Representative images of longitudinal spinal-cord sections stained with luxol fast blue from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 100 μm. Demyelinated are indicated with white arrows. Right: Average ± SEM of myelin area per field ( n = 3 for mice receiving untreated lymphocytes, n = 5 for mice receiving 1 μM PAPTP-treated lymphocytes). p -value of Student’s t test. ( E ) Representative images of longitudinal spinal-cord sections stained with Bielschowsky staining from mice receiving MOG 35–55 activated untreated or 1 μM PAPTP-treated lymphocytes. Scale bar, 200 μm. Right upper panel: enlarged image from the lower EAE AT untreated sample shown on the left. Right lower panel: enlarged image from the upper EAE AT + PAPTP sample shown on the left. Please note damaged axon fibers in the EAE. ( F ) Quantification of axon damage from longitudinal sections of Blieschowsky-stained spinal cords from EAE AT and EAE AT + PAPTP animals. Quantification was performed following the method used for Fig. (left panel) and according to (Theotokis et al, ). ( G ) Representative images of a brain section of a wild-type mouse injected with MOG 35–55 activated untreated lymphocytes, stained with the indicated antibodies. The sections are from the same experiment shown in Fig. . ( H ) Average ± SEM of the number of GFAP + cells per field in brain slices of healthy animals (Ctrl) and mice receiving untreated or 1 μM PAPTP-treated lymphocytes ( n = 3 for each group). On the right, representative immunohistochemical images of GFAP + in brain slices from mice of the indicated groups. The images were taken from the same region for each animal. The scale bar corresponds to 100 μm. p -value from one-way ANOVA. ( I ) Additional examples of Klüver-Barrera dual staining performed as in Fig. . Red arrows indicate infiltrated/demyelinated zones. Please note also vacuolation, as e.g., in (Morales et al, ) in the enlarged image.

Article Snippet: MOG 35–55 , MedchemExpress , HY P1240.

Techniques: Labeling, Purification, Column Chromatography, Thin Layer Chromatography, Solvent, Suspension, Synthesized, Confocal Microscopy, Isolation, Control, Staining, CRISPR, Transfection, Incubation, Blocking Assay, Flow Cytometry, Construct, Plasmid Preparation, Injection, Immunohistochemical staining

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Confocal microscopy image showing the accumulation of fluorescent PAPTP (PAPTP-fluor) in Mitotracker Red stained mitochondria of CD4 + CD25 - Tconv cells isolated from healthy mice. T conv were treated with 100 nM PAPTP-Fluor for 30 min. The scale bar is 10 μm. The same cells shown in this representative image are also shown in Fig. at lower magnification. ( B ) Scheme showing the strategy for the generation of Kv1.3 knockout cells expressing the mitochondrial Kv1.3 form. Splenocytes were isolated from MOG 35–55 immunized mice and Kv1.3 was deleted using CRISPR/Cas9 technology. Cells were then sorted for Kv1.3 expression, removing splenocytes still expressing Kv1.3, and transfected with EYFP-mitoKv1.3 construct. Finally, cells were treated with 1 μM PAPTP for 48 h. Apoptosis was detected in Mito-Kv1.3 + and MitoKv1.3 - cells by flow cytometry. ( C ) Percentage of Annexin V + Mito-Kv1.3 + and Annexin V + Mito-Kv1.3 - cells after 1 μM PAPTP treatment for 48 h. Populations were gated on total cells (Kv1.3 - cells) ( n = 6 for each group). Data represent average ± SEM with superimposed individual data points for each animal. Indicated p -values refer to Mann-Whitney test. See also Fig. . ( D ) Scheme of the EAE Adoptive Transfer Model: Mice underwent immunization via subcutaneous injection of MOG 35–55 in complete Freund’s adjuvant. Ten days post-immunization, spleens were harvested, and single-cell suspensions were prepared. These splenocytes were treated for 3 days with IL-12, IL-23, and anti-IFN-γ. Subsequently, the splenocytes were subjected to a 3-day treatment with 1 μM PAPTP, or left untreated. Antigen-specificity of the cells was confirmed by additional proliferation assays of isolated donor lymphocytes (see Methods). Lymphocytes were isolated, and the induction of apoptosis was assessed using flow cytometry. Additionally, these lymphocytes were transferred into wild-type recipient animals, and the animals’ clinical scores were monitored daily for 10 days. Following the observation period, the animals were euthanized for further analysis. ( E ) Percentage of Naive, T CM and T EM cells of all sorted CD3 + T lymphocytes ( n = 6 for each group). ( F ) Percentage of apoptotic Annexin V + cells of all sorted CD3 + T cells. The Trypan Blue staining gave the same result ( n = 6 for each group). ( G ) Percentage of cell death in CD4 + CD44 - CDL62 + Naive, CD44 + CDL62 + T CM and CD44 + CDL62 - T EM subsets ( n = 6 for each group). ( H ) Data represent average ± SEM of disease scores by daily scoring of mice receiving the indicated group of lymphocytes. Days 1–6: All mice had a score of 0 ( n = 6 for each group). p -values from two-way ANOVA test are shown. ( I ) Data represent average ± SEM of the percentage of spinal cord infiltrated Naive, T CM , and T EM lymphocytes (determined as in ( G )) of all cells ( n = 4 for healthy controls; n = 5 for mice receiving untreated and n = 5 for mice receiving 1 μM PAPTP-treated lymphocytes). p -values from two-way ANOVA test are shown. ( J ) Representative image (of 5 sections) of brains (upper part of the pons region) stained with PE-anti-CD45, from wild-type mice intraperitoneally injected with either untreated or PAPTP 1 μM treated, MOG 35–55 activated lymphocytes. ( E–G ) Data represent average ± SEM with superimposed individual data points for each animal. Indicated p-values from Unpaired Student’s T test. .

Article Snippet: MOG 35–55 , MedchemExpress , HY P1240.

Techniques: Confocal Microscopy, Staining, Isolation, Knock-Out, Expressing, CRISPR, Transfection, Construct, Flow Cytometry, MANN-WHITNEY, Adoptive Transfer Assay, Injection, Adjuvant, Single Cell

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Treatment scheme for wild-type mice immunized via subcutaneous injection of MOG 35–55 : upon the manifestation of initial symptoms, treatment commenced. The treatment regimen was administered every 48 h, totaling 3 injections. Mice were euthanized a few hours after the last injection for subsequent analysis of brains and spinal cords. ( B ) Data represent average ± SEM of disease scores of mice of the indicated group ( n = 4 for controls; n = 4 for EAE, n = 3 for EAE + PAPTP). PAPTP treatment was started after disease onset, when indicated. p -values of two-way ANOVA test are shown. ( C ) Representative images of transversal lumbar spinal-cord sections stained with luxol fast blue, from control ( n = 4), EAE ( n = 4), and EAE + PAPTP ( n = 3) groups. Demyelinated/infiltrated areas are indicated by arrows. Scale bar, 200 μm. Right, the demyelinated area in the white matter was calculated in the EAE and EAE + PAPTP groups. ( D ) Representative images of transversal lumbar spinal-cord sections stained with Bielschowsky staining, from control ( n = 5), EAE ( n = 5), and EAE + PAPTP ( n = 3) groups. Areas with axonal loss are indicated by arrows. Scale bar, 200 μm. Right, the axonal loss in the white matter was calculated in the EAE and EAE + PAPTP groups. ( E ) Average ± SEM of the number of GFAP + cells per field in spinal cord slices of mice of the indicated groups ( n = 3). At least 5 sections per animal were analyzed. On the left, representative immunohistochemical images of GFAP + in spinal cord transversal slices from mice of the indicated groups. GFAP + cells are indicated in the figure. The images were taken from the same region for each animal. The scale bar corresponds to 100 μm. p -values of one-way ANOVA are indicated. ( C , D ) Data represent average ± SEM with superimposed individual data points for each animal. p -values of Unpaired Student’s T test are shown. .

Article Snippet: MOG 35–55 , MedchemExpress , HY P1240.

Techniques: Injection, Staining, Control, Immunohistochemical staining

Journal: EMBO Molecular Medicine

Article Title: Selective inhibition of mitochondrial Kv1.3 prevents and alleviates multiple sclerosis in vivo

doi: 10.1038/s44321-025-00307-2

Figure Lengend Snippet: ( A ) Treatment scheme for wild-type mice immunized via subcutaneous injection of MOG 35–55 : upon the manifestation of initial symptoms, typically around day 10 post-immunization, treatment commenced. The treatment regimen was administered every 48 h, totaling 11 injections. Mice were euthanized the day following the final injection for subsequent analysis of peripheral blood, brains, and spinal cords. ( B ) Data represent average ± SEM of disease scores of mice of the indicated group ( n = 9 for controls; n = 14 for EAE, n = 15 for EAE + PAPTP). PAPTP treatment was started at day 10 post immunization (DPI). p -values of two-way ANOVA test are indicated. On the right representative photos taken at 16 DPI under the indicated conditions are shown. ( C ) Quantitative analysis of hematological parameters in the blood of mice from specified experimental groups at the endpoint of the experiment. The measurements include counts of white blood cells (WBC), red blood cells (RBC), platelets (PLT), hemoglobin and hematocrit levels. As an example, WBC count is around 8 × 10 3 /µl, while RBC count is 10 × 10 6 /µl. Data represent average ± SEM ( n = 7 for control and EAE + PAPTP groups; n = 8 for EAE). ( D ) Percentages of lymphocytes (LYM), monocytes (MID), and granulocytes (GRA) in peripheral blood of mice of the indicated group at the endpoint of the experiment, evaluated using a blood counter. Data represent average ± SEM ( n = 7 for control and EAE + PAPTP groups; n = 8 for EAE). ( E ) CD3 + T cell and CD19 + B cell percentages within the total lymphocyte population in peripheral blood samples collected from mice belonging to the indicated groups at the experimental endpoint ( n = 5 for controls; n = 13 for EAE, and n = 15 for EAE + PAPTP groups). ( F ) Latency to fall (in seconds) of mice of the indicated group evaluated using the rotarod test ( n = 8 for controls and EAE mice; n = 6 for EAE + PAPTP group). ( G ) Representative Transmission Electron Microscopy images showing neuronal myelination in brain cortexes of mice from the indicated groups at the experimental endpoint. Images were taken from the same region of the brain of each animal. Scale bar indicated in the figure. On the right, quantification of the average ± SEM of myelin layers per axon. Each point represents a different axon ( n = 3 sections for controls, and n = 3 for EAE, and n = 4 for EAE + PAPTP groups). ( H ) Representative Transmission Electron Microscopy images showing neuronal myelination in spinal cords of mice from the indicated groups at the experimental endpoint. Scale bar indicated in the figure. ( I ) Upper panel: Quantification of the average ± SEM of myelin layers per axon. Each point represents a different axon ( n = 5 sections for controls, and n = 3 for EAE, and n = 4 for EAE + PAPTP groups). Lower panel: g-ratio (axon diameter/diameter of myelinated fiber) as determined from TEM images for individual axons of the spinal cord, for the indicated groups. ( J ) Average ± SEM of the number of Iba-1 + cells per field in brain slices of mice of the indicated groups ( n = 3 for each group, 6 slices/animal were analyzed). ( K ) Representative binary images of individual microglia. The scale bar corresponds to 25 μm. ( L ) Average ± SEM of the number of branches, maximal branch length, number of junctions, and end-points in Iba-1 + microglia cells in brain slices of mice of the indicated group. Each data point represents a single cell ( n = 3 for each group). ( C–F ) Data represent average ± SEM with superimposed individual data points for each animal. ( J , L ) p -values obtained in one-way ANOVA test. ( F , G , I ) p -values of Kruskal–Wallis test or of Tukey’s multiple comparison test ( I lower panel). .

Article Snippet: MOG 35–55 , MedchemExpress , HY P1240.

Techniques: Injection, Control, Transmission Assay, Electron Microscopy, Single Cell, Comparison

Journal: Journal of translational autoimmunity

Article Title: NRF2 activator A-1396076 ameliorates inflammation in autoimmune disease models by inhibiting antigen dependent T cell activation.

doi: 10.1016/j.jtauto.2020.100079

Figure Lengend Snippet: Fig. 4. A-1396076 inhibits inflammation in autoimmune models only when dosed at the time of antigen challenge. (A/B) Lewis rats were immunized with bovine type II collagen in IFA intradermally on day 0 and boosted with collagen in IFA on day 6. Paw volume was measured using a micro-controlled volume meter plethysmograph with left and right paw volumes averaged for analysis. Rats were dosed orally BID at 10 mg/kg beginning on day 5 (late prophylactic) or day 11 (therapeutic) with A-1396076. Prednisolone was used as a positive control and was dosed orally QD starting on day 11. After initial collagen immunization on day 0 in the rat CIA model, A-1396076 was administered orally at 10 mg/kg once a day beginning either on day 5 (1 day prior to antigen boost) or day 11 at the onset of clinical signs. Paw swelling was measured by plethysmograph and expressed as mean paw volume by day (A) or AUC from day 8–18 (B) (n ¼ 9; one experiment). (C/D) Female Brown Norway rats were immunized with MOG in CFA at the base of the tail. A-1396076 was administered orally once a day at 10 mg/kg beginning either on day 0 prior to immunization (prophylactic) with MOG peptide in CFA or day 7 post immunization prior to the onset of clinical signs (late prophylactic). FTY720 was administered daily (1 mg/kg PO) beginning Day 7. Animals were monitored daily for the onset of clinical signs and scored using the scale: 0 ¼ normal, 1 ¼ limp tail, 2 ¼ abnormal gait, 3 ¼ partial hind limb paralysis, 4 ¼ complete hind limb paralysis, 5 ¼ moribund or dead. Data is expressed as clinical score for each day (C) and expressed as AUC from days 0–17 (D) (n ¼ 9; one experiment). (E/F) Male DBA1/J mice were immunized intradermally at the base of the tail with an emulsion containing glucose-6-phosphate isomerase in CFA. A-1396076 was administered orally once a day at 10 mg/kg beginning either on day 0 prior to immunization with GPI (prophylactic) or 7 days post immunization (late prophylactic). Change in paw thickness was assessed by caliper measurements and reported by day (E) and expressed as AUC from days 7–17 (F) (n ¼ 15; one experiment). All data is expressed at mean SEM. * ¼ p < 0.05; ** ¼ p < 0.01; *** ¼ p < 0.001 vs vehicle by two- way ANOVA with Bonferroni post-test (A/C/E) or one-way ANOVA with Dunnett’s post-test (B/D). All data is expressed at mean SEM.

Article Snippet: An emulsion was prepared with 5 mg/mL MOG (New England Peptide, Gardner, MA) dissolved in PBS and 10 mg/mL heat-inactivated Mycobacterium tuberculosis H37Ra (Difco Labs, Detroit, MI) suspended in Incomplete Freunds Adjuvant (Difco Labs, Detroit, MI) at a 1:1 ratio (v/v).

Techniques: Positive Control, Emulsion

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Up-regulated Hallmark and Gene Ontology (GO) pathways in ID8 cell single-guide SMARCA4 (sg SMARCA4 ) compared to those in sgNTC. Ribodeplete RNA sequencing was performed. Statistical analysis was based on hypergeometric test and performed using ClusterProfiler. IL-6, interleukin-6; JAK, Janus kinase; STAT3, signal transducer and activator of transcription 3; TNFA, tumor necrosis factor–α; FDR, false discovery rate. ( B ) Gene expression heatmap of type I IFN pathway–related genes in ID8 cells. Reads per kilobase of transcript per million mapped reads values were scaled to z -score for visualization. Gene expression fold change of sg SMARCA4 versus sgNTC cells is color coded according to the legend. ( C ) Quantitative reverse transcription polymerase chain reaction (qRT-PCR) validation results for IFN genes in ID8 cells (sgNTC and four sg SMARCA4 clones). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control (sgNTC). n = 3 independent experiments. ( D ) MHC1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. ( E ) PD-L1 expression in ID8 cells with or without IFN-ɣ by flow cytometry. MFI, median fluorescence intensity. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001; n.s., not significant. Error bars represent ± SEM. Samples in duplicates [(A) and (B)] and triplicates [(D) and (E)]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: RNA Sequencing, Gene Expression, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Biomarker Discovery, Clone Assay, Expressing, Control, Flow Cytometry, Fluorescence, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Changes in genomic site accessibility in sg SMARCA4 (KO1) versus sgNTC cells. Log 2 fold change and FDR-adjusted P value (Wald test P values from DESeq2 with Benjamini-Hochberg correction). ( B ) Gene set enrichment analysis (GSEA) of immune pathways with increased accessibility in sg SMARCA4 versus sgNTC cells. Kolmogorov-Smirnov statistic with Benjamini-Hochberg correction. Exact q values indicated in each panel. ( C ) Changes in chromatin accessibility at transcription start sites (TSSs) of ISGs (CXCL10 and CCL2) in sg SMARCA4 versus sgNTC cells. ( D ) Motifs enriched in open chromosomal regions affected by SMARCA4 deficiency. P value and binomial test were performed using Homer2. Experiments performed in duplicates. GO, Gene Ontology; KO, knockout; KO_1, target exon 14 clone 1; NTC, non-target control; TF, transcription factor.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) IFNAR-1 neutralizing assay in ID8 single-guide NTC (sgNTC) compared to that in sg SMARCA4 cells (four sg SMARCA4 clones). Cells were treated with Mock or IFNAR-1 antibody for 48 hours at 10 μg/ml. ( B ) IRF3 expression levels in sg SMARCA4 versus sgNTC cells by qRT-PCR. ( C ) IRF3 expression in doxycycline-inducible short hairpin NTC (shNTC) and shIRF3-transfected sg SMARCA4 cells by qRT-PCR. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC and sg SMARCA4 cells. The latter were transfected with shIRF3 or shNTC (with and without POLYI:C). ( E ) Enriched IRF motif at TSSs of ISG locus from ATAC-seq analysis of ID8 sgNTC versus sg SMARCA4 cells. Experiments performed in duplicates. ( F ) Analysis of publicly available ChIP-Atlas data of IRF3 DNA binding sites on ISGs in murine immune cell lines. Statistical analysis was performed using two-tailored unpaired t test [(A) to (D)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. n = 3 independent experiments in (A) to (D). Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sgNTC_1 in (A), (B), and (D) and sg SMARCA4 transfected with shNTC in (C), as indicated]. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_3, target exon 23 clone 3; KO_4, target exon 23 clone 4; LPS, lipopolysaccharide stimulated, NTC, non-target control; NTC_1 = NTC clone 1; POLYI:C, polyinosinic-polycytidylic acid stimulated.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Neutralizing Assay, Clone Assay, Expressing, Quantitative RT-PCR, Transfection, Binding Assay, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Volcano plots for differential expression of TEs in ID8 cells. ( B ) Heatmap of long terminal repeats (LTR) with adjusted P < 0.05. ( C ) Representative pictures (top) of double-stranded RNA (dsRNA) identification by immunofluorescence (IF) in sg SMARCA4 and sgNTC ID8 cells with quantification (bottom). RNAse, ribonuclease. ( D ) qRT-PCR quantification of ISGs in ID8 sgNTC cells and sg SMARCA4 cells transfected with shMAVS or shNTC. ( E ) MAVS expression in doxycycline-inducible shNTC and shMAVS-transfected sg SMARCA4 cells by qRT-PCR. Expression levels were normalized to β-actin expression, and comparisons of mRNA expression levels were performed relative to control [sg SMARCA4 transfected with shNTC in (C) and sgNTC_1 in (D), as indicated]. Statistical analysis was performed using two-tailored unpaired t test [(C) to (E)]. * P < 0.05, ** P < 0.01, *** P < 0.001, and **** P < 0.0001. Error bars represent ± SEM. Samples in duplicates [for (A) and (B)]. n = 3 independent experiments [in (C) and (D)]. Red line in (A) represents a cutoff of an adjusted P value of <0.05. KO, knockout; KO_1, target exon 14 clone 1; KO_2, target exon 14 clone 2; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Quantitative Proteomics, Immunofluorescence, Quantitative RT-PCR, Transfection, Expressing, Control, Knock-Out

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the OC tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated intraperitoneally with 10 million ID8 sgNTC or sg SMARCA4 ( KO_4 ) tumor cells, and spectral flow cytometry was performed 21 days later on harvested ascites samples. ip, intraperitoneal. ( B ) Frequency of tumor PD1 + CD4 + T cells and PD1 + CD8 + T cells. ( C ) Frequency of NK1.1 + cells expressing Granzyme B + . ( D ) Frequency of tumor dendritic cells (of CD45 + cells) and MHCII-expressing dendritic cells. ( E ) Frequency of tumor macrophages (of CD45 + cells) and PD-L1–expressing macrophages. ( F ) In vivo bioluminescence imaging of tumor burden in sg SMARCA4 versus sgNTC tumors (unpaired t test of area under the curve). Statistical analysis was performed using two-tailored unpaired t test [(B) to (F)]. * P < 0.05, ** P < 0.01 and **** P < 0.0001. Error bars represent ± SEM. n = 10 mice per group. KO, knockout; KO_4, target exon 23 clone 4; NTC, non-target control; NTC_1, NTC clone 1.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, In Vivo, Imaging, Knock-Out, Control

Journal: Science Advances

Article Title: Interferon response and epigenetic modulation by SMARCA4 mutations drive ovarian tumor immunogenicity

doi: 10.1126/sciadv.adk4851

Figure Lengend Snippet: ( A ) Workflow for the B16-F10 tumor model. C57BL/6 (Cg)–Tyr c-2J /J mice were inoculated subcutaneously with 150,000 shNTC, sgEV, sh SMARCA4_1 , sh SMARCA4_2 , or sg SMARCA4 tumor cells, and spectral flow cytometry was performed 21 days later on harvested tumor samples. ( B ) Frequency of tumor CD8 + T cells and MFI of ICOS + -expressing CD8 + T cells in B16-F10 model. ( C ) Frequency of NK1.1 + cells in B16-F10 knockout and knockdown models (of CD45 + cells). ( D ) MFI of MHCII in macrophages in B16-F10 SMARCA4 knockout and knockdown models. ( E ) Tumor volume of sh SMARCA4 versus shNTC B16 tumors (unpaired t test of final time points). n = 5 mice per group, three biological replicates. ( F ) Frequency of NK1.1 + cells in sh SMARCA4 versus shNTC tumors (of live cells) in RAG2 −/− mice. ( G ) MFI of MHCII-expressing macrophages in B16-F10 sh SMARCA4 versus shNTC tumors in RAG2 −/− mice. ( H ) Tumor volumes of sh SMARCA4 versus shNTC B16 tumors in RAG2 −/− mice (unpaired t test of final time points). n = 3 to 5 mice per group, three biological replicates. Statistical analysis was performed using two-tailored unpaired t test. * P < 0.05, ** P < 0.01, *** P < 0.001. Error bars represent ± SEM. Dox, doxycycline; EV, empty vector; NTC, non-target control; SC, subcutaneous.

Article Snippet: Knockouts of STING were generated using gene-specific CRISPR-Cas9–knockout plasmids for STING (no. SC428364, Santa Cruz Biotechnology) and for NTC (SC-421688, Santa Cruz Biotechnology).

Techniques: Flow Cytometry, Expressing, Knock-Out, Knockdown, Plasmid Preparation, Control