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Tocris nu9056

a Forward locomotion in WT and ChI-D2RKO mice on the final day of daily chronic treatment with vehicle or <t>NU9056</t> (2.5 mg/kg i.p.) 1-h prior to saline or cocaine (15 mg/kg i.p). Motor activity was recorded for 1-h following saline or cocaine administration (N = 9–12/group). Two-way ANOVA, genotype: F (1,21) = 43.11, P < 0.0001; treatment: F (1.481,26.65) = 161.8, P < 0.0001; genotype × treatment: F (3,54) = 45.50, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. b Left: Bar graph showing fold change in expression of Kat5/GAPDH by Western blot in NAcc extracts following chronic treatment. Fold changes shown are relative to WT saline (N = 6–8/group). Two-way ANOVA, genotype: F (1,14) = 6.200, P = 0.0260; treatment: F (2.573,29.16) = 10.65, P = 0.0001; genotype × treatment: F (3,34) = 10.96, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0109; WT sal/coc vs WT NU9056/coc: P = 0.0025; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0004. Right: Representative Western Blot images of samples from Left . c Representative images of fluorescent in situ hybridization (FISH) experiments showing D1R (green), D2R (red), and Kat5 (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. d Quantification of the number of Kat5 + puncta in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 52.85, P = 0.0019; treatment: F (1.988,7.954) = 100.5, P < 0.0001; genotype × treatment: F (3,12) = 44.78, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0191; WT sal/coc vs WT NU9056/coc: P = 0.0157; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0011. e Quantification of the number of Kat5 + puncta in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 7.627, P = 0.0503; treatment: F (1.809,7.235) = 11.97, P = 0.0056; genotype × treatment: F (3,12) = 4.595, P = 0.0231. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.1881; WT sal/coc vs WT NU9056/coc: P = 0.2970; WT sal/coc vs ChI-D2RKO data are provided as a file.

Nu9056, supplied by Tocris, used in various techniques. Bioz Stars score: 93/100, based on 35 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/nu9056/pmc12689792-242-11-12?v=Tocris
Average 93 stars, based on 35 article reviews

nu9056 - by Bioz Stars, 2026-07

93/100 stars

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1) Product Images from "Epigenetic regulation of cocaine intake through dopaminergic control of cholinergic interneurons in male mice"

Article Title: Epigenetic regulation of cocaine intake through dopaminergic control of cholinergic interneurons in male mice

Journal: Nature Communications

doi: 10.1038/s41467-025-65958-8

a Forward locomotion in WT and ChI-D2RKO mice on the final day of daily chronic treatment with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to saline or cocaine (15 mg/kg i.p). Motor activity was recorded for 1-h following saline or cocaine administration (N = 9–12/group). Two-way ANOVA, genotype: F (1,21) = 43.11, P < 0.0001; treatment: F (1.481,26.65) = 161.8, P < 0.0001; genotype × treatment: F (3,54) = 45.50, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. b Left: Bar graph showing fold change in expression of Kat5/GAPDH by Western blot in NAcc extracts following chronic treatment. Fold changes shown are relative to WT saline (N = 6–8/group). Two-way ANOVA, genotype: F (1,14) = 6.200, P = 0.0260; treatment: F (2.573,29.16) = 10.65, P = 0.0001; genotype × treatment: F (3,34) = 10.96, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0109; WT sal/coc vs WT NU9056/coc: P = 0.0025; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0004. Right: Representative Western Blot images of samples from Left . c Representative images of fluorescent in situ hybridization (FISH) experiments showing D1R (green), D2R (red), and Kat5 (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. d Quantification of the number of Kat5 + puncta in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 52.85, P = 0.0019; treatment: F (1.988,7.954) = 100.5, P < 0.0001; genotype × treatment: F (3,12) = 44.78, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0191; WT sal/coc vs WT NU9056/coc: P = 0.0157; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0011. e Quantification of the number of Kat5 + puncta in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 7.627, P = 0.0503; treatment: F (1.809,7.235) = 11.97, P = 0.0056; genotype × treatment: F (3,12) = 4.595, P = 0.0231. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.1881; WT sal/coc vs WT NU9056/coc: P = 0.2970; WT sal/coc vs ChI-D2RKO data are provided as a file.

Figure Legend Snippet: a Forward locomotion in WT and ChI-D2RKO mice on the final day of daily chronic treatment with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to saline or cocaine (15 mg/kg i.p). Motor activity was recorded for 1-h following saline or cocaine administration (N = 9–12/group). Two-way ANOVA, genotype: F (1,21) = 43.11, P < 0.0001; treatment: F (1.481,26.65) = 161.8, P < 0.0001; genotype × treatment: F (3,54) = 45.50, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. b Left: Bar graph showing fold change in expression of Kat5/GAPDH by Western blot in NAcc extracts following chronic treatment. Fold changes shown are relative to WT saline (N = 6–8/group). Two-way ANOVA, genotype: F (1,14) = 6.200, P = 0.0260; treatment: F (2.573,29.16) = 10.65, P = 0.0001; genotype × treatment: F (3,34) = 10.96, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0109; WT sal/coc vs WT NU9056/coc: P = 0.0025; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0004. Right: Representative Western Blot images of samples from Left . c Representative images of fluorescent in situ hybridization (FISH) experiments showing D1R (green), D2R (red), and Kat5 (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. d Quantification of the number of Kat5 + puncta in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 52.85, P = 0.0019; treatment: F (1.988,7.954) = 100.5, P < 0.0001; genotype × treatment: F (3,12) = 44.78, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0191; WT sal/coc vs WT NU9056/coc: P = 0.0157; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0011. e Quantification of the number of Kat5 + puncta in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 7.627, P = 0.0503; treatment: F (1.809,7.235) = 11.97, P = 0.0056; genotype × treatment: F (3,12) = 4.595, P = 0.0231. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.1881; WT sal/coc vs WT NU9056/coc: P = 0.2970; WT sal/coc vs ChI-D2RKO data are provided as a file.

Techniques Used: Saline, Activity Assay, Expressing, Western Blot, In Situ Hybridization

a Representative images of combination FISH-immunofluorescence experiments showing D1R (green), D2R (red), and H4K8ac (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. b Quantification of the fluorescent intensity (RFU) of H4K8ac in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 15.18, P = 0.0176; treatment: F (3,12) = 15.28, P = 0.0002; genotype × treatment: F (3,12) = 6.787, P = 0.0063. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. c Quantification of the fluorescent intensity (RFU) of H4K8ac in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 11.82, P = 0.0263; treatment: F (3,12) = 3.255, P = 0.0597; genotype × treatment: F (3,12) = 0.4714, P = 0.7079. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0201; WT sal/coc vs WT NU9056/coc: P = 0.0833; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0387. Values shown are mean ± SEM.

Figure Legend Snippet: a Representative images of combination FISH-immunofluorescence experiments showing D1R (green), D2R (red), and H4K8ac (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. b Quantification of the fluorescent intensity (RFU) of H4K8ac in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 15.18, P = 0.0176; treatment: F (3,12) = 15.28, P = 0.0002; genotype × treatment: F (3,12) = 6.787, P = 0.0063. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. c Quantification of the fluorescent intensity (RFU) of H4K8ac in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 11.82, P = 0.0263; treatment: F (3,12) = 3.255, P = 0.0597; genotype × treatment: F (3,12) = 0.4714, P = 0.7079. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0201; WT sal/coc vs WT NU9056/coc: P = 0.0833; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0387. Values shown are mean ± SEM.

Techniques Used: Immunofluorescence, Expressing

Image Search Results

Journal: Nature Communications

Article Title: Epigenetic regulation of cocaine intake through dopaminergic control of cholinergic interneurons in male mice

doi: 10.1038/s41467-025-65958-8

Figure Lengend Snippet: a Forward locomotion in WT and ChI-D2RKO mice on the final day of daily chronic treatment with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to saline or cocaine (15 mg/kg i.p). Motor activity was recorded for 1-h following saline or cocaine administration (N = 9–12/group). Two-way ANOVA, genotype: F (1,21) = 43.11, P < 0.0001; treatment: F (1.481,26.65) = 161.8, P < 0.0001; genotype × treatment: F (3,54) = 45.50, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. b Left: Bar graph showing fold change in expression of Kat5/GAPDH by Western blot in NAcc extracts following chronic treatment. Fold changes shown are relative to WT saline (N = 6–8/group). Two-way ANOVA, genotype: F (1,14) = 6.200, P = 0.0260; treatment: F (2.573,29.16) = 10.65, P = 0.0001; genotype × treatment: F (3,34) = 10.96, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0109; WT sal/coc vs WT NU9056/coc: P = 0.0025; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0004. Right: Representative Western Blot images of samples from Left . c Representative images of fluorescent in situ hybridization (FISH) experiments showing D1R (green), D2R (red), and Kat5 (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. d Quantification of the number of Kat5 + puncta in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 52.85, P = 0.0019; treatment: F (1.988,7.954) = 100.5, P < 0.0001; genotype × treatment: F (3,12) = 44.78, P < 0.0001. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0191; WT sal/coc vs WT NU9056/coc: P = 0.0157; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0011. e Quantification of the number of Kat5 + puncta in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 7.627, P = 0.0503; treatment: F (1.809,7.235) = 11.97, P = 0.0056; genotype × treatment: F (3,12) = 4.595, P = 0.0231. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.1881; WT sal/coc vs WT NU9056/coc: P = 0.2970; WT sal/coc vs ChI-D2RKO data are provided as a file.

Article Snippet: Cocaine HCl (Sigma; Cat# C57761G), Tropicamide HCl (Tocris; Cat# 0909), and NU9056 (Tocris; Cat# 9043) were dissolved in sterile saline (0.9% NaCl, pH 7.4).

Techniques: Saline, Activity Assay, Expressing, Western Blot, In Situ Hybridization

Journal: Nature Communications

Article Title: Epigenetic regulation of cocaine intake through dopaminergic control of cholinergic interneurons in male mice

doi: 10.1038/s41467-025-65958-8

Figure Lengend Snippet: a Representative images of combination FISH-immunofluorescence experiments showing D1R (green), D2R (red), and H4K8ac (white) expression in the NAcc of WT and ChI-D2RKO mice (N = 3/group) treated with vehicle or NU9056 (2.5 mg/kg i.p.) 1-h prior to cocaine (15 mg/kg i.p.) for 7 days (blue: DAPI). Scale bar: 10 μm. b Quantification of the fluorescent intensity (RFU) of H4K8ac in D1R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 15.18, P = 0.0176; treatment: F (3,12) = 15.28, P = 0.0002; genotype × treatment: F (3,12) = 6.787, P = 0.0063. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P < 0.0001; WT sal/coc vs WT NU9056/coc: P < 0.0001; WT sal/coc vs ChI-D2RKO sal/coc: P < 0.0001. c Quantification of the fluorescent intensity (RFU) of H4K8ac in D2R + MSNs in the NAcc. Two-way ANOVA, genotype: F (1,4) = 11.82, P = 0.0263; treatment: F (3,12) = 3.255, P = 0.0597; genotype × treatment: F (3,12) = 0.4714, P = 0.7079. Tukey post-hoc test, WT sal/sal vs WT sal/coc: P = 0.0201; WT sal/coc vs WT NU9056/coc: P = 0.0833; WT sal/coc vs ChI-D2RKO sal/coc: P = 0.0387. Values shown are mean ± SEM.

Article Snippet: Cocaine HCl (Sigma; Cat# C57761G), Tropicamide HCl (Tocris; Cat# 0909), and NU9056 (Tocris; Cat# 9043) were dissolved in sterile saline (0.9% NaCl, pH 7.4).

Techniques: Immunofluorescence, Expressing

MG149 treatment reduces PINK1/Parkin-dependant mitophagy initiation. ( A ) Representative images of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 (KAT5 inhibitor) or 100 μM MG149 (KAT5/KAT8 inhibitor). for 1, 3, 24, and 48 h and subsequently treated with DMSO or 1 μM O/A for 3 h to induce mitophagy (representative images show a 3 h. timepoint). Cells were then immuno-stained anti-Tom20 (yellow) and anti-pUb(Ser65) (green), with Hoechst nuclear counterstain (blue). Scale bar: 20 μm. ( B ) Quantification of integrated pUb(Ser65) signal density in A, compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalized to control O/A (specifically the 48 h DMSO control pre-treatment and 3 h O/A treatment timepoint). ( C ) Representative immunoblots of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for MFN2, pUb(Ser65), pParkin(Ser65) and GAPDH. ( D ) Quantification of MFN2, pUb(Ser65), pParkin signal density in C, compared to control conditions (n = 3–5, two-way ANOVA with Dunnett’s correction). Data normalized to control O/A and GAPDH used as a loading control. Data shown as mean + /− SD.

Journal: Scientific Reports

Article Title: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy

doi: 10.1038/s41598-024-60602-9

Figure Lengend Snippet: MG149 treatment reduces PINK1/Parkin-dependant mitophagy initiation. ( A ) Representative images of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 (KAT5 inhibitor) or 100 μM MG149 (KAT5/KAT8 inhibitor). for 1, 3, 24, and 48 h and subsequently treated with DMSO or 1 μM O/A for 3 h to induce mitophagy (representative images show a 3 h. timepoint). Cells were then immuno-stained anti-Tom20 (yellow) and anti-pUb(Ser65) (green), with Hoechst nuclear counterstain (blue). Scale bar: 20 μm. ( B ) Quantification of integrated pUb(Ser65) signal density in A, compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalized to control O/A (specifically the 48 h DMSO control pre-treatment and 3 h O/A treatment timepoint). ( C ) Representative immunoblots of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for MFN2, pUb(Ser65), pParkin(Ser65) and GAPDH. ( D ) Quantification of MFN2, pUb(Ser65), pParkin signal density in C, compared to control conditions (n = 3–5, two-way ANOVA with Dunnett’s correction). Data normalized to control O/A and GAPDH used as a loading control. Data shown as mean + /− SD.

Article Snippet: mitoSRAI POE SH-SY5Y cells were seeded in a 96-well plate (PhenoPlate 96-well microplate, PerkinElmer, 6055302), pre-treated with MG149 or NU9056 for 3 h according to desired concentrations, and subsequently with DMSO (Santa Cruz, sc-202581) or O/A for 3 h. Equal volumes of DMSO (Santa Cruz, sc-202581) were used as no treatment controls.

Techniques: Staining, Control, Western Blot

MG149 treatment initiates mitochondrial delivery to the lysosome. ( A ) Live time-course imaging of mitoKeima POE SH-SY5Y cells simultaneously treated with DMSO, 10 μM NU9056 or 100 μM MG149 and with . either DMSO or 1 μM O/A to induce mitophagy (representative images show a 6 h time-point). The neutral Keima-green signal (green) is shown alongside the acidic lysosomal Keima-red signal (red). Scale bar: 20 μm. ( B ) Quantification of the Keima mitophagy index in A, calculated as the ratio of the area of lysosomal mitoKeima signal to total mitoKeima signal (sum of lysosomal and neutral mitoKeima signal), compared to control conditions (n = 4, two-way ANOVA with Dunnett’s correction). Data normalised to control 6 h O/A. ( C ) Representative images of mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 or 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cell nuclei were counterstained with DRAQ5 (red). The TOLLES fluorophore (cyan) shows total mitochondria while the Ypet fluorophore (orange) shows the non-lysosomal mitochondria. Scale bar: 20 μm. ( D ) Quantification of the SRAI mitophagy index in C, calculated as the ratio of the lysosomal mitochondria (TOLLES area without Ypet signal) to the total mitochondrial area (total TOLLES area) and compared to control conditions (n = 6, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. ( E ) Representative images of mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cells were immuno-stained for p62 (yellow) and counterstained with DRAQ5 (red) (TOLLES fluorophore in cyan). Scale bar: 20 μm. ( F ) Quantification of p62 recruitment to the mitochondria in E, calculated as the ratio of mitochondrial to cytoplasmic p62 intensity (ratio of p62 signal intensity inside vs. outside TOLLES), compared to control conditions (n = 4, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. Data shown as mean + /− SD.

Journal: Scientific Reports

Article Title: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy

doi: 10.1038/s41598-024-60602-9

Figure Lengend Snippet: MG149 treatment initiates mitochondrial delivery to the lysosome. ( A ) Live time-course imaging of mitoKeima POE SH-SY5Y cells simultaneously treated with DMSO, 10 μM NU9056 or 100 μM MG149 and with . either DMSO or 1 μM O/A to induce mitophagy (representative images show a 6 h time-point). The neutral Keima-green signal (green) is shown alongside the acidic lysosomal Keima-red signal (red). Scale bar: 20 μm. ( B ) Quantification of the Keima mitophagy index in A, calculated as the ratio of the area of lysosomal mitoKeima signal to total mitoKeima signal (sum of lysosomal and neutral mitoKeima signal), compared to control conditions (n = 4, two-way ANOVA with Dunnett’s correction). Data normalised to control 6 h O/A. ( C ) Representative images of mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 or 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cell nuclei were counterstained with DRAQ5 (red). The TOLLES fluorophore (cyan) shows total mitochondria while the Ypet fluorophore (orange) shows the non-lysosomal mitochondria. Scale bar: 20 μm. ( D ) Quantification of the SRAI mitophagy index in C, calculated as the ratio of the lysosomal mitochondria (TOLLES area without Ypet signal) to the total mitochondrial area (total TOLLES area) and compared to control conditions (n = 6, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. ( E ) Representative images of mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cells were immuno-stained for p62 (yellow) and counterstained with DRAQ5 (red) (TOLLES fluorophore in cyan). Scale bar: 20 μm. ( F ) Quantification of p62 recruitment to the mitochondria in E, calculated as the ratio of mitochondrial to cytoplasmic p62 intensity (ratio of p62 signal intensity inside vs. outside TOLLES), compared to control conditions (n = 4, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. Data shown as mean + /− SD.

Techniques: Imaging, Control, Staining

The mitochondrial clearance initiated in MG149 treated cells is dependant on PINK1. ( A ) Representative images of PINK1 KO mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cells were immuno-stained for p62 (yellow) and nuclei counterstained with DRAQ5 (red) (TOLLES fluorophore in cyan). Scale bar: 20 μm. ( B ) Quantification of p62 recruitment to the mitochondria in E, calculated as the ratio of mitochondrial to cytoplasmic p62 intensity (ratio of p62 signal intensity inside vs. outside TOLLES), compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. ( C ) Representative images of PINK1 KO mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cell nuclei were counterstained with DRAQ5 (red). The TOLLES fluorophore (cyan) shows total mitochondria while the Ypet fluorophore (orange) shows the non-lysosomal mitochondria. Scale bar: 20 μm. ( D ) Quantification of the SRAI mitophagy index in C, calculated as the ratio of the lysosomal mitochondria (TOLLES area without Ypet signal) to the total mitochondrial area (total TOLLES area), and compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. Data shown as mean + /− SD.

Journal: Scientific Reports

Article Title: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy

doi: 10.1038/s41598-024-60602-9

Figure Lengend Snippet: The mitochondrial clearance initiated in MG149 treated cells is dependant on PINK1. ( A ) Representative images of PINK1 KO mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cells were immuno-stained for p62 (yellow) and nuclei counterstained with DRAQ5 (red) (TOLLES fluorophore in cyan). Scale bar: 20 μm. ( B ) Quantification of p62 recruitment to the mitochondria in E, calculated as the ratio of mitochondrial to cytoplasmic p62 intensity (ratio of p62 signal intensity inside vs. outside TOLLES), compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. ( C ) Representative images of PINK1 KO mitoSRAI POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Cell nuclei were counterstained with DRAQ5 (red). The TOLLES fluorophore (cyan) shows total mitochondria while the Ypet fluorophore (orange) shows the non-lysosomal mitochondria. Scale bar: 20 μm. ( D ) Quantification of the SRAI mitophagy index in C, calculated as the ratio of the lysosomal mitochondria (TOLLES area without Ypet signal) to the total mitochondrial area (total TOLLES area), and compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. Data shown as mean + /− SD.

Techniques: Staining, Control

MG149 effectively reduces mitochondrial membrane potential. ( A ) Representative images of TMRM (yellow) in POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 or 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h to induce mitophagy. Nuclei counterstained with Hoechst (blue) Scale bar: 20 μm. ( B ) Quantification of TMRM spot intensity in A, compared to control conditions (n = 5, two-way ANOVA with Dunnett's correction). Data normalised to control DMSO. ( C ) Representative immunoblot of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for OPA1 and GAPDH. ( D ) Quantification of OPA1 signal intensity in C, calculated as the ratio of the signal intensity of L-OPA1 to S-OPA1, and compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalized to control DMSO, and GAPDH used as a loading control. Data shown as mean + /− SD.

Journal: Scientific Reports

Article Title: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy

doi: 10.1038/s41598-024-60602-9

Figure Lengend Snippet: MG149 effectively reduces mitochondrial membrane potential. ( A ) Representative images of TMRM (yellow) in POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056 or 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h to induce mitophagy. Nuclei counterstained with Hoechst (blue) Scale bar: 20 μm. ( B ) Quantification of TMRM spot intensity in A, compared to control conditions (n = 5, two-way ANOVA with Dunnett's correction). Data normalised to control DMSO. ( C ) Representative immunoblot of POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for OPA1 and GAPDH. ( D ) Quantification of OPA1 signal intensity in C, calculated as the ratio of the signal intensity of L-OPA1 to S-OPA1, and compared to control conditions (n = 3, two-way ANOVA with Dunnett’s correction). Data normalized to control DMSO, and GAPDH used as a loading control. Data shown as mean + /− SD.

Techniques: Membrane, Control, Western Blot

$MG149 attenuates PINK1 autophosphorylation. ( A ) Quantification of PINK1 mRNA levels in POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h, compared to control conditions (n = 6, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. B. Representative immunoblots of mitochondrial fractions from POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for PINK1, Hsp60, PDHE1a, and Tim23. C. Quantification of PINK1 signal intensity in B, compared to controls (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A, and the PDHE1a used as a loading control. ( D ) Representative immunoblots of mitochondrial fractions from PINK1-HA SH-SY5Y cells pre-treated with DMSO and 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for pPINK1, PINK1, Hsp60, PDHE1a, and Tim23 ( E ) Quantification of pPINK1 signal intensity in D, calculated as the ratio of pPINK1 to PINK1 signal intensity, compared to controls (n = 3, two-way ANOVA with Sídák correction). Data normalized to control O/A and PDHE1a used as a loading control. Data shown as mean + /− SD.$

Journal: Scientific Reports

Article Title: KAT8 compound inhibition inhibits the initial steps of PINK1-dependant mitophagy

doi: 10.1038/s41598-024-60602-9

Figure Lengend Snippet: MG149 attenuates PINK1 autophosphorylation. ( A ) Quantification of PINK1 mRNA levels in POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h, compared to control conditions (n = 6, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A. B. Representative immunoblots of mitochondrial fractions from POE SH-SY5Y cells pre-treated with DMSO, 10 μM NU9056, 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for PINK1, Hsp60, PDHE1a, and Tim23. C. Quantification of PINK1 signal intensity in B, compared to controls (n = 3, two-way ANOVA with Dunnett’s correction). Data normalised to control O/A, and the PDHE1a used as a loading control. ( D ) Representative immunoblots of mitochondrial fractions from PINK1-HA SH-SY5Y cells pre-treated with DMSO and 100 μM MG149 for 3 h and subsequently treated with DMSO or 1 μM O/A for 3 h. Blots were probed for pPINK1, PINK1, Hsp60, PDHE1a, and Tim23 ( E ) Quantification of pPINK1 signal intensity in D, calculated as the ratio of pPINK1 to PINK1 signal intensity, compared to controls (n = 3, two-way ANOVA with Sídák correction). Data normalized to control O/A and PDHE1a used as a loading control. Data shown as mean + /− SD.

Techniques: Control, Western Blot

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