Journal: Stem Cell Research & Therapy

Article Title: hucMSC-derived exosomes targeting macrophage polarization attenuate systemic inflammation in T1DM via INS/SOD1 delivery

doi: 10.1186/s13287-025-04521-0

Figure Lengend Snippet: Anti-inflammatory protein components in hucMSC-EXOs. ( A-F ) qPCR was performed to compare the effects of heat-inactivated hucMSC-EXOs and untreated hucMSC-EXOs on the expression levels of inflammatory cytokines in macrophages. ( G ) Topological analysis of hucMSC-EXOs proteins (top 200 by IBAQ value) via the STRING database, identifying the top 40 hub proteins ranked by interaction degree. Their degrees of interaction are visualized as a concentric circle plot. ( H-K ) ELISA was used to detect the INS ( H-I ) content and SOD1 ( J-K ) content of the hucMSCs culture supernatant and hucMSC-EXOs. (L‒O) qPCR analysis the effects of EXO alone, EXO combined with S961, and EXO combined with ATN-224 on the expression of inflammatory factors in macrophages. ( P ) WB experiment analysis the influence of EXO alone, EXO combined with S961, and EXO combined with ATN-224 treatment on PI3K/AKT and P65 phosphorylation in macrophages. * P < 0.05; ** P < 0.01 ; *** P < 0.001

Article Snippet: THP-1 cells were differentiated into M0 macrophages with 100 nM phorbol myristate acetate (PMA; HY-18739, MCE, US) for 24 h, and then divided into six experimental groups: PMA control, lipopolysaccharide (LPS; 50 ng/mL; L2880, Sigma, US), LPS + 5 nM INS (GC2646, Genxion, China), LPS + 1 μg SOD1 (HY-P71048A, MCE, US), LPS + hucMSC-EXOs (8.5 × 10^5 particles), LPS + heat-inactivated hucMSC-EXOs (8.5 × 10^5 particles), LPS + hucMSC-EXOs + 5 nM INS receptor antagonist S961 (HY-P2093B, MCE, US), and LPS + hucMSC-EXOs + 20 nM SOD1 inhibitor ATN-224 (HY-16074, MCE, US).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Phospho-proteomics

Journal: Nature Communications

Article Title: The motor neuron m6A repertoire governs neuronal homeostasis and FTO inhibition mitigates ALS symptom manifestation

doi: 10.1038/s41467-025-59117-2

Figure Lengend Snippet: a Timeline of MN differentiation in control (Ctrl) and ALS ( SOD1 +/L144F , C9ORF72 exp~800 G4C2 , TDP43 G298S ) iPSC lines. CPA (cyclopiazonic acid) was applied to accelerate MN degeneration (annotated as basal time point day 0). NF neurotrophic factors. b – d MN degeneration index and m 6 A RNA methylation levels of Ctrl and ALS iPSC~MNs. m 6 A percentages of Ctrl/ALS types at indicated time points were normalized to those at day 0. ALS iPSC~MNs undergo dramatic degeneration from day 7 to day 21 post-CPA treatment, while a significant reduction in m 6 A level was already exhibited at day 4. The degeneration index measures the neurite fragmentation. e – g Quantification of the m 6 A ratio in mRNAs from day 4 post-CPA treatment of Ctrl and ALS ( SOD1 +/L144F , C9ORF72 exp~800 G4C2 , TDP43 G298S ) iPSC lines. The mRNAs were extracted by poly(A) purification. The panels at the right show representative images of an m 6 A dot blot and methylene blue staining (for loading controls) ( b – g: n = 3 independent experiments). h Timeline of STM2457 (20 μM)-mediated inhibition of m 6 A modification during MN differentiation of wild-type iPSC lines. STM2457 was applied for 6 days to accelerate MN degeneration. i and j Inhibition of METTL3-mediated m 6 A modification in human wild-type iPSC~MNs results in a sharp decline in m 6 A levels after four days of treatment for STM2457, as assayed by m 6 A ELISA in mRNA ( i ) and mRNA m 6 A dot blot ( j ) ( i , j : n = 3 independent experiments). m 6 A methylation was normalized to the vehicle (DMSO), which served as a non-stressed control. Note that dramatic neurite degeneration was observed on day 6 in k upon STM2457 treatment. Scale bar, 200 µm. l Quantification of the degeneration index at an indicated time point normalized to the vehicle control ( n = 6 independent experiments). Illustrations in a and h created in BioRender. Chen, J. (2025) https://BioRender.com/3nizfu7 . All Data are presented as mean ± SD, significant P values from two-tailed t -tests. N.S. non-significant. Source data are provided as a Source data file.

Article Snippet: Mice carrying the mutant human SOD1 G93A transgene (B6SJL-Tg(SOD1*G93A)1Gur/J) were purchased from the Jackson Laboratory (JAX:002726).

Techniques: Control, Methylation, Purification, Dot Blot, Staining, Inhibition, Modification, Enzyme-linked Immunosorbent Assay, Two Tailed Test

Journal: Nature Communications

Article Title: The motor neuron m6A repertoire governs neuronal homeostasis and FTO inhibition mitigates ALS symptom manifestation

doi: 10.1038/s41467-025-59117-2

Figure Lengend Snippet: a Schematic illustration of the m 6 A biogenesis pathway and the applied FTO inhibitor (FB23-2) with their corresponding targeting pathways. Created in BioRender. Chen, J. (2025) https://BioRender.com/3nizfu7 . b Representative images of FB23-2 rescuing the MN degeneration associated with ALS. Scale bar, 200 µm. Quantifications of m 6 A mRNA methylation levels ( c ) and degeneration index values ( d ) at an indicated time point and compared to the CPA treatment. Note the significant rescue of the degeneration index upon applying FB23-2 to CPA-stressed C9ORF72 exp , SOD1 +/L144F , TDP43 G298S , and sALS iPSC~MNs. Data are presented as mean ± SD, n = 3, significant P values from two-way ANOVA. N.S. non-significant. e Heatmaps of normalized expression level between stress-treated (CPA) ALS-relevant lines with or without subsequent FB23-2 treatment, revealing restorations of many ALS risk genes with m 6 A modifications (highlighted with rectangles) to control (vehicle) levels for the FB23-2-treated groups. A z-score normalization was performed on the normalized read counts across samples for each gene after stress treatment (CPA) with or without subsequent FB23-2 treatment. Samples were normalized to the vehicle control to reveal the normalized expression level. Notably, following FTO inhibitor treatment, these genes were restored to levels comparable to controls among those ALS iPSC~MNs are highlighted in bold purple. Source data are provided as a Source data file.

Article Snippet: Mice carrying the mutant human SOD1 G93A transgene (B6SJL-Tg(SOD1*G93A)1Gur/J) were purchased from the Jackson Laboratory (JAX:002726).

Techniques: Methylation, Expressing, Control

Journal: Nature Communications

Article Title: The motor neuron m6A repertoire governs neuronal homeostasis and FTO inhibition mitigates ALS symptom manifestation

doi: 10.1038/s41467-025-59117-2

Figure Lengend Snippet: a , b Overview of the experimental strategy. Created in BioRender. Chen, J. (2025) https://BioRender.com/3m2vxum . c Western blot reveals that Fto protein level is reduced in mouse lumbar spinal cords after scAAV9-sh Fto injection ( n = 3 mice). d and e Kaplan-Meier survival curves with log-rank test revealing prolongation of the onset of weight decline in SOD1 G93A mice (from ∼140 to ∼155 days), with lifespans extended by ∼10% (from ∼170 days to ∼187 days), following scAAV9-sh Fto injection. m 6 A levels is upregulated ( f ), MN number is rescued ( f , with quantification in h ), and gliosis is reduced ( g , with quantification in i ) after scAAV9-sh Fto injection of SOD1 G93A mice at P140. Scale bars, 100 µm. (mean ± SD, n = 4 mice; two-tailed t -tests). j The CMAP amplitude is reduced in SOD1 G93A mice at P60 and gradually declines further over time, whereas scAAV9-sh Fto treatment significantly ameliorates neuromuscular function at P160 (mean ± SD, Ctrl: n = 5/1, 4/3, 3/3, and 4/3 mice, Ctrl; scAAV9-sh Fto : n = 5/1, 5/2, 5/2, and 5/2 mice; SOD1 G93A : n = 2/3, 5/6, 4/5, and 4/3 mice; SOD1 G93A ; scAAV9-sh Fto : n = 3/2, 5/3, 4/3, and 4/3 mice at P60, 120, 140, and 160 from males/females respectively; two-tailed t -tests) (right). k , l Motor coordination and muscle strength are enhanced by scAAV9-sh Fto injection, as assayed by rotarod test at P60~P160 ( k ) (mean ± SD, Ctrl: n = 6/2, 4/3, 4/3, and 4/3 mice, Ctrl; scAAV9-sh Fto : n = 5/1, 5/2, 5/2, and 5/2 mice; SOD1 G93A : n = 3/4, 5/6, 5/6, and 5/5 mice; SOD1 G93A ; scAAV9-sh Fto : n = 5/2, 5/3, 5/3, and 5/3 mice at P60, 120, 140, and 160 from males/females respectively; two-tailed t -tests), and by grip strength test ( l ) (mean ± SD, Ctrl: n = 6/2, 4/3, 4/3, and 4/1 mice, Ctrl; scAAV9-sh Fto : n = 5/1, 5/2, 5/2, and 5/2 mice; SOD1 G93A : n = 4/4, 5/6, 5/6, and 4/3 mice; SOD1 G93A ; scAAV9-sh Fto : n = 5/2, 5/3, 5/3, and 5/3 mice at P60, 120, 140, and 160 from males/females respectively; two-tailed t -tests). N.S. non-significant. Illustrations in j – l were created in BioRender. Chen, J. (2025) https://BioRender.com/mrtnwlk . Source data are provided as a Source data file.

Article Snippet: Mice carrying the mutant human SOD1 G93A transgene (B6SJL-Tg(SOD1*G93A)1Gur/J) were purchased from the Jackson Laboratory (JAX:002726).

Techniques: Western Blot, Injection, Two Tailed Test

Journal: Nature Communications

Article Title: The motor neuron m6A repertoire governs neuronal homeostasis and FTO inhibition mitigates ALS symptom manifestation

doi: 10.1038/s41467-025-59117-2

Figure Lengend Snippet: a Representative image illustrating H3K9me3 marks (yellow arrowheads) in the ventral horn of different sets of Ctrl and SOD1 G93A mice, with scAAV9-sh Fto intrathecal injections. b Quantifications of lumbar H3K9me3 on MNs (Ctrl: n = 7 mice, Ctrl; scAAV9-sh Fto : n = 3 mice, SOD1 G93A : n = 4 mice, SOD1 G93A ; scAAV9-sh Fto : n = 4 mice, quantification for all MN nuclei from all views of captured images; Scale bars, 20 µm). Data are presented as mean ± SD with significant P values from two-way ANOVA. N.S. non-significant. Source data are provided as a Source data file.

Article Snippet: Mice carrying the mutant human SOD1 G93A transgene (B6SJL-Tg(SOD1*G93A)1Gur/J) were purchased from the Jackson Laboratory (JAX:002726).

Techniques:

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: Description of SOD1 G93A mice censured from therapeutic efficacy study.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: Drug discovery, Infection

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: Establishing the effect of CET combination therapy on cell survival of NSC-34 cells expressing SOD1 G93A -EGFP. (a) Heatmaps of the 3D checkerboard treatment of NSC-34 cells expressing SOD1 G93A -EGFP. The colour scale indicates the degree to which treatment improves survival, and numbers indicate the fold increase in survival relative to the vehicle control. (b) The CET ratios that resulted in the greatest improvements in SOD1 G93A -EGFP cell survival (≥2.7 fold) from (a) were compared against CuATSM treatment alone. (c) A ratio of 1:80:250 for the CET combination therapy was selected to move forward for further testing using a range of CuATSM concentrations in NSC-34 cells expressing SOD1 G93A -EGFP. The CET combination therapy with a CuATSM concentration of 0.15 μM and 0.2 μM improved the survival of SOD1 G93A -expressing cells compared to the equivalent concentration of CuATSM alone (determined by a repeated one-way ANOVA with Tukey’s multiple comparisons post-test. All data represent mean ± SD ( n = 3 individual experiments).

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: Expressing, Control, Concentration Assay

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: CET combination therapy is more effective than CuATSM treatment in increasing the survival of cells expressing SOD1 G93A and reducing SOD1 G93A inclusion formation. (a) NSC-34 cells expressing SOD1 G93A -EGFP were treated with either CuATSM (0.15 μM), ebselen (12 μM) and telbivudine (37.5 μM) (CET) combination therapy or CuATSM alone and imaged every 3 h for 48 h on an IncuCyte S3 automated fluorescent microscope and GFP cell counts quantified. (b) The survival of SOD1 G93A -EGFP expressing cells was determined by measuring the area under the GFP count curves (a) . Solid lines represent mean values with shaded areas representing ± SD (n ≥ 5 technical replicates from at least 3 separate experiments) One-way ANOVA with a Tukey’s multiple comparisons post-test was used to compare statistical significance between treatments. (c) The number of cells containing SOD1 G93A -EGFP inclusions was determined by semi-automated analysis of the 48 h time point images from the IncuCyte survival assay. One-way ANOVA with a Tukey’s multiple comparisons post-test was used to compare statistical significance between treatments.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: Expressing, Microscopy, Clonogenic Cell Survival Assay

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: CET polytherapy improves SOD1 G93A folding but does not alter SOD1 activity in NSC-34 cells. (a) The proportion of disulfide-bonded SOD1 was determined by SDS-PAGE migration of SOD1 G93A -EGFP lysates treated with either CET or CuATSM under reducing conditions (+β-merc) and nonreducing conditions (−β-merc). Arrows indicate the position of disulfide-bonded SOD1 (SS) and reduced SOD1 (SH) on the immunoblot. (b) Quantification of the immunoblots from (a) by densitometry. Data shown are mean ± SD (n = 3) and statistical significance was determined by one-way ANOVA with a Tukey’s multiple comparisons post-test. (c) Native-PAGE (top) and in-gel zymography (bottom) of SOD1 G93A -tdTomato cell lysates treated with either CET or CuATSM. The position of SOD1 dimers, monomers and endogenous mouse SOD1 are indicated with arrows. (d) Quantification of the tdTomato signal from native-PAGE to determine the proportion of SOD1 dimer present following CET or CuATSM treatment. Data shown are mean ± SD (n = 3) and statistical significance was determined by one-way ANOVA with a Tukey’s multiple comparisons post-test. Asterisks indicate significant difference to the vehicle control. (e) SOD1 activity (normalised to SOD1 protein levels) was measured in cells treated with either CET or CuATSM by quantifying the achromatic bands corresponding to the SOD1 G93A dimer from in-gel zymography and normalising to the corresponding tdTomato fluorescence signal from the native-PAGE gel. Data represent mean ± SD (n = 3). One-way ANOVA was used to determine significant differences in SOD1 activity between treatments.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: Activity Assay, SDS Page, Migration, Western Blot, Clear Native PAGE, Zymography, Control, Fluorescence

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: CET polytherapy delays disease onset, modifies neurological scoring and extends survival in SOD1 G93A mice. The effect of daily oral gavage administration of either vehicle, CuATSM or CET on (a) body weight, (b) age of peak body weight, (c) neurological score, (d) age of disease onset (defined as attaining a neurological score of 1), (e) latency to fall during rotarod task, (f) Kaplain–Meier plot and (g) survival (a, c and e) Solid lines represent mean values with shaded areas representing ± SEM ( n = 21–24 per treatment group). (b and d) Data is shown as mean ± SD ( n = 20–24 per treatment group). (g) Data is shown as median ± IQR.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques:

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: CET treatments increases in vivo SOD1 maturation and reduces misfolded SOD1 accumulation in SOD1 G93A mice. (a and b) The relative levels of SOD1 protein and (c) the proportion of disulfide SOD1 (reduced = SH, oxidised intact = SS) in lumbar spinal cord homogenates of vehicle-, CuATSM- and CET-treated mice as determined by non-reducing immunoblot. (d) SOD1 in-gel zymography and (e) quantification of SOD1 activity normalised to soluble SOD1 levels in lumbar spinal cord homogenates of vehicle-, CuATSM- and CET-treated mice. (f) C4F6 dot blots of lumbar spinal cord homogenates and (g) relative misfolded SOD1 (C4F6) levels in lumbar spinal cord homogenates of vehicle-, CuATSM- and CET-treated mice. Data shown are mean ± SD ( n = 11–12/treatment). Data are from 3 technical replicates. One-way ANOVA was used to compare relative differences between vehicle-, CuATSM- and CET-treated SOD1 G93A mice.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: In Vivo, Western Blot, Zymography, Activity Assay

Journal: eBioMedicine

Article Title: A polytherapy approach demonstrates therapeutic efficacy for the treatment of SOD1 associated amyotrophic lateral sclerosis

doi: 10.1016/j.ebiom.2025.105692

Figure Lengend Snippet: CuATSM and CET treatment provides neuroprotection. (a) Representative micrographs of thionin acetate-stained ventral horn lumbar spinal cord sections and (b) motor neuron counts from vehicle-, CuATSM- and CET-treated SOD1 G93A mice. Data shown are mean ± SD ( n = 9–10/treatment). One-way ANOVA was used to compare relative differences between vehicle-, CuATSM- and CET-treated SOD1 G93A mice.

Article Snippet: Male and female heterozygous human SOD1 G93A mice (B6-Tg(SOD1-G93A)1GUr/j); The Jackson Laboratory Stock Number: 004435] were maintained on a C57BL/6J background and bred at the Australian Bioresources Animal Facility (Moss Vale, Australia).

Techniques: Staining