Journal: bioRxiv

Article Title: Sialidases derived from Gardnerella vaginalis remodel the sperm glycocalyx and impair sperm function

doi: 10.1101/2025.02.01.636076

Figure Lengend Snippet: A) Schematic describing lectin flow cytometry and representative glycan epitopes. B, C, D) Sperm were exposed to sialidases (AUS 20 units or GvNanH2 160 units) for 1 hour, fixed in formalin, and stained with MAL-II-biotin/Neutravidin-Alexa Fluor 488, SNA-Cy5, or PSA-FITC respectively. Each point represents the median fluorescence intensity from one donor. Statistics represent a mixed model comparison with post-hoc Holm-Šidák tests. E) Schematic representing change in zeta potential after removal of negatively charged sialic acid on sperm surface. F) Sperm were treated with sialidase at various doses for 1 hour, and zeta potential was measured via dynamic light scattering. Each measurement represents the average of seven technical replicates, and statistics represent a mixed model comparison with post hoc Holm-Šidák tests.

Article Snippet: Biotinylated Maackia Amurensis Lectin II (MAL II, Cat #:B-1265-1) and Cy5-conjugated Sambucus Nigra Lectin (SNA, Cat #: CL-1305-1) were purchased from Vector Labs. Human Contraception Antibody (HCA), was a gift from ZabBio.

Techniques: Flow Cytometry, Staining, Fluorescence, Comparison, Zeta Potential Analyzer

Journal: Neurobiology of disease

Article Title: Inhibition of GSK-3 ameliorates the pathogenesis of Huntington's disease.

doi: 10.1016/j.nbd.2021.105336

Figure Lengend Snippet: Fig. 1. Amounts of mHtt aggregates are regulated by the autophagy- lysosome pathway. A. SH-SY5Y cells were transfected with GFP-Q23 or GFP-mHtt plasmids, and cells expressing GFP-mHtt were starved for amino acids (−aa, 6 h), or treated with vinblastine (20 nM, 6 h). Fixed cells were stained with DAPI and visualized by fluorescence microscopy (left panel). The percentage of transfected cells containing mHtt aggregates is shown at the middle panel. Numbers of mHtt aggregates/cell are shown in Fig. S1. Western blot analysis of cells expressing GFP-mHtt that were starved or not are shown in the right panel showing mHtt, phospho-S6K-1 (Thr389), and autophagic markers. Densitometry analysis of mHtt is show at the right. B. MEF cells (WT) or TSC1/2−/−cells were transfected with GFP-mHtt plasmid, and TSC1/2−/−cells were also subjected to aa starvation. Fixed cells stained with DAPI were visualized by fluorescence microscopy. Numbers of mHtt aggregates/cell is presented in the top panel. Representative Western blot analysis of WT and TSC1/2−/−cells showing mHtt, TSC2, phospho-mTOR (Ser2448), and autophagy markers are shown in the right panel. Densitometry analysis of mHtt is shown in the bottom. C. MEF cells (WT) or ATG5−/−cells were transfected with mCherry-mHtt. Fixed cells stained with DAPI were visualized by fluorescence microscopy. The percentage of transfected cells containing mHtt aggregates is shown in the bottom panel. Western blot analysis of WT and ATG5−/−cells showing mHtt and autophagic/lysosomal markers is shown in the right panel D. SH-SY5Y cells were co-transfected with mCherry-mHtt and GFP-LAMP2 plasmids, and live cells were imaged by confocal microscopy. White square shows magnification of mHtt in lysosome. Treatment with CQ (30 μM, 4 h) is shown at the right end panel. E. Lysosomes were isolated by density-gradient ultracentrifugation from SH-SY5Y cells expressing GFP-mHtt. The lysosome enriched fraction (fraction 1) and the subsequent fractions were immunoblotted for mHtt and CatD. ‘Total’ refers to preparation before organelle fractionation. F. WT and TSC1/2−/−cells were co-transfected with mCherry-mHtt and GFP-LAMP2 plasmids and imaged by confocal microscopy. It is shown that the major portion of mHtt is not localized within lysosomes in the TSC1/2−/−cells. For all panels, gels and data represent 3 independent experiments and results are mean ± SEM, *p < 0.05, ****p < 0.0001, by two-tailed student’s t-test.

Article Snippet: The following antibodies were used: anti-phopsho-S6K-1 (Thr389) (cat#9205), anti-phopsho-mTOr (Ser 2448) (cat#5536), anti-S6 (cat#2217), anti-LC3 (cat#4108), anti-GAPDH (cat#2118), and anti- phospho-AKT (Ser473) (cat#9271) were from Cell Signaling Technologies (Beverly, MA, USA).

Techniques: Transfection, Expressing, Staining, Fluorescence, Microscopy, Western Blot, Plasmid Preparation, Confocal Microscopy, Isolation, Fractionation, Two Tailed Test

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 3. The effect of FUNDC1 on neuronal apoptosis in the spinal cord tissue of SOD1G93A mice. (A, B, and C) shAAV9-EGFP and shAAV9-FUNDC1-infected spinal cord tissues of SOD1G93A mice were subjected to immunoblotting to determine the protein levels of BAX and BCL-2 (A), along with quantitative analysis results (B and C) (n = 4). (D, E, and F) AAV9-EGFP and AAV9-FUNDC1-infected spinal cord tissues of SOD1G93A mice were subjected to immunoblotting to determine the protein levels of BAX and BCL-2 (D), along with quantitative analysis results (E and F) (n = 4). (G, H, I, and J) In SOD1G93A mouse spinal cord tissues infected with AAV9/ shAAV9-FUNDC1, (G and H) representative Nissl-stained sections of the spinal cord anterior horn and quantitative analysis of surviving neurons, as well as (I and J), representative TUNEL-stained sections of the spinal cord anterior horn and quantitative analysis of the number of neurons undergoing apoptosis. (scale bar = 50 μm). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05, ** P < 0.01, *** P < 0.001. NS indicates no sig nificance (P > 0.05). AAV9-EGFP, AAV overexpression virus negative control; AAV9-FUNDC1, AAV overexpression FUNDC1 virus.

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Infection, Western Blot, Staining, TUNEL Assay, Two Tailed Test, Over Expression, Virus, Negative Control

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 5. The effect of FUNDC1 on autophagy flux in hSOD1G93A N2a neuronal cells. (A, B, and C) Evaluation of autophagy flux in hSOD1G93AN2a cells using the lysosomal inhibitor bafilomycin A1 (Baf, 100 nM). Baf-induced accumulation of LC3B-II and P62 in siCtrl or siFUNDC1-transfected cells (n = 3) (D, E, F, G, H, I, and J) siCtrl and siFUNDC1-transfected hSOD1G93AN2a cells were subjected to immunoblotting to detect FUNDC1, TOM20, COXIV, HSP60, BAX, and BCL-2 protein levels (D), along with quantitative analysis results (E, F, I, J (n = 4)) (G, H(n = 3)). (K, L, and M)Evaluation of autophagy flux in hSOD1G93AN2a cells using the lysosomal inhibitor bafilomycin A1 (Baf, 100 nM). Baf-induced accumulation of LC3B-II and P62 in adctrl or adFUNDC1-transfected cells (n = 3). (N, O, P, Q, R, S, and T) hSOD1G93A N2a cells transfected with adctrl and adFUNDC1 were subjected to immunoblotting to detect FUNDC1, TOM20, COXIV, HSP60, BAX, and BCL-2 protein levels (H), along with quantitative analysis results O-T) (O, P, S and T(n = 4))(Q, R(n = 3)). (U, V, W, and X) hSOD1G93A N2a cells transfected with si/adFUNDC1 and GFP-LC3 were observed for the colocalization of autophagosomes GFP-LC3 (green fluorescence) and Mito-Tracker Red (red fluorescence) (U), along with quantitative analysis of average mitochondrial area, perimeter and mitophagosome (V, W, and X) (n = 20) (scale bar = 10 um). (Y, Z) hSOD1G93A N2a cells transfected with si/ adFUNDC1 were subjected to TUNEL staining (Z) and JC-1 staining (Y) (scale bar = 20 um). The results are presented using an unpaired, two-tailed Student's t-test and expressed as the mean ± SD. * P < 0.05, ** P < 0.01, *** P < 0.001. NS indicates no significant difference (P > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Transfection, Western Blot, Fluorescence, TUNEL Assay, Staining, Two Tailed Test

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 6. Effect of FUNDC1 on neuronal apoptosis and mitophagy in hSOD1G93A N2a cells. (A) Transfected hSOD1G93A N2a cells with adCtrl and adFUNDC1, treated with or without 10 μM CQ, and observed autophagic vesicles GFP-LC3 (green) and DAPI (blue) (scale bar = 10 μm). (B) Transfected hSOD1 G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM carbonyl cyanide m-chlorophenylhydrazone (CCCP), colocalization of GFP-LC3 (green fluorescence) with Mito-Tracker Red (red fluorescence) was observed, along with quantitative analysis of average mitochondrial area, perimeter and mitophagosome (D, E, and F) (n = 15) (scale bar = 10 um). (C and G) Transfected hSOD1G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM CCCP, TUNEL-positive cells (green), DAPI (blue) (C), and results of quantitative analysis of the number of neuronal apoptosis positives (G) (scale bar = 50 um) were observed. (H, I, J, and K) Transfected hSOD1G93A N2a cells with adCtrl and adFUNDC1, treated with or without 10 μM CQ, and analyzed the protein levels of LC3B-II, TOM20, and BAX by immunoblotting (H), with quantitative analysis results (I, J, and K) (n = 4). (L, M, N, and O) Transfected hSOD1G93A N2a cells with siCtrl and siFUNDC1, treated with or without 10 μM CCCP, and analyzed the protein levels of LC3B-II, TOM20, and BAX by immunoblotting (L), with quantitative analysis results (M, N, and O) (n = 4). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05 or ** P < 0.01 indicates significance, whereas NS indicates no significant difference (P > 0.05). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Transfection, Fluorescence, TUNEL Assay, Western Blot, Two Tailed Test

Journal: Neurobiology of disease

Article Title: FUDNC1-dependent mitophagy ameliorate motor neuron death in an amyotrophic lateral sclerosis mouse model.

doi: 10.1016/j.nbd.2024.106534

Figure Lengend Snippet: Fig. 7. Effects of FOXD3 on FUNDC1-associated neuroapoptosis in SOD1G93A mice and cells. (A and B) FOXD3 protein expression in the spinal cord tissues of SOD1G93A mice (A) Quantitative analysis of FOXD3 protein expression levels (B) (n = 4). (C) A dual fluorescein reporter gene assay to detect the transcriptional regulation of FUNDC1 by FOXD3 (n = 4). (D, E, and F) FOXD3, FUNDC1 protein expression in adCtrl and adFOXD3 transfected hSOD1G93A N2a cells (D), quantitative analysis of FOXD3, FUNDC1 protein expression levels (E and F) (n = 4). (G, H, I, J, and K) Protein levels of LC3B-II, P62, TOM20, and BAX in hSOD1G93AN2a cells transfected with adCtrl, adFOXD3, and adFOXD3 + siFUNDC1, respectively (G), and quantitatively analyzed for LC3B-II, P62, TOM20, and BAX protein expression levels (H, I, J, and K) (n = 4). Results are demonstrated as mean ± SD with statistics using an unpaired two-tailed Student's t-test. * P < 0.05 or ** P < 0.01. NS indicates no significance (P > 0.05).

Article Snippet: The following primary antibodies were used for western blotting: FUNDC1 (1:1000, CST), LC3B-II (1:1000, CST, #49240), BAX (1:1000, Proteintech, Cat No. 60267-1-Ig), BCL-2 (1:1000, Proteintech, Cat No. 60178-1-Ig), GAPDH (1:5000, Proteintech, Cat No. 10494-1-AP), beta actin (1:5000, Cat No. 81115-1-RR), P62 (1:1000, Proteintech, Cat No. 18420-1-AP), HSP60 (1:1000, Zenbio, 222340), COXIV (1:1000, Zenbio, 200147), and FOXD3 (1:1000, Zenbio, 220508).

Techniques: Expressing, Reporter Gene Assay, Transfection, Two Tailed Test