Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: A) Images show co-localized immunofluorescence signals for glucagon and Stmn2 (top row), glucagon and the lysosomal marker LAMP1 (middle row) and glucagon and the secretory granule membrane docking protein syntaxin-1A (bottom row). Insets show magnified areas outlined in green. Yellow arrowheads indicate the spatial localization of immunofluorescence signals in peripheral and intracellular regions. B) Plot profile analyses of colocalized immunofluorescence signals in the intracellular or peripheral regions in response to vehicle (open circle), 1 nM insulin+25 μM GABA (open squares), or 400 nM SST (x). Bars represent the averaged colocalized fluorescence intensities ± SEM (n=3) and symbols represent measurements from individual cells. Comparisons were made between and within cellular regions using a two-way ANOVA, followed by a post-hoc test. ***p<0.001, ****p<0.0001.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Immunofluorescence, Marker, Membrane, Fluorescence

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Transfected cells were immunostained using primary antibodies against Stmn2 (green) and glucagon (red). a) Cells transfected with GFP alone show co-localized signals mainly at the cell periphery (yellow arrowheads). b) In Stmn2-KD cells, there is diminished Stmn2 immunofluorescence with glucagon localized largely to the periphery (yellow arrowheads). c) After Stmn2 OE, there is increased Stmn2 fluorescence intensity colocalizing strongly with glucagon in the intracellular region (yellow arrowheads). d) Changes in integrated Stmn2 fluorescence intensity in the Stmn2-KD and OE compared to the control (GFP-alone), normalized to endogenous Stmn2. Values were expressed as average integrated fluorescence intensity ±SEM (n=6) and compared among groups using a one-way ANOVA, followed by a post-hoc test. **p<0.01, ****p<0.0001. e) Glucagon distribution under control conditions (dark grey bars), and in response to Stmn2-KD (black bars) or OE (light grey bars) with values expressed as average glucagon fluorescence intensity ±SEM (n=6), compared among groups and cellular regions using a two-way ANOVA, followed by a post-hoc test. **p<0.01, ****p<0.0001.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Immunofluorescence, Fluorescence, Control

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Transfected cells were treated with 1 nM insulin for 24 hours, then immunostained using primary antibodies against Stmn2 and glucagon. a) Cells transfected with GFP vector and treated with 1 nM insulin showed Stmn2 (green) and glucagon (red) redistributing intracellularly. b) The Stmn2-KD model diminished Stmn2 fluorescence and resulted in glucagon in the periphery, even after 1 nM insulin treatment. c) The Stmn2-OE model increased Stmn2 fluorescence intensity that colocalized strongly with glucagon in the intracellular region after 1 nM insulin treatment. d) Quantification of glucagon distribution in response to Stmn2-KD and OE with values expressed as average glucagon fluorescence intensity ±SEM (n=6), compared among groups and cellular regions using a two-way ANOVA, followed by a post-hoc test. ****p<0.0001.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Transfected cells were treated with 400 nM somatostatin for 24 hours. Cells were then immunostained using primary antibodies against Stmn2 (green) and glucagon (red). a) Cells transfected with GFP vector and treated with 400 nM somatostatin resulted in Stmn2 and glucagon distributing mostly in the intracellular region (yellow arrowheads). b) The Stmn2-KD model diminished Stmn2 fluorescence intensity, and glucagon was present in the periphery after somatostatin treatment. c) The Stmn2-OE model increased Stmn2 fluorescence intensity while colocalizing strongly with glucagon in the intracellular region after somatostatin treatment. d) Glucagon distribution in response to Stmn2-KD (red bars) and OE (green bars) with values expressed as average glucagon fluorescence intensity ±SEM (n=6) and compared among groups and cellular regions using a two-way ANOVA, followed by a post-hoc test. ****p<0.0001.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Plasmid Preparation, Fluorescence

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Cells (n=3) were co-transfected with LAMP1-RFP and GFP vector or Stmn2-GFP, or with scrambled siRNA or siRNA against stathmin-2 (Stmn2-KD) and live images were captured 24 hours post-transfection. a) LAMP1-RFP is present in the cell periphery and the intracellular region (yellow arrowheads) after co-transfection with GFP alone as a negative control (upper panel). After co-transfection with Stmn2-GFP, (lower panel), LAMP1-RFP appears exclusively in the intracellular region (yellow arrowheads). b) LAMP1-RFP is present in the cell periphery and the intracellular region (yellow arrowheads) after co-transfection with scrambled siRNA sequences as a negative control for Stmn2-KD (upper panel). Transfection with Stmn2 siRNAs (KD) resulted in LAMP1-RFP exclusively in the cell periphery (yellow arrowheads) (lower panel). c) Transcription factor EB (TFEB) is not translocated to the nucleus upon overexpression of Stmn2-GFP. Immunofluorescence images of fixed cells show the subcellular distribution of TFEB, GFP alone (upper panel), Stmn2-GFP (lower panel) and cell nuclei (DAPI). Box plots show the ratio of nuclear to cytoplasmic TFEB immunofluorescence. Values were expressed as the average nuclear/cytoplasmic TFEB intensity ±SEM (n=4). Data points in colour represent the average ratio of 8-16 cells per coverslip; black points represent ratios from individual cells. d) Stmn2 regulates the lysosomal transport protein Arl8. Immunofluorescence images of fixed cells show the presence of Arl8 (orange) in cells transfected with scrambled siRNA or siRNA against Stmn2. Quantification of fluorescence (left) shows that knockdown of Stmn2 (Stmn2-KD) significantly increased the fluorescence intensity of Arl8. Values are means ±SEM (n=6). Data points in colour represent the average fluorescence intensities in 8-16 cells per coverslip; black points represent values from individual cells.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Plasmid Preparation, Cotransfection, Negative Control, Over Expression, Immunofluorescence, Fluorescence, Knockdown

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Live images of αTC1-6 cells transfected with LAMP1-RFP (n=3) were captured in the same cell before, during, and five minutes after treatment with 1 nM insulin. a) LAMP1-RFP redistributed intracellularly (yellow arrowheads) in response to 1 nM insulin treatment in control cells transfected with scrambled siRNA. b) LAMP1-RFP distribution was not changed after 1 nM insulin treatment after siRNA-mediated knockdown of Stmn2 (Stmn2-KD) (yellow arrowheads). Graphs show the distribution of LAMP1-RFP fluorescence intensities in the intracellular and peripheral regions before (pre-INS) and after (post-INS) treatment with 1 nM insulin. Values are means ±SEM (n=3). * p<0.05

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Control, Knockdown, Fluorescence

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: αTC1-6 cells (n=3) were transfected overnight. Live images were captured in the same cell before, during, and five minutes after treatment with 400 nM somatostatin. a) LAMP1-RFP redistributed intracellularly (yellow arrowheads) in response to 400 nM somatostatin treatment in control cells transfected with scrambled siRNA. b) LAMP1-RFP distribution was not changed after 400 nM somatostatin treatment after siRNA-mediated knockdown of Stmn2 (Stmn2-KD) (yellow arrowheads). Graphs show means ±SEM (n=3). * p<0.05

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Control, Knockdown

Journal: bioRxiv

Article Title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Schematic structure of Gpr151 gene in wild type control (black) or targeted knockout (KO) mice (blue). Gpr151 gene was disrupted by the insertion of LacZ -cassette in the CDS region. (b) Schematic diagram of timeline illustrating the pre-conditioning injury and sample preparation. Sciatic nerves of wild type control or KO mice were injured by crushing the nerves with forceps and dissected at 3 days after injury. The sciatic nerves were prepared as cryosections and subjected to immunohistochemistry with anti-SCG10 and βIII tubulin antibodies. L4,5 DRGs were subjected to RT-qPCR analysis (ÄÄCt) or adult DRG neuron cultures. (c) RT- qPCR analysis of mouse L4,5 DRGs. Average of normalized levels of wild type control and KO mRNA levels at 3 days after introducing sciatic nerve crush injury. The averages of relative Gpr151 mRNA levels from uninjured KO, injured control and KO samples was compared to the average level from control uninjured samples (set to 1). (n=6 for each condition; *** p <0.001, ANOVA followed by Tukey test). (d) Representative longitudinal sections of immunohistochemistry of sciatic nerves from in vivo axon regeneration assay. The longitudinal cryosections were immunostained with anti-SCG10 (white; top and yellow converted; bottom), a marker protein specifically labelling regenerating axon. βIII tubulin antibody was used for counterstaining to visualize the nerve sections (blue; bottom). The red dotted arrow line indicates the crush site. Scale bar, 500 μm. (e) Average of normalized SCG10 intensity from immunostained sections in (d). Immunostained SCG10 fluorescence intensity was acquired by measurement windows with 100 pixel-width (equivalent to 87.7 μm) from crush sites to distal parts of nerves sections at an every 100-pixel distance from ImageJ . The acquired intensity from every measurement window was normalized to the intensity of the crush site (n=7 for control mice and n=5 for KO mice; * p <0.05 by t -test; mean ± SEM). (f) Regeneration index calculated from (e). Regeneration index was defined as a distance of an indicated SCG10 intensity. Average distance of normalized SCG10 intensity 0.5 or 0.2 was calculated from (e) and presented as regeneration index (mm). ( # p = 0.59, ## p = 0.05 by t -test). (g) Mouse L4,5 DRG tissues were dissected at 3 days with (+SNI) or without (-SNI) sciatic nerve crush injury (SNI). DRG neurons were plated and cultured to monitor pre-conditioning effect-induced neurite outgrowth. Scale bar, 100 μm. (h) Average of the longest axon length from (g) (three independent biological replicates; total 6 mice; total 304, 313, 291, 283 cells for each condition, -SNI Control, -SNI KO, +SNI Control, +SNI KO; *** p <0.001 by ANOVA followed by Tukey tests). (i) Cumulative frequency of the longest axon length from (g). (j) Percentage of neurons in three categories of the longest axon length.

Article Snippet: Anti-HA antibody (Abcam, ab9110; RRID: AB_307019), anti-GPR151 antibody (Aviva Systems Biology, OAAF06441), anti-α tubulin antibody (Santa Cruz Biotechnology, sc-53030; RRID: AB_2272440), anti-c-Jun antibody (Cell Signaling Technology, 9165; RRID: AB_2130165), anti-SCG10 antibody (Novus Biologicals, NBP1-49461; RRID: AB_10011569), anti-βIII tubulin antibody (TUJ1, Abcam, ab41489, RRID: AB_727049), anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233; RRID: AB_627679), anti-FLAG antibody (Cell Signaling Technology, 14793; RRID: AB_2572291).

Techniques: Control, Knock-Out, Sample Prep, Immunohistochemistry, Quantitative RT-PCR, In Vivo, Marker, Fluorescence, Cell Culture

Journal: bioRxiv

Article Title: Promoting axon regeneration by enhancing the non-coding function of the injury-responsive coding gene Gpr151

doi: 10.1101/2021.02.19.431965

Figure Lengend Snippet: (a) Schematic diagram of AAV-control and AAV-5’UTRm adeno-associated virus vector and experimental timeline of in vivo axon regeneration assay in sciatic nerves (wk, weeks). U6, U6 promoter; Scrambled shRNA sequence 48-mer; T, transcription termination sequence; CMV, human cytomegalovirus immediate early enhancer/promoter; GFP, enhanced green fluorescence protein CDS. (b) Representative image of adult DRG neurons. L4,5 DRGs were dissected from AAV-injected mice and cultured for 12 hours and immunostained with βIII tubulin antibody. GFP fluorescence signal indicated AAV-mediated in vivo gene delivery and expression of target genes. Scale bar, 100 μm. (c) In vivo axon regeneration assay from crushed sciatic nerves. Representative longitudinal sections of sciatic nerves from AAV-control or AAV-5’UTRm- injected mice. Red dotted arrows indicate the injury site. Scale bar, 500μm. (d) Normalized SCG10 intensity measured from SCG10 immunostained sections in (c). Measurement window (100-pixel width, equivalent to 87.7 μm) was placed at the crush site and intensities were acquired at every 100-pixel to the distal part of the sections. (e) Regeneration index calculated from (d). (n=8 for AAV-control, 10 for AAV-5’UTRm; ** p <0.01, **** p <0.0001 by t -test; mean±SEM). (f) Experimental timeline of in vivo axon regeneration assay in optic nerves (wk, weeks). (g) Representative longitudinal sections of optic nerves from AAV-control or AAV-5’UTRm-injected mice. Regenerating axons were labeled by Alexa594-conjugated CTB injection. Red dotted arrows indicate the injury site. Red bars indicated the locations analyzed the estimation of regenerating axon numbers in (h). Scale bar, 200μm. (h) Estimated numbers of regenerating axons (n=5 for AAV-control, 6 for AAV-5’UTRm; * p <0.05, ** p <0.01, *** p <0.001 by one-way AVONA with Bonferroni test; mean±SEM). (i) Relative expression level of Gpr151 and Atf3 after injury in mouse retina tissues (injured / uninjured). RT-qPCR analysis of Gpr151 and Atf3 from mouse retina tissues dissected at 3 days with optic nerve crush injury (injured, purple) or without injury (uninjured, black) (n=3 for each condition; *** p <0.001, ns, not significant by t-test; mean±SEM).

Article Snippet: Anti-HA antibody (Abcam, ab9110; RRID: AB_307019), anti-GPR151 antibody (Aviva Systems Biology, OAAF06441), anti-α tubulin antibody (Santa Cruz Biotechnology, sc-53030; RRID: AB_2272440), anti-c-Jun antibody (Cell Signaling Technology, 9165; RRID: AB_2130165), anti-SCG10 antibody (Novus Biologicals, NBP1-49461; RRID: AB_10011569), anti-βIII tubulin antibody (TUJ1, Abcam, ab41489, RRID: AB_727049), anti-GAPDH antibody (Santa Cruz Biotechnology, sc-32233; RRID: AB_627679), anti-FLAG antibody (Cell Signaling Technology, 14793; RRID: AB_2572291).

Techniques: Control, Virus, Plasmid Preparation, In Vivo, shRNA, Sequencing, Fluorescence, Injection, Cell Culture, Expressing, Labeling, Quantitative RT-PCR

Journal: eLife

Article Title: Translatome analysis reveals cellular network in DLK-dependent hippocampal glutamatergic neuron degeneration

doi: 10.7554/eLife.101173

Figure Lengend Snippet:

Article Snippet: STMN2 antibodies were a mouse monoclonal anti-STMN2 (R&D Systems, MAB6930) and a rabbit polyclonal anti-STMN2 (Proteintech, 10586–1-AP).

Techniques: Knock-Out, Over Expression, Sequencing, RNAscope, TUNEL Assay, SYBR Green Assay, Bicinchoninic Acid Protein Assay, Multiplex Assay, Software

Journal: bioRxiv

Article Title: Stathmin-2 Mediates Paracrine Hormone Regulation of Glucagon Through Lysosomal Trafficking in αTC1-6 cells

doi: 10.64898/2026.04.02.715646

Figure Lengend Snippet: Cells (n=3) were co-transfected with LAMP1-RFP and GFP vector or Stmn2-GFP, or with scrambled siRNA or siRNA against stathmin-2 (Stmn2-KD) and live images were captured 24 hours post-transfection. a) LAMP1-RFP is present in the cell periphery and the intracellular region (yellow arrowheads) after co-transfection with GFP alone as a negative control (upper panel). After co-transfection with Stmn2-GFP, (lower panel), LAMP1-RFP appears exclusively in the intracellular region (yellow arrowheads). b) LAMP1-RFP is present in the cell periphery and the intracellular region (yellow arrowheads) after co-transfection with scrambled siRNA sequences as a negative control for Stmn2-KD (upper panel). Transfection with Stmn2 siRNAs (KD) resulted in LAMP1-RFP exclusively in the cell periphery (yellow arrowheads) (lower panel). c) Transcription factor EB (TFEB) is not translocated to the nucleus upon overexpression of Stmn2-GFP. Immunofluorescence images of fixed cells show the subcellular distribution of TFEB, GFP alone (upper panel), Stmn2-GFP (lower panel) and cell nuclei (DAPI). Box plots show the ratio of nuclear to cytoplasmic TFEB immunofluorescence. Values were expressed as the average nuclear/cytoplasmic TFEB intensity ±SEM (n=4). Data points in colour represent the average ratio of 8-16 cells per coverslip; black points represent ratios from individual cells. d) Stmn2 regulates the lysosomal transport protein Arl8. Immunofluorescence images of fixed cells show the presence of Arl8 (orange) in cells transfected with scrambled siRNA or siRNA against Stmn2. Quantification of fluorescence (left) shows that knockdown of Stmn2 (Stmn2-KD) significantly increased the fluorescence intensity of Arl8. Values are means ±SEM (n=6). Data points in colour represent the average fluorescence intensities in 8-16 cells per coverslip; black points represent values from individual cells.

Article Snippet: Stmn2-GFP was obtained as the Stmn2 mouse tagged ORF clone (OriGene, cat#NM_025285) and pcDNA3-eGFP was used as the vector control.

Techniques: Transfection, Plasmid Preparation, Cotransfection, Negative Control, Over Expression, Immunofluorescence, Fluorescence, Knockdown

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: Human spinal SMA iPSC-derived MNs showed STMN2 dysregulation. A Experimental strategy used to assess the cellular effect of STMN2 modulation by JNK-inhibitor SP600125 (15 µM) or by lentivirus delivering STMN2 cDNA (MOI 5) in SMA MNs differentiated from iPSCs via embryoid bodies (EBs). Created with BioRender.com. B Representative immunofluorescence images of Ctrl and SMA d18 MNs stained for STMN2 (red), β-tubulin (green), and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. C Quantification of STMN2 level intensity ( n = 3 cell lines/group) in B reporting separately soma, axon, and the ratio axon/soma data. STMN2 levels were normalized to SMA average values. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Derivative Assay, Immunofluorescence, Staining, Microscopy

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: JNK-inhibitor SP600125 protected SMA MNs from degeneration and ameliorated axon length and outgrowth defects in vitro. A Representative immunofluorescence images of untreated (SMA) and SP-treated (SMA + SP) d18 MNs, labeled for STMN2 (red), β-tubulin (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. B Intensity quantification of STMN2 in A ( n = 3 cell lines/group) analyzing separately soma, axon, and the ratio axon/soma. STMN2 levels were normalized to untreated SMA average values. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. C Representative immunofluorescence images of Ctrl, SMA, and SMA + SP d18 MNs stained for TUNEL (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. D Representative immunofluorescence images of Ctrl, SMA and SMA + SP d18 MNs stained for β-tubulin (black) and acquired with a confocal microscope at 63X magnification. Scale bar = 50 μm. E Quantification of TUNEL-positive cells ( n = 3 cell lines/group) in C , expressed as percentage of apoptotic cells on the total amount of nuclei. Values were normalized to untreated SMA average. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. F , G Measurement of axon length ( F ) of D , expressed as normalized values to untreated SMA average, and dendritic complexity performed with Sholl analysis ( G ) of D , expressed as number of intersections at different distances from soma ( n = 3 cell lines/Ctrl; n = 4 cell lines/SMA and /SMA + SP). Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. ** P < 0.01, *** P < 0.001, **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: In Vitro, Immunofluorescence, Labeling, Microscopy, Staining, TUNEL Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: STMN2 modulation by lentivirus specifically restored axon length and outgrowth defects in SMA MNs. A Representative immunofluorescence images of null vector (indicated as SMA) and STMN2 -LV (indicated as SMA + STMN2 -LV) treated d21 MNs, labeled with STMN2 (red), β-tubulin (green) and DAPI (blue) and acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. B Intensity quantification of STMN2 ( n = 3 cell lines/group) in A analyzing separately soma, axon, and the ratio axon/soma. STMN2 levels were normalized to SMA MNs treated with the null vector. Values show means ± SEM from 3 independent experiments. Statistical significance was determined by Student’s t -test. C Representative immunofluorescence images of Ctrl, SMA and SMA + STMN2 -LV d21 MNs ( n = 3 cell lines/group) stained for TUNEL (green) and DAPI (blue) acquired with a confocal microscope at 40X magnification. Scale bar = 50 μm. D Representative immunofluorescence images of Ctrl, SMA and SMA + STMN2 -LV d21 MNs stained for β-tubulin (black) and acquired with a confocal microscope at 63X magnification. Scale bar = 50 μm. E Quantification of TUNEL-positive cells ( n = 3 cell lines/group) in C expressed as percentage of apoptotic cells on the total amount of nuclei. Values were normalized to the average of SMA treated with the empty vector. Values show means ± SEM from 3 independent experiments. F , G Measurement of axon length ( F ) of D , expressed as normalized on average of SMA treated with the null vector values, and dendritic complexity performed with Sholl analysis ( G ) of D , expressed as number of intersections at different distances from soma ( n = 3 cell lines/Ctrl; n = 4 cell lines/SMA and /SMA + STMN2 -LV). Values show means ± SEM from 3 independent experiments. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. *** P < 0.001, **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Immunofluorescence, Plasmid Preparation, Labeling, Microscopy, Staining, TUNEL Assay

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: AAV9:: Stmn 2 treatment improved survival and neuromuscular functions in SMA mice. A Representative Western blot of STMN2 in the brain, spinal cord, and sciatic nerves of SMA mice treated with AAV9:: Stmn2 or AAV9::null at P10. B Densitometric quantification of STMN2/actin levels in A normalized to SMA + AAV9::null ( n = 4 animals/group). The values are represented as mean value ± SEM. Statistical significance was determined by Student’s t -test. C Kaplan-Meier survival curves of WT ( n = 20), SMA + AAV9::null ( n = 23) and SMA + AAV9:: Stmn2 ( n = 18). Statistical significance was determined by the Log-rank test. D Postnatal mean body weight ± SEM expressed in grams at different times of WT ( n = 20), SMA + AAV9::null ( n = 23) and SMA + AAV9:: Stmn2 ( n = 18). Statistical significance was determined by multiple unpaired t -test. E Righting response score expressed as percentage of mice that can perform the test (WT n = 20; SMA + AAV9::null n = 23, SMA + AAV9:: Stmn2 n = 18). The values are represented as mean value ± SEM. Statistical significance was determined by two-way ANOVA followed by Dunnett’s post-hoc test. F Macroscopic phenotype of WT, SMA + AAV9::null and SMA + AAV9:: Stmn2 at P8. * P < 0.05, *** P < 0.001, **** P < 0.0001

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Western Blot

Journal: Cellular and Molecular Life Sciences: CMLS

Article Title: Targeting STMN2 for neuroprotection and neuromuscular recovery in Spinal Muscular Atrophy: evidence from in vitro and in vivo SMA models

doi: 10.1007/s00018-024-05550-3

Figure Lengend Snippet: AAV9:: Stmn2 ameliorated muscle-related features in SMA mice. A Representative immunofluorescence images of gastrocnemius muscle of WT, AAV9::null and AAV9:: Stmn2 -treated SMA mice at P10 stained for α-bungarotoxin (α-BTX, red) and neurofilament-M (NF-M, green) and acquired with a confocal microscope at 63X magnification. Scale bar = 100 μm. B Representative gastrocnemius muscle fibers of WT, AAV9::null and AAV9:: Stmn2 -treated SMA mice at P10 stained with hematoxylin/eosin and acquired with an optical microscope with a magnification of 20X. Scale bar = 200 μm. C Respective quantification of the percentage of innervated NMJs on the total number ( n = 6 animals/group). Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. D , E Relative fiber CSA quantification expressed in µm 2 ( D ) and fibrotic/necrotic tissue quantification expressed as non-muscular area percentage ( E ) were measured ( n = 6 animals/group). The values are represented as mean value ± SEM. Statistical significance was determined by one-way ANOVA followed by Tukey’s post-hoc test. **** P < 0.0001. All inserts are 4X magnification

Article Snippet: In particular, cells were treated for 72 hours (h) after seeding at day 15 with 15 μM SP600125 (Cell Signaling, #8177), an anthrapyrazolone ATP-competitive inhibitor of JNK, or transduced with a lentivirus- STMN2 (MOI 5; Origene RC202914L4V, accession number NM_007029) or with a null vector as the negative control.

Techniques: Immunofluorescence, Staining, Microscopy

Journal: Scientific Reports

Article Title: Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1 A4V mutation

doi: 10.1038/s41598-023-31720-7

Figure Lengend Snippet: Antibodies/Reagent used for immunofluorescence staining.

Article Snippet: Stathmin-2 (STMN2) , Early regeneration marker , Novus , 1:2000.

Techniques: Immunofluorescence, Staining, Marker

Journal: Scientific Reports

Article Title: Enhanced axonal regeneration of ALS patient iPSC-derived motor neurons harboring SOD1 A4V mutation

doi: 10.1038/s41598-023-31720-7

Figure Lengend Snippet: SOD1 +/+ and SOD1 +/A4V axons regenerate similarly 24 h following axotomy. ( A ) Mean axon length 24 h post-axotomy (SOD1 +/+ n = 13; SOD1 +/A4V n = 12). Each data point represents 50–100 traced axons from 1 individual axonal compartment. ( B ) Frequency distribution of axon length 24 h post-axotomy. ( C ) SOD1 +/+ (left) and SOD1 +/A4V (right) regenerating axons 24 h post axotomy stained for GAP-43. Scale bar = 50 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ m. ( D-E ) Growth cones 24 h post axotomy stained for smi-31 (green), F-actin (white), stathmin-2 (red). Scale bar = 2 \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\upmu$$\end{document} μ m. ( D ) SOD1 +/+ (top panels) ( E ) SOD1 +/A4V (bottom panels). ( F ) Average growth cone area 24 h post-axotomy. (G) Average STMN2 fluorescence intensity in growth cone area 24 h post-axotomy. Bars represent mean ± SEM. ns indicates p > 0.05.

Article Snippet: Stathmin-2 (STMN2) , Early regeneration marker , Novus , 1:2000.

Techniques: Staining, Fluorescence

Journal: Nature communications

Article Title: The landscape of multiscale transcriptomic networks and key regulators in Parkinson's disease.

doi: 10.1038/s41467-019-13144-y

Figure Lengend Snippet: Fig. 3 pHluorin assay in cultured DA neurons with Stmn2 knockdown. a SV endocytosis and exocytic fraction in midbrain DA neurons with or without Stmn2 knockdown. Two-sided Student’s t test for two-group comparison. t = −2.6572, df = 19, p-value = 0.01556 for endocytosis time constant, which is the τ of the fitted one-phase exponential decay indicated by the blue dash line; t = 0.87198, df = 19, p = 0.3941 for exocytic fraction, which is the normalized peak height. b SV exocytosis in midbrain DA neurons with or without Stmn2 knockdown. The exocytosis time constant is the τ of the fitted one- phase exponential decay indicated by the blue dash line. This is a subset of neurons recorded in (a). Two-sided Student’s t test for two group comparison. t = −1.6213, df = 9, p-value = 0.1394. c SV endocytosis during stimulation in midbrain DA neurons with or without Stmn2 knockdown as indicated by the arrows in c. This is a subset of neurons recorded in (a) and the same neurons in (b). Two-sided Student’s t test for two group comparison. t = 3.6414, df = 9, p-value = 0.005389. N indicated in each experiment. d Immunostaining of VMAT2-pHluorin, RFP tag of Stmn2-shRNA plasmid, and TH of recorded DA neurons treated with scrambled or Stmn2-shRNA. Arrows pointed to representative boutons. e Quantification of bouton sizes in scrambled and Stmn2- shRNA-treated TH + neurons. Two-sided Student’s t test for two group comparison. t = 3.2224, df = 16, p-value = 0.008433. All data are present as mean ± SEM. Source data are provided as a Source Data file

Article Snippet: The Stmn2 shRNA plasmids were purchased from Origene, MD, USA (TF511127), in which the Stmn2 shRNA expression was driven by a universal U6 promoter with an RFP tag driven by CMV promoter on the same construct.

Techniques: Cell Culture, Knockdown, Comparison, Immunostaining, shRNA, Plasmid Preparation

Journal: Nature communications

Article Title: The landscape of multiscale transcriptomic networks and key regulators in Parkinson's disease.

doi: 10.1038/s41467-019-13144-y

Figure Lengend Snippet: Fig. 4 Validation of the PD MEGENA network by Stmn2 perturbation. a Volcano plot shows genes that are significantly differentially expressed in the midbrain between the mice received scrambled AAV and Stmn2-shRNA AAV. b The genes in M4 are significantly enriched for the downregulated DEGs identified in the midbrain of the Stmn2 knockdown mice. Nodes with burgundy labels are key hubs of M4. The left-side half circle of each node indicates the DEGs in human between the PD and control groups while the right-side represents the DEGs in mice by Stmn2 knockdown. c The two-layer network neighborhood of STMN2 is significantly enriched for the DEG downregulated by Stmn2 knockdown. Nodes with burgundy labels are key hubs in the MEGENA network. The left upper part of the circle represents the DEG in human between the PD and control groups while the right upper part of the circle represents the correlation with STMN2 in PD patients. The bottom part of the circle indicates the DEGs in mice by Stmn2 knockdown

Article Snippet: The Stmn2 shRNA plasmids were purchased from Origene, MD, USA (TF511127), in which the Stmn2 shRNA expression was driven by a universal U6 promoter with an RFP tag driven by CMV promoter on the same construct.

Techniques: Biomarker Discovery, shRNA, Knockdown, Control

Journal: Nature communications

Article Title: The landscape of multiscale transcriptomic networks and key regulators in Parkinson's disease.

doi: 10.1038/s41467-019-13144-y

Figure Lengend Snippet: Fig. 6 Striatal alterations in mice with AAV-shRNA-mediated Stmn2 knockdown. a The content of dopamine (DA) and its major metabolites (3-MT, DOPAC, and HVA) were measured in the contralateral and ipsilateral striatal tissues isolated from the mice that received scrambled AAV and Stmn2- shRNA AAV injections, N = 7 in each group. The biogenic monoamine levels of the ipsilateral striatum were normalized to those of the contralateral side in each individual mouse, and the comparison between mice with scrambled and Stmn2-shRNA AAV injection was analyzed by two-sided Student’s t test. DA: t = 4.4367, df = 12, p-value = 0.0008; DOPAC: t = 2.8365, df = 12, p-value = 0.01499; 3-MT: t = 2.614, df = 12, p-value = 0.02263; HVA: t = 3.6415, df = 12, p-value = 0.003379. b Anti-DAT antibody was used to detect DAT + terminals in the striatum in mice that received scrambled AAV and Stmn2- shRNA AAV injections. RFP serves as an indicator of transfected terminals. The yellow line separated the dorsal and ventral striatum. c The quantitative analysis of DAT + immunofluorescent intensity in the two groups of mice. The DAT fluorescence intensity of ventral striatum without injection was used as the baseline to normalize the DAT intensity of the rest regions. N = 7 in each group. Two-way ANOVA, F = 25.689, df = 25, p = 3.12E-05. All data are present as mean ± SEM. Source data are provided as a Source Data file

Article Snippet: The Stmn2 shRNA plasmids were purchased from Origene, MD, USA (TF511127), in which the Stmn2 shRNA expression was driven by a universal U6 promoter with an RFP tag driven by CMV promoter on the same construct.

Techniques: shRNA, Knockdown, Isolation, Comparison, Injection, Transfection