sprague dawley rat primary cortical neurons  (Dawley Inc)

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induced oxidative stress in neuronal cells. ( A ) SH-SY5Y cells were treated with indicated concentrations of Cd for 24 h, and subsequently, ROS levels were analyzed by flow cytometry. ( B ) SH-SY5Y cells were incubated with 5 μM Cd for various time periods. The formation of ROS was measured by flow cytometry. ( C ) SH-SY5Y cells were exposed to 5 μM Cd and 2.5 mM NAC alone or together for 24 h; subsequently, ROS levels were analyzed by flow cytometry. ( D , E ) SH-SY5Y cells were exposed to 5 μM Cd at the indicated times. Western Blot analysis was performed to determine SOD2 protein expression, and a CuZn/Mn-SOD Assay Kit was used to measure SOD2 activity. ( F ) Rat cerebral cortical neurons were incubated with 5 μM Cd for various time periods, and subsequently, ROS levels were analyzed by fluorescence microscopy. All results are representative of three independent experiments. The provided scale bar in the merged image represents 200 μm. * p < 0.05, ** p < 0.01 versus the control group; ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Flow Cytometry, Incubation, Western Blot, Expressing, Activity Assay, Fluorescence, Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: Cd induces the nuclear expression of FoxO3a in neuronal cells. SH-SY5Y cells were treated with 5 μM Cd for various time periods. ( A ) The cell lysates were subjected to Western Blot analysis to measure FoxO3a protein expression; ( B ) nuclear and cytoplasmic extracts were subjected to Western Blot analysis to measure FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h, and confocal laser scanning microscopy was performed to determine FoxO3a localization. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. The provided scale bar in the merged image represents 20 μm. * p < 0.05, ** p < 0.01 versus the control group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: p38 regulates the Cd-induced nuclear expression of FoxO3a. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods, and phosphorylated and total MAPK proteins were detected by Western Blot analysis. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine FoxO3a expression. ( C , D ) SH-SY5Y cells and rat cerebral cortical neurons were exposed to 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Confocal laser scanning microscopy was performed to determine FoxO3a expression. FoxO3a is shown in green, and nuclei were counterstained with DAPI (blue). ( E ) SH-SY5Y cells were treated with 5 μM Cd for various time periods. Western Blot analysis was performed to determine phosphorylation of FoxO3a-Ser7. ( F – H ) SH-SY5Y cells and rat cerebral cortical neurons were incubated with 5 μM Cd for 6 h after preincubation with 10 μM SB203580 for 1 h. Western Blot analysis and confocal laser scanning microscopy were performed to determine phosphorylation of FoxO3a-Ser7 expression. The provided scale bar in the merged image represents 20 μm. ( G – H ) p-FoxO3a is shown in red, and nuclei were counterstained with DAPI (blue). All results are representative of three independent experiments. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Expressing, Western Blot, Confocal Laser Scanning Microscopy, Phospho-proteomics, Incubation, Control

Journal: International Journal of Molecular Sciences

Article Title: p38 Regulates FoxO3a-Mediated SOD2 Expression to Prevent Cd-Induced Oxidative Stress in Neuronal Cells

doi: 10.3390/ijms262210919

Figure Lengend Snippet: The effects of the p38/FoxO3a pathway on the Cd-induced oxidative stress. ( A ) SH-SY5Y cells were treated with 5 μM Cd for various time periods after being pretreated with 10 μM SB203580 for 1 h. Western Blot analysis was performed to determine SOD2 protein expression. ( B ) SH-SY5Y cells were treated with 5 μM Cd for 6 h after preincubating with 10 μM SB203580 for 1 h. A CuZn/Mn-SOD Assay Kit was used to measure SOD2 activity. SH-SY5Y cells and cerebral cortical neurons were preincubated with 10 μM SB203580 for 1 h, followed by treatment with 5 μM Cd for various time periods. ROS levels were analyzed by flow cytometry or fluorescence microscopy ( C , D ); cells were exposed to 5 μM Cd for 24 h after preincubation with SB203580, and subsequently, cell viability of SH-SY5Y cells and rat cerebral cortical neurons was analyzed by CCK8 ( E , F ). All results are representative of three independent experiments. The provided scale bar in the merged image represents 200 μm. * p < 0.05, ** p < 0.01 versus the control group; # p < 0.05, ## p < 0.01 versus the Cd group.

Article Snippet: Primary rat cerebral cortical neurons were obtained from the cerebral cortex of one-day-old Sprague–Dawley rats, with a typical yield of approximately 3–4 × 10 6 viable neurons per pup.

Techniques: Western Blot, Expressing, Activity Assay, Flow Cytometry, Fluorescence, Microscopy, Control

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Z-stacked images show sections of primary rat cortical neurons that were stained with the AZ marker ELKS. Cells were also immunostained with Synapsin-1 to confirm the presynapse and MAP2 to localise staining along sections of dendrite. In the colour merged images shades of white indicate colocalisation between ELKS and Synapsin-1, highlighted by arrows. (B) Orthogonal views are enlarged regions taken from the dashed yellow boxes indicated in (A) to show depth of the acquired stacks. The region of colocalisation is also given by arrows. Scale bar: (A) 5 µm; (B) 0.5 µm in XY, 1 µm in Z. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Staining, Marker

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Z-stack images of rat cortical neurons immunostained for MYCBP2. MYCBP2 was coimmunostained with the presynaptic marker Bassoon to help identify presynaptic localisation. Cells were also coimmunostained with MAP2 to highlight areas of dendrite. In the merged image MYCBP2 can frequently be observed to associate closely with Bassoon as indicated by the solid arrows. Additionally, several areas are observed to colocalise as white in the merged image indicated by the hollow arrows. (B) Orthogonal views of the inset dashed yellow boxes in (A) to show the depth of the acquired images. Multiple MYCBP2 puncta surround the Bassoon puncta with a small region of colocalisation seen as white in the merged image. Scale bar: (A) 5 µm; (B) 0.5 µm in XY, 1 µm in Z. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Marker

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Example images of analysed regions of boutons along the axon. Rat cortical neurons were transfected with α-Syn-A30P. (B) An enlarged section of the inset dashed yellow box in (A) showing a single synaptic bouton. (C) A simplified demonstration of how the boutons and axons were analysed in ImageJ. Once the appropriate synaptic bouton is identified (1) a ROI is drawn around the bouton (2). The centre of the bouton is approximated and two lengths of 1.5 µm are drawn from the centre along the path of the axon (3). A whole ROI is then drawn (4). The bouton and the whole ROIs are overlaid on the channel of interest and the puncta are identified (5, 6). The immunostaining is then approximated in the axon regions post-acquisition by finding the difference between the whole regions and the bouton (7). Scale bar: (A) 10 µm; (B, C) 1 µm. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Transfection, Immunostaining

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with eGFP, α-Synuclein-WT or α-Synuclein-A30P mutant and subsequently immunostained with GFP. The boutons were identified as indicated with the yellow lines. (B) No significant difference was found between any of the groups, F(2,33)=0.40, p=0.6762. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. N=2-4 boutons from 36 cells. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Transfection, Mutagenesis

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were overexpressed with either eGFP tagged α-Synuclein-WT or -A30P mutant plasmids and analysed for GFP puncta count (B-D) and size (E-G). (B, C) In boutons, GFP tagged A30P puncta significantly increased in number compared to WT, U(31.5), p=0.0018. GFP puncta also increased significantly in adjacent axons in A30P compared to WT, t(25)=4.47, p=0.0001. (D) Counts were normalised to bouton area, and demonstrated a significant interaction between presynaptic regions and plasmid used, F(1.0,50.0)=7.11, p=0.0103. (E, F) The GFP puncta area also showed a significant reduction in A30P compared to WT within boutons, U(12.0), p=0.0001 but there was no significant change in the axon region. (G) Log transformed values for puncta area compared across bouton and axon regions and plasmid used demonstrated a significant interaction, F(1.0,45.0)=14.44, p=0.0004. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. Error bars: (D, G) SD. N=2-4 boutons from 25-27 cells. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Mutagenesis, Plasmid Preparation, Transformation Assay

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Synuclein-WT or α-Synuclein-A30P. The cells were immunostained for both GFP and MYCBP2. Hollow arrows indicate MYCBP2 lying within the bouton regions. The solid arrows show MYCBP2 puncta distributed within the axon region. The bottom pseudo-coloured panels of MYCBP2 demonstrate the signal intensity of the MYCBP2 within the above fields of view. (B-D) The characteristics of MYCBP2 in boutons were analysed as follows: (B) MYCBP2 puncta area significantly altered by both α-Syn-WT and -A30P mutant overexpressions compared to control F(2,32)=5.85, p=0.0068; (C) MYCBP2 count within boutons did not significantly alter; (D) Relative intensity of MYCBP2 puncta was significantly reduced in the α-Syn-WT and -A30P conditions F(2,34)=6.00, p=0.0057. (E-G) The characteristics of MYCBP2 in axon segments were similarly analysed for (E) area size, (F) count and (G) intensity, however, no significant differences were observed in these conditions. (H-J) Two-way ANOVA analysis was conducted to examine interaction effects within the following conditions: (H) There was a significant interaction between the region measured and the puncta area F(2,61)=5.09, p=0.0091; (I) Significant interaction was not identified between the region measured and the puncta count; (J) The puncta intensity did not show a significant interaction with the region measured. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 μm. Error bars: (H-J) SD. N=2-4 boutons from 32-37 cells. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Transfection, Mutagenesis, Control

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Syn-WT or -A30P. The cells were immunostained for both GFP and ELKS. Hollow arrows indicate ELKS lying within the bouton regions. The solid arrows show ELKS puncta distributed within the axon region. The bottom pseudo-coloured panels of ELKS demonstrate the signal intensity of the ELKS within the above fields of view. (B-D) The characteristics of ELKS in boutons between overexpression conditions were analysed as follows: (B) ELKS puncta area showed no significant alterations; (C) ELKS puncta count within boutons also showed no significant change; (D) The relative intensity of ELKS in the bouton was significantly altered in the α-Syn-WT and -A30P conditions F(2,32)=5.28, p=0.0104. (E-G) The characteristics of ELKS in axon segments were similarly analysed for (E) area size, (F) count and (G) intensity, however, no significant differences were observed in these conditions. (H-J) Two-way ANOVA analysis was conducted to examine interaction effects between the area analysed and the following conditions: (H) Puncta area; (I) Puncta count; (J) Puncta intensity. No significant interaction was observed in any of these conditions. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 μm. Error bars: (H-J) SD. N=2-4 boutons from 35-37 cells. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Transfection, Over Expression

Journal: bioRxiv

Article Title: Overexpression of α-Synuclein Alters The Nanoscopic Organisation of Presynaptic Proteins

doi: 10.1101/2025.10.10.681706

Figure Lengend Snippet: (A) Rat cortical neurons were transfected with either eGFP, α-Syn-WT or -A30P overexpression plasmids. The regions that were measured are identified in the GFP panel with yellow arrows. The hollow yellow arrows indicate the bouton regions analysed, and the solid yellow arrows demonstrate the axon regions analysed. The last pseudo-coloured panels show MYCBP2 immunostaining in green and ELKS immunostaining in magenta. The overlap of these colours shows as white indicating sites of colocalisation. The solid arrows indicate colocalisation within the bouton regions, whereas the hollow arrows identify colocalised puncta in the axon regions. (B-C) There was no significant difference to the colocalisation count of MYCBP2 and ELKS in neither the boutons (B) or the axon regions (C). (D) There was no significant interaction between the region analysed and the type of overexpression on the colocalisation count of MYCBP2 and ELKS. Box plots: dashed lines indicate mean; colours indicate biological repeats; diamonds indicate data outside 1.5x IQR. Scale bar: 1 µm. Error bars: (D) SD. N=2-4 boutons from 36-37 cells. Images acquired on iSIM.

Article Snippet: Primary rat cortical neurons were prepared from E18 Sprague-Dawley embryos as previously described ( ).

Techniques: Transfection, Over Expression, Immunostaining

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) PC12 cells co-transfected with GFP and empty vector (EV) or BASP1, either wild-type (WT) or phosphomutants at the phosphosites identified by MS (T31, S40, and S170) were analyzed for the number of branches and longest neurite length. N>300 cells per condition. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. *q<0.05 comparison between phosphomimetic and phosphoablative mutants, ▴ q<0.05 comparison between phosphomutants and WT. (B) Representative western blot from primary embryonic rat cortical neurons transduced with either GFP alone or BASP1-GFP constructs: WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Blots were probed with anti-GFP to detect expression of the BASP1-GFP constructs and with anti-actin as a loading control. (C) Quantification of ATP levels from conditions in (B). N>30. One-way Anova with post hoc Tukey’s test. (D) Representative confocal images of MAP2-immunostained primary embryonic rat cortical neurons cultured under the conditions described in (B). Scale bar = 100 µm. (E) Quantification of dendrite complexity in the cultures shown in (D), including measurements of the maximal secondary neurite length, secondary and tertiary dendritic arborization. N≥30. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (F) Representative confocal images of F-actin stained with BODIPY™ 558/568 Phalloidin in primary embryonic rat cortical neurons cultured under the conditions described in (B). Neurons were treated with 2 µM latrunculin B (LatB) for 10 min, followed by washout and a 10 min recovery period to allow actin re-polymerization. Scale bar = 20 µm. (J) Quantification of the levels of F-actin stained with BODIPY™ 558/568 Phalloidin in primary embryonic rat cortical neurons cultured under the conditions described in (B). Mean fluorescent intensities were measured before latrunculin B treatment (Pre-LatB), 10 min after LatB exposure (LatB), and at 10 min and 20 min following LatB washout (10′ and 20′ Post-LatB). N>30. One-way Anova with Tukey’s or Games-Howell’s post-hoc tests (depending on the condition of homoscedasticity and heteroscedasticity), * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Transfection, Plasmid Preparation, Comparison, Western Blot, Transduction, Construct, Expressing, Control, Cell Culture, Staining

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Schematic representation of the immunoprecipitation (IP) procedure used to isolate BASP1-GFP WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutant interactors from transduced primary embryonic rat cortical neurons. GFP alone was used as a control. Biorender software was used to create the figure under an academic license. (B) Venn diagram showing all mass spectrometry hits from (A) that met the selection criteria detailed in the main text. (B) Venn diagram showing all mass spectrometry hits from (A) that met the selection criteria detailed in the main text. (C) Reactome pathway analysis for molecular processes for the common BASP1 interactors from (B). (D) Heat map representation of CaN-dependent phosphoproteomic hits associated with BASP1 after normalization to BASP1 WT. Each row corresponds to a protein for which a phosphorylated peptide was identified. One-way ANOVA followed by a multiple comparison test with a two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli. ( E) Reactome pathway analysis for molecular processes from the CaN-dependent phosphoproteomic hits for BASP1.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Immunoprecipitation, Mutagenesis, Control, Software, Mass Spectrometry, Selection, Comparison

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Representative Western blot of GFP immunoprecipitates (IP) from HeLa cells transfected with GFP alone or BASP1-GFP constructs: wild-type (WT), phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Blots were probed with anti-GFP and anti-prohibitin 1 (Phb1). (B) MS quantification of prohibitin 1 (Phb1) co-immunoprecipitated using anti-GFP beads from primary embryonic rat cortical neurons transduced with GFP alone or BASP1-GFP constructs: WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (C) Representative Western blot of BASP1 from HeLa cells co-transfected with GFP alone or BASP1-GFP constructs: BASP1 WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants, along with either a 3’UTR-targeting siRNA against human BASP1 or non-targeting control siRNA. (D) Quantification of BASP1 signal normalized to GAPDH (loading control) from (C), with values further normalized to the non-targeting siRNA control within each experiment. N=3. One-way Anova with post hoc Dunnett’s test, * P <0.05. (E-G) Mitochondrial length (E) and number (G) analyzed in HeLa cells from (C) using MitoTracker Deep Red-assisted confocal microscopy (F). Scale bar = 10 µm. N>20. One-way Anova with post hoc Tukey’s test, * P <0.05, ** P <0.01, *** P <0.001, **** P <0.0001. (H) MS quantification of actin co-immunoprecipitated using anti-GFP beads from primary embryonic rat cortical neurons cultured under the conditions described in (B). One-Way Anova followed by multiple comparisons with two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, *q<0.05. (I) Representative Western blot of GFP co-immunoprecipitates from HeLa cells cultured under the conditions described in (A). Blots were probed with anti-GFP and anti-actin. (J) Representative confocal pictures of mitochodnira and actin from HeLa cells cultured under the conditions described in (C) and co-transfected with LifeAct-mScarlet to visualize actin. Time-lapse imaging was performed at 150-second intervals to monitor actin localization at mitochondria following 4 µM ionomycin stimulation. Scale bar = 10 µm. (K) Quantification of actin fluorescence intensity from (J) over time following 4 µM ionomycin treatment. (L) Representative confocal pictures of HeLa cells cultured in conditions described in (C) under 4 µM Ionomycin treatment. Actin was visualized by transient expression of LifeAct-mScarlet and mitochondria were labeled with MitoTracker Deep Red. Time-lapse imaging was performed at 15-second intervals. Arrows indicate actin recruitment to mitochondria with BASP1-GFP WT, phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants followed by fission events only in BASP1 WT and D,D. Scale bar = 2 µm.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Western Blot, Transfection, Construct, Immunoprecipitation, Transduction, Control, Confocal Microscopy, Cell Culture, Imaging, Fluorescence, Expressing, Labeling

Journal: bioRxiv

Article Title: BASP1 Couples Ca 2+ Signaling and Actin Polymerization to Mitochondrial Fission Essential for Neurite Outgrowth

doi: 10.1101/2025.09.05.674494

Figure Lengend Snippet: (A) Representative confocal images of β-III-tubulin staining in DIV23 primary embryonic rat cortical neurons, transduced with either GFP alone or BASP1-GFP constructs: wild-type (WT), phosphoablative (S40A/S170A; A,A), and phosphomimetic (S40D/S170D; D,D) double mutants. Neurons were subjected to mechanical injury in a scratch assay, and the staining was performed 24 hours post-injury. Scale bar = 50 µm. (B) Quantification of neurite outgrowth for the cultures in (A) using ImageJ/Fiji. N>950. One-Way Anova with Games-Howell’s post-hoc test, **** P <0.0001 (C) Representative immunofluorescence images of primary embryonic rat cortical neurons cultured under the conditions described in (A) showing immunostaining for mitochondrial markers TOMM20 and prohibitin 1 (Phb1). Scale bar = 5 µm. (D) Quantification of mitochondrial length in neurites for the cultures in (C) N>550. One-way Anova with Games-Howell’s post-hoc test, **** P <0.0001. (E) Representative live-cell images of primary embryonic rat cortical neurons cultured under the conditions described in (A) and stained with the fluorescent mitochondrial dye tetramethylrhodamine methyl ester (TMRM) acquired at 3-min intervals. Scale bar = 5 µm. (F) Quantification of TMRM intensities for the cultures in (E), normalized to GFP alone within each experiment. N=3. One-way Anova with post hoc Tukey’s test. (G) Quantification of α-syn in neurites from neurons cultured under the conditions described in (A). N>80. One-way Anova with Games-Howell’s post-hoc test, * P <0.05, ** P <0.01, *** P <0.001. (H) Representative immunofluorescence images of primary embryonic rat cortical neurons cultured under the conditions described in (A) showing staining for α-synuclein (α-syn). Scale bar = 5 µm.

Article Snippet: Primary embryonic rat cortical neurons were isolated from euthanized pregnant Sprague–Dawley rats at embryonic day 18 as described elsewhere ( ).

Techniques: Staining, Transduction, Construct, Wound Healing Assay, Immunofluorescence, Cell Culture, Immunostaining