primary embryonic rat cortical neurons  (Dawley Inc)

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