Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: CRISPR/Cas9-mediated knockout of Mcu in RBL-2H3 cells. A , exon structure of rat Mcu indicating the target sites of guide RNA1 and guide RNA2. B , quantitative real-time PCR analysis of Mcu mRNA expression in wild-type (WT) RBL-2H3 cells and Mcu knockout (KO) clones A4 and D10 (n = 30 wells; 3 biological replicates). Medians ( bars ) with 95% confidence intervals (error bars ) are displayed on the scatter dot plots. C , immunoblot analysis of WT and Mcu KO clones A4 and D10 using MCU antibodies raised against either the C terminus or N terminus. Vinculin served as a loading control. Molecular weights are indicated in kilodaltons (kDa). MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: CRISPR, Knock-Out, Real-time Polymerase Chain Reaction, Expressing, Clone Assay, Western Blot, Control

Journal: The Journal of Biological Chemistry

Article Title: Deletion of mitochondrial calcium uniporter enhances calcium signals by slowing calcium clearance and triggers adaptive transcriptomic remodeling

doi: 10.1016/j.jbc.2026.111473

Figure Lengend Snippet: Mcu knockout by CRISPR/Cas9 causes widespread and divergent transcriptomic changes across clones generated using distinct guide RNAs. A and B , volcano plots showing differentially expressed genes (DEGs) in Mcu −/− clones A4 ( A ) and D10 ( B ) relative to WT cells. Red and blue dots represent significantly upregulated and downregulated genes, respectively. Mcu is shown as a green dot . Gray dots indicate unchanged genes, either with below absolute fold change 2.0 cutoff ( light gray ) or having false discovery rate(FDR) higher than 0.05 ( dark gray ). C , volcano plot comparing A4 and D10 clones directly. D and E , Venn diagrams of downregulated genes ( D ) and upregulated genes ( E ) in A4 versus WT and D10 versus WT, highlighting both overlapping and clone-specific expression changes. F , ingenuity pathway analysis (IPA) of the 213 DEGs common to both clones (64 downregulated and 149 upregulated) with a z-score cutoff of 1.0. G , bubble plot displays enriched canonical pathways predicted by IPA. The y -axis on the right shows enriched pathway names ordered according to hierarchical clustering of their z-scores across two conditions (A4 versus WT and D10 versus WT), with the corresponding dendrogram displayed on the left . The x -axis labels the two conditions. Each pathway is represented by two circles , one for each condition. Circle size corresponds to the gene ratio for that pathway in the given condition. The fill color of each circle reflects the z-score: red for positive z-scores (activation), blue for negative z-scores (inhibition), transparent for values near zero, and gray where the z-score is NA. Threshold of z-score was set to absolute z-score ≥ 1.5. Below cutoff p values ( i.e. , p value > 0.05) are indicated by small lines on the right of the bubble. MCU, mitochondrial Ca 2+ uniporter.

Article Snippet: Two different guide (g)RNAs targeting rat Mcu (Ensembl Gene ID: ENSRNOG00000045920 ; chromosomal location chr20 29,038,480—29,199,224 [-]; genome: Rnor_6.0 (rn6, Rattus norvegicus )) were designed using the Benchling CRISPR design webtool ( https://benchling.com/crispr ).

Techniques: Knock-Out, CRISPR, Clone Assay, Generated, Expressing, Activation Assay, Inhibition

Journal: Life

Article Title: CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

doi: 10.3390/life11010013

Figure Lengend Snippet: The mechanism of action of clustered, regularly interspaced, short palindromic repeats–associated Cas9 (CRISPR-Cas9). The guide RNA (gRNA) molecule directs Cas9 protein to the target DNA and Cas9 cleaves genomic DNA 3–4 bp upstream of the PAM (protospacer-adjacent motif) site.

Article Snippet: Some available webtools include, CRISPy ( www.crispy.secondarymetabolites.org ) [ ], CRISPR-ERA ( www.crispr-era.stanford.edu ) [ ], E-CRISPR ( www.e-crisp.org ) [ ], Benchling ( www.benchling.com ), or ATUM gRNA design tool ( www.atum.bio ).

Techniques: CRISPR

Journal: Life

Article Title: CRISPR-Cas9: A Powerful Tool to Efficiently Engineer Saccharomyces cerevisiae

doi: 10.3390/life11010013

Figure Lengend Snippet: Some CRISPR-based applications in Saccharomyces cerevisiae . ARS: autonomously replicating sequence; CEN: yeast centromere.

Article Snippet: Some available webtools include, CRISPy ( www.crispy.secondarymetabolites.org ) [ ], CRISPR-ERA ( www.crispr-era.stanford.edu ) [ ], E-CRISPR ( www.e-crisp.org ) [ ], Benchling ( www.benchling.com ), or ATUM gRNA design tool ( www.atum.bio ).

Techniques: CRISPR, Sequencing, Expressing, Transformation Assay, Plasmid Preparation, Activation Assay

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Schematic of STMN2 -NLuc models. WT and TDP-43 mutant (N352S) SH-SY5Y cells were CRISPR-Cas9 edited to express Nanoluciferase at the C-terminus of both endogenous STMN2 alleles. (B) PCR of edited lines using primers outside and within the repair template homology arms confirms homozygosity of the insertion. (C) RT-PCR using primers spanning the entire STMN2 cDNA demonstrate no unusual splice variants after NLuc CRISPR-Cas9 insertion. (D) Immunoblot of WT STMN2 -NLuc line demonstrates that the sensitivity to STMN2/TDP-43 siRNA-mediated disruption is maintained after editing. (E) WT STMN2-NLuc and TDP-43 mutant (N352S) lines demonstrate linear luciferase response up to 20k plated cells proportional to TDP-43 status. (F) Luciferase expression in WT STMN2 -NLuc line is inversely proportional to cell death (CellTox Green cytotoxicity assay, Promega) after treatment with high-doses of hygromycin. (G) Small molecules libraries used for pharmacological screen in STMN2 -NLuc TDP-43 mutant (N352S) SH-SY5Y cells. (H-I) Pearson correlation of replicates (H) and Z-score dot plot (I) of experimental compounds across the screen. Points in both graphs represent average of technical duplicates. (J) Electromobility shift assay (EMSA) using HDJ2 antibody demonstrates the inhibition of HDJ2 farnesylation following cerivastatin, but not pravastatin, treatment which can be visualised as an accumulation of the unprenylated protein and upward shift on immunoblot. (K) Schematic for WT and TDP-43 STMN2 -mGreenLantern lines. (L) PCR using primers within and outside the repair template homology arms confirm correct insertion of mGreenLantern at the C-terminal of STMN2 after CRISPR-Cas9 mediated editing. (M) Exons and functional domains schematic of STMN2 transcript and protein showing known post-translational modifications. (N) RT-qPCR for STMN2 cryptic exon (Ex2A) expression in TDP-43 mutant (N352S) SH-SY5Y treated with cerivastatin. Points represent mean of technical replicates from independent experiments. Unpaired t-test.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Mutagenesis, CRISPR, Reverse Transcription Polymerase Chain Reaction, Western Blot, Disruption, Luciferase, Expressing, CellTox Assay, Cytotoxicity Assay, Electro Mobility Shift Assay, Inhibition, Functional Assay, Quantitative RT-PCR

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Immunoblot with antibodies against STMN2 and TDP-43 in WT and TDP-43 mutant (N352S) SH-SY5Y cells edited to express a Nanoluciferase tag (NLuc) fused to the STMN2 C-terminus on both endogenous alleles. GAPDH used as loading control. (B) Luciferase assay in edited lines measuring STMN2 protein level in TDP-43 mutant compared to WT cells. Points represent technical replicates from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons correction. (C) Top 10 compounds identified through screening of 11,895 small molecules and validated by dose-response analysis. EC50 of each compound as listed. (D) Statin dose-response in TDP-43 mut STMN2 -NLuc SH-SY5Y demonstrates that lipophilic (cerivastatin, simvastatin, pitavastatin), but not hydrophilic (rosuvastatin, pravastatin) statins promote STMN2 expression. Points represent mean of technical replicates in three independent experiments. Error bars = SEM. (E) Nanoluciferase assay in WT and TDP-43 mutant SH-SY5Y demonstrates that simvastatin and cerivastatin return STMN2 expression to WT baseline at ∼2μM and 0.25μM respectively. Points represent mean of technical replicates from three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons correction. (F) Immunoblot and quantification of STMN2 protein in WT and TDP-43 mutant SH-SY5Y treated with DMSO, 2μM simvastatin, 1μM cerivastatin, or 3μM pravastatin. Points represent technical replicates from three independent experiments. GAPDH used as loading control. Kruskal-Wallis test, asterisks indicate comparisons to DMSO control. (G) Immunoblot for the heat-shock protein HDJ2 demonstrates that the lipophobic statins cerivastatin and pravastatin, but not pravastatin, inhibit farnesylation (demonstrated by a migration shift) in SH-SY5Y cells. GAPDH used as loading control. (H) Luciferase assay shows significant increase in STMN2 expression upon 1μM cerivastatin treatment of WT-SH-SY5Y STMN2 -NLuc with siRNA-mediated TDP-43 suppression. Points represent mean of technical replicates from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons. (I) Immunoblot with STMN2 and TDP-43 antibodies under same conditions as in (H) . GAPDH used as loading control. Points represent technical replicates from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons correction. (J) Both the lactone and salt formulations of 1μM cerivastatin promote STMN2 expression equally via luciferase assay. One-way ANOVA with Tukey’s multiple comparisons correction. Points represent mean of technical replicates from independent experiments. One-way ANOVA with Tukey’s multiple comparisons. (K) WT-SH-SY5Y edited to express mGreenLantern on one endogenous STMN2 allele co-cultured with 50% unedited cells exhibit increased endogenous fluorescence upon statin exposure. Points represent individual wells. Unpaired t-test. Scale bar = 50μm. (L) Acyl-biotin exchange assay demonstrates no change in relative palmitoylation status of STMN2 after 1μM cerivastatin treatment. Points represent technical replicates from two independent experiments. Unpaired t-test. (M) RT-qPCR demonstrating 2μM simvastatin and 1μM cerivastatin treatment returns STMN2 mRNA expression to WT baseline in TDP-43 mutant (N352S) SH-SY5Y. Points represent mean of technical triplicates from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (N) RT-qPCR of statin-treated iPSC demonstrates a dose-dependent increase in STMN2 RNA expression. One-way ANOVA with Tukey’s multiple comparisons correction. (O-P) RT-qPCR (O) and immunoblot (P) of mouse N2a cells treated with 1μM cerivastatin indicates conservation of the mechanism between species. Points represent averages of technical triplicates from independent biological replicates. One-way ANOVA with Dunnett’s correction.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Western Blot, Mutagenesis, Control, Luciferase, Expressing, Migration, Cell Culture, Fluorescence, Quantitative RT-PCR, RNA Expression

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Simplified schematic illustrating key steps of the mevalonate pathway. (B-C) Luciferase assay (B) and immunoblot (C) to quantify STMN2 protein in WT SH-SY5Y cells co-treated with 1μM cerivastatin and either 30μM mevalonate or 10μM GGPP substrates. Points represent mean of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. Asterisks indicate comparison of each condition to DMSO control. (D) Immunofluorescence with STMN2 (red/black) and TUJ1 (green) antibodies of WT SH-SY5Y treated with statin and mevalonate/GGPP substrates. Scale bar = 25μm. (E) Newly synthesized isoform of pitavastatin (MGB06) bearing modified hydroxyl side-chain to inhibit engagement of the HMG-CoA reductase catalytic domain. (F) MGB06 Hydroxyl-modification prevents hydrogen bonding to the HMG-CoA reductase catalytic domain. Panels generated using Maestro software (Schrodinger). (G-H) MGB06 has no effect on STMN2 expression as measured by luciferase assay (G) or immunoblot (H) . Points represent mean of technical replicates in three independent experiments. Two-way ANOVA with Tukey’s multiple comparisons. Asterisks represent comparisons to DMSO control. (I-K) Enzyme-linked immunosorbent assay for STMN2 (I) and RT-qPCR for STMN2 (J) and HMGCR (K) in iPSC-derived hNIL motor neurons treated with cerivastatin and harvested at day 5 or day 9 post-doxycycline induction, normalized to mean of DMSO control values. Points represent replicates from three independent experiments. One-way ANOVA with Holm-Šídak multiple comparisons. Asterisks indicate comparison to DMSO. (L-M) Confocal image (L) and automated quantification of STMN2 immunostaining (M) in motor neurons differentiated from iPSC using a small molecules (SM)-based protocol and treated with cerivastatin. Points represent mean intensity across four independent wells. Unpaired t-test. (N) RT-qPCR for STMN2 in small-molecule differentiated motor neurons. One-way ANOVA with Holm-Šídak multiple comparisons. Asterisks indicate comparison to DMSO.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Luciferase, Western Blot, Comparison, Control, Immunofluorescence, Synthesized, Modification, Generated, Maestro Software, Expressing, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Derivative Assay, Immunostaining

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A-B) Luciferase assay in WT STMN2-NLuc cells (A) and RT-qPCR in WT SH-SY5Y (B) treated with DMSO, 5mg/ml cholesterol or 1mM cerivastatin alone or co-treated with cerivastatin and cholesterol. Points represent mean of technical replicates from three independent experiments. One-way ANOVA with Tukey’s multiple comparisons. (C) RT-qPCR quantifying siRNA-mediated knockdown of HMGCR in WT SH-SY5Y cells compared to control siRNA. Points represent mean of technical replicates from three independent biological replicates. Unpaired t-test. (D) Immunofluorescent staining 11 days post-doxycycline induction of NGN2 -dCas9 cortical neurons using antibodies targeting NeuN (red), vGlut1 (green), and TUJ1 (purple). Scale bar = 25mm. (E) Branching analysis conducted on WT-GFP, retinoic acid differentiated SH-SY5Y after treatment with indicated compounds. Comparisons represented to DMSO control. Points represent average of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Luciferase, Quantitative RT-PCR, Knockdown, Control, Staining

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Luciferase assay for STMN2 in WT SH-SY5Y cells treated with siRNA against HMGCR and control. Points represent mean of three technical replicates. Unpaired t-test. (B) Pooled CRISPRi-single-cell RNA-seq data from Tian et al (2019) demonstrates that inhibition of the GGTase I beta-subunit ( PGGT1B) promotes STMN2 expression in iPSCs. Asterisks indicate comparison to non-targeting. (C) RT-qPCR for PGGT1B and STMN2 transcripts in WT SH-SY5Y cells treated with ASOs targeting PGGT1B . Points represent average of technical replicates from independent biological replicates. Unpaired t-tests. (D) RT-qPCR for RHOB and STMN2 transcripts in WT SH-SY5Y cells treated with ASOs targeting RHOB . Points represent average of technical replicates from independent experiments. Unpaired t-tests. (E) Experimental scheme for CRISPRi of targets using NGN2-dCas9 cortical neurons. iPSC were transduced using lentiviral constructs bearing dual-guide RNA at day -3, selected using blasticidin, and NGN2 expression was induced through the addition of doxycycline. Cells were harvested at day 11 post-induction for RT-qPCR. (F) Immunofluorescence imaging of NGN2 -dCas9-BFP iPSC neurons. dCas9 is localized to the nucleus and used to repress indicated genes after lentiviral-mediated expression of dual gRNA. (G) CRISPR inhibition of MVD and GGPS1 in iPSC-derived cortical neurons results in a knockdown efficiency of 60% and 40% respectively via RT-qPCR. Points represent average of technical replicates from independent experiments. Unpaired t-tests. (H) RT-qPCR for STMN2 upon CRISPR inhibition of MVD or GGPS1 in iPSC-derived cortical neurons. Points represent average of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (I) RT-qPCR of selected targets in day 32 iPSC-derived cortical neurons following 10 days of treatment with ASOs targeting TARDBP, PGGT1B , GGPS1 or MVD . Points represent independent biological replicates. Unpaired t-tests. (J) RT-qPCR for STMN2 expression following ASO knockdowns in (I) . Points represent independent biological replicates. One-way ANOVA with Dunnett’s multiple comparisons. (K) Immunofluorescent staining using STMN2 (red) and TUJ1 (green) antibodies in non-differentiated WT SH-SY5Y cells demonstrate an upregulation in STMN2 expression, including at the nascent growth cone (arrow). Right panels are insets as indicated. Scale bar = 25μm. (L) Immunofluorescent staining of WT SH-SY5Y using TUJ1 antibody (green) indicates neurite length extension following statin treatment. Scale bars = 25μm. (M) GFP overexpression in TDP-43 mutant SH-SY5Y also reveals neurite extension following 1μM cerivastatin treatment. (N) Automated image analysis of retinoic acid differentiated WT-GFP SH-SY5Y demonstrates dose-response increase in neurite length upon treatment with lipophilic cerivastatin and simvastatin, but not hydrophilic pravastatin. Points represent average of technical replicates from independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (O) Image analysis of TUJ1 staining to measure neurite length and branching in NGN2 iPSC-derived cortical neurons after statin treatment. Unpaired t-tests. Points represent average values from nine images across each well, across two independent experiments. (P) Automated quantification of neurite length in iPSC-derived motor neurons treated with cerivastatin and immunostained with a TUJ1 antibody. Points represent average of measurements from individual wells. One-way ANOVA with Dunnett’s multiple comparisons.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Luciferase, Control, Single Cell, RNA Sequencing, Inhibition, Expressing, Comparison, Quantitative RT-PCR, Construct, Immunofluorescence, Imaging, CRISPR, Derivative Assay, Knockdown, Staining, Over Expression, Mutagenesis

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Volcano plot from RNAseq analysis of simvastatin or DMSO treated WT SH-SY5Y cells showing significantly downregulated (blue) and upregulated (red) genes. Dot size is inversely proportional to DEseq adjusted p-value. Genes shown: Transcripts Per Million (TPM) 1-15000, Fold change (FC) 0.12-15. (B) Heatmaps of normalized TPM of all up/downregulated genes following statin treatment. Samples treated with simvastatin and mevalonate are included as control. Normalization for each point was to mean TPM of DMSO condition. (C) TPM normalized to DMSO average for STMN2 and TARDBP transcripts upon simvastatin or simvastatin + mevalonate treatment. One-way ANOVA with Dunnett’s multiple comparisons. (D) Top gene ontology terms identified using ShinyGO as enriched in upregulated genes upon statin treatment. (E-F) Normalized TPM for selected cytoskeletal genes (E) and Rho-GTPases (F) . Asterisks indicate adjusted p-values from comparison between DMSO and Simvastatin treatments using DESeq2. (G) Immunoprecipitation using a RhoA/B/C antibody cocktail revealed an upregulation of total Rho, but downregulation GTP-bound Rho, after 1μM cerivastatin treatment. (H) Normalized TPM for selected transcription factors. Asterisks indicate adjusted p-values from comparison between DMSO and Simvastatin treatments using DESeq2. (I) Immunoblot with antibodies against STMN2 and c-jun in WT SH-SY5Y cells treated with 1μM cerivastatin alone or with the addition of mevalonate. GAPDH is included as loading control. Points represent technical replicates from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (J) Normalized TPM for selected cholesterol biosynthesis gene. (K) Examples of genes upregulated both after simvastatin treatment and CRISPR inhibition of PGGT1B . Asterisks indicate p-value of transcriptional changes in iPSC with PGGT1B gRNA compared to control gRNA .

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: RNA sequencing, Control, Comparison, Immunoprecipitation, Western Blot, CRISPR, Inhibition

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A-B) RT-qPCR for UNC13A mRNA in WT SH-SY5Y (A) and hNIL motor neurons (B) treated with 1mM or 2mM cerivastatin, respectively. Points represent mean of technical replicates from three independent experiments. Unpaired t-tests. (C) Levels of UNC13B transcripts detected by RNAseq in WT SH-SY5Y treated with DMSO, simvastatin or simvastatin + mevalonate. TPM normalized to DMSO average. Asterisks indicate adjusted p-values obtained using DESeq2. (D) Top gene ontology terms identified as enriched using ShinyGO in downregulated genes upon simvastatin treatment. (E) Quantification of Total Rho and pull-down Rho-GTP from . WT SH-SY5Y treated with indicated compounds for 24hrs. Points represent replicates from independent experiments. Both graphs one-way ANOVA with Dunnett’s multiple comparisons. (F) Immunofluorescent staining of WT SH-SY5Y with c-jun (red) and TUJ1 (green) antibodies following DMSO or 1mM cerivastatin treatment. (G) Schematic denoting predicted AP-1 binding sites in the proximal human STMN2 promoter. (H) Top genes (ranked by Log2 FC) upregulated by both statin treatment and CRISPRi- PGGT1B. RHOB, JUN, STMN2, and KLF6 were among the most upregulated in both datasets, with significant upregulations of RHOB and KLF6 maintained in mature iPSC-neurons following PGGT1B knockdown .

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Quantitative RT-PCR, RNA sequencing, Staining, Binding Assay, Knockdown

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) RT-qPCR for ATF3 in differentiated ReNVM neurons treated with DMSO or 2μM cerivastatin. Points represent the mean of technical replicates from three independent experiments. Unpaired t-test. (B) RT-qPCR for ATF3 in hNIL motor neurons treated with DMSO or 0.5 and 2μM cerivastatin at day 5 or day 9 of differentiation. Points represent average of technical replicates from three independent experiments. One-way ANOVA with Dunnett’s multiple comparisons. (C) RT-qPCR for ATF3 in SH-SY5Y CRISPR-edited to inactivate ATF3 compared to SH-SY5Y cells treated with a non-targeting sgRNA. Points represent mean of technical replicates from independent biological replicates. Two-way ANOVA with Tukey’s multiple comparisons correction. (D-E) RT-qPCR (D) and ELISA (E) for STMN2 RNA and protein, respectively, in ATF3 KO and control lines after 1μM cerivastatin treatment. Points represent average of technical replicates from independent biological replicates. Two-way ANOVA with multiple comparisons Tukey’s correction. (F) Schematic of in vivo experimental design in Atf3 conditional knockout mice where expression was removed from motor neurons using Chat-Cre expression. Control (n=7) and Atf3 cKO mice (n=7) underwent surgical sciatic nerve crush and were euthanized 7 days post-injury for tissue analyses. (G) Quantification of VAChT+ motor neurons in the lumbar spinal cord. Large points = individual animals, small points = individual neurons. Error bars = mean with SEM. Unpaired t-test. (H) Immunofluorescence of the lumbar spinal cord with an Atf3 antibody demonstrates increase of Atf3 expression in the injured motor neurons of control animals but not Atf3 cKO mice. Scale bar inset = 100μm, zoom out = 500μm. (I) Quantification of immunofluorescence staining for Atf3 from confocal microscopy shown in (H) . Large points = individual animals, small points = individual neurons. Error bars = mean with SEM. Unpaired t-test. (J) Immunofluorescent staining with STMN2 and VAChT antibodies of sections from the lumbar spinal cord from control and Atf3 cKO mice after sciatic nerve crush. Injured and uninjured pools of motor neurons are labelled on the ipsilateral injured side. Scale bars on insets = 100μm, whole spinal cord section = 250μm. Insets of the ventral horns ipsilateral and contralateral to the sciatic nerve injury are highlighted by white dashed boxes on the whole spinal cord sections. (K) RNA-FISH of lumbar spinal cord with probes against Stmn2 , Atf3 and Chat of sections from the lumbar spinal cord from control and Atf3 cKO mice after sciatic nerve crush. Injured and uninjured pools of motor neurons are labelled on the ipsilateral injured side. Scale bar on insets = 100μm, whole spinal cord section = 250μm. Insets of the ventral horns ipsilateral and contralateral to the sciatic nerve injury are highlighted by red dashed boxes on the whole spinal cord sections. (L) Quantification of STMN2 protein levels from immunofluorescence staining in (J) . Values of STMN2 intensity in injured motor neurons are normalized to uninjured motor neurons. Small points = individual neurons, large points = individual animals (n=7/condition). Error bar = median. Unpaired t-test. (M) Quantification of Stmn2 RNA levels from FISH staining in (K) . Values of Stmn2 intensity in injured motor neurons are normalized to uninjured motor neurons. Small points = individual neurons, large points = individual animals (n=4/condition). Error bar = median. Unpaired t-test.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Quantitative RT-PCR, CRISPR, Enzyme-linked Immunosorbent Assay, Control, In Vivo, Knock-Out, Expressing, Immunofluorescence, Staining, Confocal Microscopy

Journal: bioRxiv

Article Title: Statins and genetic inhibition of the mevalonate pathway activate an ATF3-STMN2 regenerative program

doi: 10.64898/2026.02.23.707492

Figure Lengend Snippet: (A) Cas9 and a gRNA targeting ATF3 Exon 1 were used to generate a clone of WT SH-SY5Y with a deletion of 1bp, resulting in a frameshift and premature stop codon upstream of the bZIP DNA-binding domain of ATF3 protein. (B) Next-generation sequencing of the targeted locus confirmed the deletion in ∼98% of reads after clonal selection. Sequencing was repeated after characterization of the line to re-confirm the indel. (C) RT-qPCR of known ATF3 targets reveals a downregulation of their RNA in ATF3 KO line. RNA levels are normalized to values in WT SH-SY5Y generated using a non-targeting sgRNA as control. Points represent mean of technical replicates from independent experiments. Asterisks indicate unpaired t-test comparisons to control cells. (D) Quantification of Stmn2 RNA-FISH in uninjured and injured motor neuron pools from both control and Atf3 cKO mice. Small points represent individual motor neurons; large points represent average for each animal. Bar represents median value.

Article Snippet: A single guide RNA (sgRNA) targeting STMN2 was designed (Benchling webtool) and cloned into pSpCas9–2A-green fluorescent protein (GFP) plasmid (px458-Addgene) using the BbsI restriction site.

Techniques: Binding Assay, Next-Generation Sequencing, Selection, Sequencing, Quantitative RT-PCR, Generated, Control