Dataset DOI: 10.5061/dryad.6m905qgdg

Description of the data and file structure

In this study, we discovered how a molecule called poly(ADP-ribose) (PAR), which signals cells to die under toxic conditions, moves from the nucleus to the cytosol to trigger cell death. This form of cell death, known as parthanatos, is activated when the enzyme PARP-1 in the nucleus produces PAR. However, how PAR exits the nucleus was previously unknown. We found that a nuclear protein, histone H1.2, binds to and carries PAR out of the nucleus. When histone H1.2 was reduced or deleted in human neurons, PAR could no longer move to the cytosol after exposure to harmful stimuli such as NMDA or oxygen-glucose deprivation. We also showed that another protein, Iduna (RNF146), tags histone H1.2 with ubiquitin to control its movement and the release of PAR. In mice, deleting histone H1.2 protected the brain from damage after stroke and improved recovery. Together, these findings reveal histone H1.2 as a key carrier that transports PAR from the nucleus to the cytosol, uncovering a crucial step in the parthanatos cell death pathway.

Files are labeled by figure and include imaging files [.jpg, .tif and .czi (image data file format developed by ZEISS),  data sets in excel and graphs in GraphPad Prism (.pzf and .pzfx). All files included in this submission are raw data files generated from experiments performed for this manuscript.

The following abbreviations are retained as published, with clarified and expanded definitions for better reader intuition:

Chemicals and Ischemia model

NMDA or N+: N-methyl-D-aspartate (a common excitatory neurotransmitter receptor)

OGD or O+: Oxygen-Glucose Deprivation (an in vitro model of ischemia)

MCAO: Middle Cerebral Artery Occlusion (an in vivo model of ischemic stroke)

PAR: Poly(ADP-ribose) (a key signaling polymer in the DNA damage response)

DPQ: PARP1 inhibitor (3,4-Dihydro-5-[4-(1-piperidinyl)butoxy]-1(2H)-isoquinolinone)

PDD00017273: PARG inhibitor (a specific inhibitor of Poly(ADP-ribose) glycohydrolase)

XAV-939: Tankyrase Inhibitor (a specific inhibitor of Tankyrase, a PARP family member) 

Genetic Status and Tools

WT: Wild Type (the normal, non-mutated genetic state)

Het: Heterogeneous (used here likely for Heterozygous, having two different alleles for a gene)

sg or sgRNA: Single Guide RNA for CRISPR-Cas9 targeting

KD: Knock Down (reduced expression of a gene/protein)

KO: Knockout (complete removal of a gene/protein function)

OE: Over Expression (artificially increased expression of a gene/protein)

sgRNAres: Guide RNA insensitive/resistant (a genetic construct or cell line not targeted by a specific sgRNA)

Cas9: Cas9 only treatment (used to denote a condition where the Cas9 enzyme is present without an active sgRNA)

Specific Protein Constructs and Mutants

Iduna-GFP: RNF146/Iduna wildtype protein fused with Green Fluorescent Protein

C60A-GFP: RNF146/Iduna C60A mutant (Cysteine to Alanine substitution at position 60) fused with Green Fluorescent Protein

YRAA-GFP: RNF146/Iduna YRAA mutant (substitutions at a Tyr−Arg−Ala−Ala motif) fused with Green Fluorescent Protein

K213A: H1.2 K213A mutant (Lysine to Alanine substitution at position 213 of Histone H1.2)

C1: Truncated form of H1.2 mutant (amino acids 1 through 202)

C2: Truncated form of H1.2 mutant (amino acids 1 through 190)

Assays and Instrumentation

MSD: Electrochemiluminescent system developed by Meso Scale Diagnostics

IVUA: In Vitro Ubiquitination Assay

File structure

The archived files are organized into a hierarchical structure to ensure easy access and verification of the data presented in the manuscript.

Main Figure folder contains all files corresponding to Figures 1 through 6.

Supplementary Figure folder contains all files corresponding to Figures S1 through S13.

Individual data files are stored within designated subfolders under these major categories.

The full path to each archived file is explicitly listed in the separated file named Jing_et_al_Nat_Comms_data_path.xlsx

Files and variables

File: Jing_et_al_Nat_Comms_data.zip

Description: 

Main figure

1. Figure 1: Histone H1.2 physically interacts with PAR and translocates from the nucleus to the cytosol and mitochondria in response to NMDA or OGD stimuli. The data illustrate the stimulus-induced redistribution of H1.2 and its association with PAR signaling.

Fig1A: Immunoblot showing the interaction of H1.2 with PAR in human cortical neurons after NMDA stimulation (Immunoprecipitation with Anti-PAR antibody).

Fig1B: Immunoblot showing the interaction of PAR with H1.2 in human cortical neurons after OGD stimulation (Immunoprecipitation with Anti-H1.2 antibody).

Fig1C: Representative immunofluorescence images showing the co-translocation of H1.2 and PAR from nucleus to cytosol following NMDA challenge, with or without DPQ/XAV-939 treatment.

Fig1D: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR following NMDA stimulation (Data from Fig. 1C).

Fig1E: Representative immunofluorescence images showing the co-translocation of H1.2 and PAR from nucleus to cytosol following OGD challenge, with or without DPQ/XAV-939 treatment.

Fig1F: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR following OGD stimulation (Data from Fig. 1E).

Fig1G: Immunoblot of subcellular fractionation showing the time-dependent shift of

H1.2 and PAR after NMDA challenge, with or without DPQ treatment.

Fig1H: Quantification of the post-nuclear fraction (Cytosol/Mitochondria) shift of H1.2 and PAR after NMDA stimulation (Ratio to GAPDH, data from Fig. 1G).

Fig1I: Immunoblot of subcellular fractionation showing the time-dependent shift of H1.2 and PAR after OGD challenge, with or without DPQ treatment.

Fig1J: Quantification of the post-nuclear fraction (Cytosol/Mitochondria) shift of H1.2 and PAR after OGD stimulation (Ratio to GAPDH, data from Fig. 1I).

2. Figure 2: Deletion of H1.2 reduces PAR/H1.2 translocation from the nucleus and protects human neurons from NMDA- and OGD-induced cell death. The data support a functional role for H1.2 in PAR-dependent neuronal injury.

Fig2A: Representative immunofluorescence images demonstrating H1.2 and PAR translocation in human neurons after NMDA challenge, comparing GFP control, Cas9 control, H1.2 knockdown (sgRNA #1/#2), and sgRNA-resistant H1.2 rescue.

Fig2B: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR under various genetic conditions following NMDA stimulation (Data from Fig. 2A).

Fig2C: Representative immunofluorescence images demonstrating H1.2 and PAR translocation in human neurons after OGD challenge, comparing GFP control, Cas9 control, H1.2 knockdown (sgRNA #1/#2), and sgRNA-resistant H1.2 rescue.

Fig2D: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR under various genetic conditions following OGD stimulation (Data from Fig. 2C).

Fig2E: Representative images of PI/Hoechst staining used to assess neuronal cell death 24 hours after NMDA or OGD insults in control (Cas9 only) and H1.2 knockdown cells.

Fig2F: Quantification of the percentage of neuronal cell death following NMDA insult in control and H1.2 knockdown cells (Assessed via PI/Hoechst staining).

Fig2G: Quantification of the percentage of neuronal cell death following OGD insult in control and H1.2 knockdown cells (Assessed via PI/Hoechst staining).

3. Figure 3: Inhibiting nuclear export with Leptomycin B (LMB) diminishes PAR/H1.2 translocation following NMDA or OGD and lowers neuronal death. The results indicate that nuclear export contributes to H1.2-mediated injury signaling.

Fig3A: Representative immunofluorescence images showing the co-translocation of H1.2 and PAR following NMDA challenge under various conditions, including Leptomycin B (LMB) and specific inhibitors.

Fig3B: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR after NMDA stimulation and various drug treatments (Data from Fig. 3A).

Fig3C: Representative immunofluorescence images showing the co-translocation of H1.2 and PAR following OGD challenge under various conditions, including LMB and specific inhibitors.

Fig3D: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 and PAR after OGD stimulation and various drug treatments (Data from Fig. 3C).

Fig3E: Quantification of the percentage of neuronal cell death following NMDA insult, showing the protective effect of LMB (Assessed via PI/Hoechst staining).

Fig3F: Quantification of the percentage of neuronal cell death following OGD insult, showing the protective effect of LMB (Assessed via PI/Hoechst staining).

Fig3G: Immunoblot of Cytosol (Cyto) and Nuclear (Nu) fractions showing H1.2 and PAR separation following NMDA challenge with or without LMB.

Fig3H: Immunoblot of Cytosol (Cyto) and Nuclear (Nu) fractions showing H1.2 and PAR separation following OGD challenge with or without LMB, along with the corresponding quantification.

4. Figure 4: Overexpression of Iduna, but not its mutant forms, reduces PAR and H1.2 translocation and protects neurons from NMDA- and OGD-induced injury. The data highlight Iduna’s role as a protective regulator of PAR-dependent cell death pathways.

Fig4A: Representative immunofluorescence images of neurons overexpressing Iduna-GFP (Wild-Type RNF146) following NMDA challenge, with or without DPQ treatment.

Fig4B: Representative immunofluorescence images of neurons overexpressing C60A-GFP (Iduna Mutant) following NMDA challenge, with or without DPQ treatment.

Fig4C: Representative immunofluorescence images of neurons overexpressing YRAA-GFP (Iduna Mutant) following NMDA challenge, with or without DPQ treatment.

Fig4D: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR from Fig 4A-4C after NMDA challenge, comparing Wild-Type Iduna and its mutants.

Fig4E: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 from Fig 4A-4C after NMDA challenge, comparing Wild-Type Iduna and its mutants.

Fig4F: Quantification of neuronal Cell Death (PI/Hoechst ratio) after NMDA challenge in neurons overexpressing Iduna-GFP, C60A-GFP, or YRAA-GFP.

Fig4G: Representative immunofluorescence images of neurons overexpressing Iduna-GFP following OGD challenge, with or without DPQ treatment (Same experimental setup as Fig 4A).

Fig4H: Representative immunofluorescence images of neurons overexpressing C60A-GFP following OGD challenge, with or without DPQ treatment (Same experimental setup as Fig 4B).

Fig4I: Representative immunofluorescence images of neurons overexpressing YRAA-GFP following OGD challenge, with or without DPQ treatment (Same experimental setup as Fig 4C).

Fig4J: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR after OGD challenge, comparing Wild-Type Iduna and its mutants (Same experimental setup as Fig 4D).

Fig4K: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 after OGD challenge, comparing Wild-Type Iduna and its mutants (Same experimental setup as Fig 4E).

Fig4L: Quantification of neuronal Cell Death (PI/Hoechst ratio) after OGD challenge in neurons overexpressing Iduna-GFP, C60A-GFP, or YRAA-GFP (Same experimental setup as Fig 4F).

5. Figure 5: The carboxyl-terminal region of H1.2 is required for PAR/H1.2 translocation and neuronal death triggered by NMDA and OGD. The data define specific H1.2 domains essential for Parthanatos signaling.

Fig5A: Schematic illustrating the structure of human Histone H1.2 and the locations of its key variants: the K213A point mutation and the two carboxyl-tail truncated forms (C1 and C2).

Fig5B: Representative immunofluorescence images showing PAR and H1.2 translocation following NMDA challenge in neurons rescued with V5-tagged H1.2 (WT, K213A, C1, or C2) constructs.

Fig5C: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR and H1.2 following NMDA stimulation, comparing the effects of H1.2 WT and mutant overexpression (Data from Fig5B).

Fig5D: Representative immunofluorescence images showing PAR and H1.2 translocation following OGD challenge in neurons rescued with V5-tagged H1.2 (WT, K213A, C1, or C2) constructs.

Fig5E: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR and H1.2 following OGD stimulation, comparing the effects of H1.2 WT and mutant overexpression (Data from Fig5D).

Fig5F: Representative images of PI/Hoechst staining used to assess neuronal cell death after NMDA or OGD insults in neurons overexpressing H1.2 WT or its K213A, C1, or C2 mutants.

Fig5G: Quantification of the percentage of neuronal cell death following NMDA insult, comparing the effects of overexpressing H1.2 WT versus its K213A, C1, and C2 mutants.

Fig5H: Quantification of the percentage of neuronal cell death following OGD insult, comparing the effects of overexpressing H1.2 WT versus its K213A, C1, and C2 mutants.

6. Figure 6: Partial or complete deletion of H1.2 reduces neuronal death in mouse cortical cultures exposed to NMDA or OGD and mitigates injury in an MCAO stroke model. The data indicate that H1.2 contributes to ischemic and excitotoxic neurodegeneration in vivo.

Fig6A: Representative images of TTC-stained wild-type (WT) and H1.2−/− mouse brain slices 24 hours after a 1-hour transient MCAO (Middle Cerebral Artery Occlusion).

Fig6B: Pooled data showing the total brain infarct volume percentage in the whole hemisphere 24 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6C: Pooled data showing the total brain infarct volume percentage in the striatum 24 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6D: Pooled data showing the total brain infarct volume percentage in the cortex 24 hours post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6E: Quantification of the coronal section infarct volume percentage in the whole hemisphere 24 hours post-MCAO across different genotypes.

Fig6F: Quantification of the coronal section infarct volume percentage in the striatum 24 hours post-MCAO across different genotypes.

Fig6G: Quantification of the coronal section infarct volume percentage in the cortex 24 hours post-MCAO across different genotypes.

Fig6H: Representative images of TTC-stained WT and H1.2−/− mouse brain slices 7 days after a 1-hour transient MCAO.

Fig6I: Pooled data showing the total brain infarct volume percentage in the whole hemisphere 7 days post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6J: Pooled data showing the total brain infarct volume percentage in the striatum 7 days post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6K: Pooled data showing the total brain infarct volume percentage in the cortex 7 days post-MCAO, comparing WT, H1.2+/−, and H1.2−/− mice.

Fig6L: Quantification of the coronal section infarct volume percentage in the whole hemisphere 7 days post-MCAO across different genotypes.

Fig6M: Quantification of the coronal section infarct volume percentage in the striatum 7 days post-MCAO across different genotypes.

Fig6N: Quantification of the coronal section infarct volume percentage in the cortex 7 days post-MCAO across different genotypes.

Fig6O: Pooled data from the Cylinder Test, assessing sensorimotor function in the three genotypes at the 7-day endpoint post-MCAO.

Fig6P: Pooled data from the Treadmill Test, assessing motor endurance in the three genotypes at the 7-day endpoint post-MCAO.

Fig6Q: Pooled data from Spontaneous Activity monitoring, assessing general motor function in the three genotypes at the 7-day endpoint post-MCAO.

7. Figure 7: Schematic illustration of roles of H1.2 in Parthanatos

Supplementary figure

1. Figure S1: Histone H1.2 directly binds PAR and can be ribosylated by PARP-1 in vitro. These data establish H1.2 as a PAR-interacting and substrate of PARP-1.

FigS1A: Electromobility gelshift assay testing the PAR binding capacity of recombinant H1.2. Increasing amounts of H1.2 were incubated with [32P]-PAR and resolved by PAGE; both bound and free [32P]-PAR signals were visualized via autoradiography.

FigS1B: In vitro ribosylation assay testing the ribosylation of H1.2 by PARP1. Ribosylated products of PARP1 (auto-ribosylation control), GST (negative control), and H1.2 were separated by SDS-PAGE and visualized by autoradiography to demonstrate that H1.2 can be modified by PARP1.

2. Figure S2: NMDA and OGD treatments trigger nuclear translocation of Parthanatos markers AIF and MIF in cortical neurons.

Representative photomicrographs showing nuclear translocation of the parthanatos markers AIF and MIF induced by NMDA or OGD treatment.

3. Figure S3: Experimental validation of the immunoprecipitation conditions used in the study. The data confirm specificity and efficiency of the IP assays applied throughout the work.

FigS3A: Immunoblots validating the H1.2-PAR interaction by showing the dependence on PAR polymer integrity. Lysates from NMDA/OGD challenged neurons were subjected to Immunoprecipitation (IP) with anti-PAR or anti-H1.2, followed by treatment with active DNase (to remove DNA) or PARG (to cleave PAR polymer) to confirm the specificity of the binding.

FigS3B: Immunoblots validating the H1.2-PAR interaction using inhibitors. Lysates from NMDA/OGD challenged neurons treated with inhibitors for PARP1, Tankyrase, or PARG were subjected to IP (anti-PAR or anti-H1.2) to confirm that the observed interaction is enzyme-dependent.

4. Figure S4: CRISPR/Cas9-mediated H1.2 knockdown reduces PAR/H1.2 translocation in neurons exposed to NMDA and OGD.

FigS4A: Representative immunofluorescence images demonstrating PAR translocation in human cortical neurons following NMDA challenge combined with DPQ (PARP1 inhibitor) or PDD00017273 (PARG inhibitor). Cells were stained for PAR (red) and DAPI (blue).

FigS4B: Representative immunofluorescence images demonstrating PAR translocation in human cortical neurons following OGD challenge combined with DPQ or PDD00017273. Cells were stained for PAR (red) and DAPI (blue).

FigS4C: Quantification of the Nuclear-to-Cytosolic PAR translocation ratio after NMDA stimulation and treatment with inhibitors (DPQ, PDD00017273).

FigS4D: Quantification of the Nuclear-to-Cytosolic PAR translocation ratio after OGD stimulation and treatment with inhibitors (DPQ, PDD00017273).

5. Figure S5: H1.2 sgRNA-mediated knock down reduces PAR/H1.2 translocation by NMDA and OGD stimulation.

FigS5A: Representative immunoblots validating the H1.2 knockdown efficiency. Lysates from neurons expressing Cas9 and H1.2-targeting sgRNAs (#1 and #2) were probed with anti-H1.2 and alpha-Tubulin (loading control). Quantification of H1.2 protein levels is shown, normalized to alpha-Tubulin.

FigS5B: Representative immunoblots of nuclear and cytosolic fractions from control (Cas9) and H1.2 knockdown (sg#1 and sg#2) neurons. The blots were probed with anti-PAR, anti-H1.2, and fraction markers (TBP for nucleus, GAPDH for cytosol).

FigS5C: Quantification of the NMDA-induced release of PAR and H1.2 from the nucleus to the cytosol, comparing control (Cas9 only) and H1.2 knockdown conditions (Data from FigS5B). Release is indicated by normalization to the cytosolic marker GAPDH.

FigS5D: Representative immunoblots of nuclear and cytosolic fractions from control (Cas9) and H1.2 knockdown (sg#1 and sg#2) neurons following OGD challenge. The blots were probed with anti-PAR, anti-H1.2, and fraction markers.

FigS5E: Quantification of the OGD-induced release of PAR and H1.2 from the nucleus to the cytosol, comparing control (Cas9 only) and H1.2 knockdown conditions (Data from FigS5D). Release is indicated by normalization to the cytosolic marker GAPDH.

6. Figure S6: CRISPR-mediated H1.2 knockdown does not increase total cellular PAR levels as confirmed by Western blot.

Western blot validation confirming the effect of CRISPR-mediated H1.2 knockdown on total cellular PAR levels. Human cortical neurons were transduced with lentiviruses carrying either a control gRNA or the H1.2-targeting gRNA #1, and the total cellular PAR levels were subsequently assessed by normalization to the Actin.

7. Figure S7: Quantification of PAR levels in neurons after NMDA or OGD using MSD assays.

FigS7A: Standard curve analysis for the PAR MSD platform. This curve establishes the linear range and sensitivity of the assay using serial dilutions of PAR calibrators.

FigS7B: Quantification of relative PAR concentration in neurons using the MSD system. Neurons expressing either Cas9 (control) or H1.2 sgRNA #1 were challenged with NMDA or OGD. PAR was captured by a coated antibody (HuPAR clone #19) and detected by a Sulfo-tagged antibody (HuPAR clone #25). Results are expressed as fold changes, demonstrating the impact of H1.2 knockdown on PAR accumulation following injury.

8. Figure S8: shRNA-mediated H1.2 knockdown reduces PAR/H1.2 translocation and neuronal death following NMDA and OGD exposure.

FigS8A: Representative immunoblots validating H1.2 knockdown efficiency using shRNA. Lysates from neurons expressing the empty vector (pSME) or H1.2-targeting shRNAs (#1 and #2) were probed with anti-H1.2 and alpha-Tubulin. Quantification of normalized H1.2 levels is shown.

FigS8B: Representative immunofluorescence image showing PAR/H1.2 translocation in control shRNA neurons following NMDA challenge 15 minutes and/or 2 hrs post challenge combined with DPQ. 

FigS8C: Representative immunofluorescence image showing PAR/H1.2 translocation in H1.2 knockdown (shRNA mixed) neurons following NMDA challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.

FigS8D: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 after NMDA stimulation, comparing control and H1.2 shRNA knockdown conditions.

FigS8E: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR after NMDA stimulation, comparing control and H1.2 shRNA knockdown conditions.

FigS8F: Representative immunofluorescence image showing PAR/H1.2 translocation in control shRNA neurons following OGD challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.

FigS8G: Representative immunofluorescence image showing PAR/H1.2 translocation in H1.2 knockdown (shRNA mixed) neurons following OGD challenge 15 minutes and/or 2 hrs post challenge combined with DPQ.

FigS8H: Quantification of the Nuclear-to-Cytosolic translocation ratio of H1.2 after OGD stimulation, comparing control and H1.2 shRNA knockdown conditions.

FigS8I: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR after OGD stimulation, comparing control and H1.2 shRNA knockdown conditions.

FigS8J: Quantification of neuronal Cell Death (PI/Hoechst ratio) 24 hours after NMDA insult, comparing control (pSME) and H1.2 shRNA knockdown conditions.

FigS8K: Quantification of neuronal Cell Death (PI/Hoechst ratio) 24 hours after OGD insult, comparing control (pSME) and H1.2 shRNA knockdown conditions.

9. Figure S9: The release of PAR polymer after MNNG treatment to isolated nuclei of cortical tissues from wild type and Histone H1.2 knockout mice

FigS9A: PAR polymer release from isolated nuclei after MNNG treatment. Representative immunoblots show lysates and post-nuclei buffer of nuclei isolated from cortical tissues of Wild Type (WT), H1.2+/− (Het), and H1.2−/− (KO) mice. Blots were probed for PAR, TBP (nuclear marker), and GAPDH (cytosolic marker).

FigS9B: Quantification of PAR release is shown as the PAR/GAPDH ratio, demonstrating the effect of H1.2 deletion on PAR nuclear efflux.

10. Figure S10: Representative ubiquitination blots and quantification for Iduna mutants. The data compare the ubiquitination status of wild-type Iduna with its mutant variants.

FigS10A: In vitro ubiquitination assay comparing the E3 ligase activity of immunoprecipitated GFP-Iduna (WT) against the mutants GFP-Iduna YRAA and GFP-Iduna C60A. The target substrates were recombinant H1.2 or Ribosylated H1.2 (Ribo-H1.2). Ubiquitination was detected by immunoblots using anti-His, anti-EGFP, and anti-GST antibodies.

FigS10B: Representative immunoblots of nuclear and cytosolic fractions from human neurons overexpressing GFP (control), Iduna-GFP (WT), C60A-GFP, or YRAA-GFP following NMDA treatment. Blots were probed for PAR, H1.2, and fraction markers (TBP and GAPDH).

FigS10C: Quantification of the NMDA-induced release of PAR and H1.2 from the nucleus, comparing WT Iduna and its mutants (Data from FigS10B). Release is calculated as the ratio of protein levels to the cytosolic marker GAPDH.

FigS10D: Representative immunoblots of nuclear and cytosolic fractions from human neurons overexpressing GFP (control), Iduna-GFP (WT), C60A-GFP, or YRAA-GFP following OGD treatment. Blots were probed for PAR, H1.2, and fraction markers.

FigS10E: Quantification of the OGD-induced release of PAR and H1.2 from the nucleus, comparing WT Iduna and its mutants (Data from FigS10D). Release is calculated as the ratio of protein levels to the cytosolic marker GAPDH. 

11. Figure S11: Treatment with MG132 in neurons overexpressing wild-type Iduna restores PAR translocation to the cytoplasm following NMDA or OGD stimulation.

FigS11A: Representative immunofluorescence images showing the translocation of PAR and H1.2 in human cortical neurons overexpressing Iduna-GFP following NMDA challenge and treated with the proteasome inhibitor MG132.

FigS11B: Representative immunofluorescence images showing the translocation of PAR and H1.2 in human cortical neurons overexpressing Iduna-GFP following OGD challenge and treated with the proteasome inhibitor MG132.

FigS11C: Quantification of the Nuclear-to-Cytosolic translocation ratio of PAR and H1.2 following NMDA stimulation, under the experimental conditions described in FigS11A.

FigS11D: Quantification of the Nuclear-to-Cytosolic Translocation Ratio of PAR and H1.2 following OGD stimulation, under the experimental conditions described in FigS11B.

12. Figure S12: Evaluation of PAR binding and ribosylation of H1.2 mutants. The data identify regions of H1.2 required for PAR interaction and modification.

FigS12A: Immunoblots confirming the expression of Flag-tagged H1.2 WT and its mutants (H213A, C1, and C2) following overexpression in human cortical neurons with prior H1.2 knockdown (gRNA targeting H1.2). Blots were probed with anti-H1.2, anti-Flag, and beta-actin (loading control).

FigS12B: Electromobility Shift Assay (EMSA) testing the PAR binding capacity of recombinant H1.2 WT versus its mutants (K213A, C1, and C2). The assay separates bound [32P]-PAR from unbound free [32P]-PAR, which are visualized by autoradiography. Recombinant H3 and BSA were used as controls.

FigS12C: Representative image of the in vitro ribosylation assay demonstrating the ability of PARP1 to ribosylate H1.2 WT versus its mutants (K213A, C1, and C2). The proteins used in the reaction were visualized by Coomassie staining, and the level of ribosylation was quantified by autoradiography from three independent experiments.

13. Figure S13: Quantification of neuronal cell death (PI/Hoechst ratio) in primary cortical cultures from H1.2 wild-type and knockout mice. H1.2−/− neurons show resistance to NMDA (Fig. S13A) and OGD (Fig. S13B) challenges, demonstrating a protective effect of H1.2 deletion.

FigS13A: Quantification of neuronal Cell Death (PI/Hoechst ratio) following NMDA insult in primary cortical neuron cultures isolated from H1.2+/+ (Wild Type) versus H1.2−/− (Knockout) mice, demonstrating resistance in knockout neurons.

FigS13B: Quantification of neuronal Cell Death (PI/Hoechst ratio) following OGD insult in primary cortical neuron cultures isolated from H1.2+/+ versus H1.2−/− mice, demonstrating resistance in knockout neurons.

14. Figure S14: PI/Hoechst staining data assessing cell death in human cortical neurons expressing pSME or H1.2 shRNAs following NMDA (Fig. S8J) or OGD (Fig. S8K) treatment.

15. Figure S15: PI/Hoechst staining data assessing cell death in human cortical neurons expressing Iduna-GFP, C60A-GFP, or YRAA-GFP following NMDA (Fig. 4F) or OGD (Fig. 4L) treatment.

16. Figure S16: PI/Hoechst staining data assessing cell death in human cortical neurons expressing Cas9/sgH1.2 and GFP-tagged H1.2 variants after NMDA (Fig. 5G) or OGD (Fig. 5H) treatment

File: Jing_et_al_Nat_Comms_data_path.csv

Description: The full path to each archived file is explicitly listed