Journal: iScience

Article Title: Ex vivo removal of pro-fibrotic collagen and rescue of metabolic function in human ovarian fibrosis

doi: 10.1016/j.isci.2025.112020

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were rabbit anti-COLIV/COL4A1 (1:50, ab217147, Abcam), mouse anti-AMH (1:30, MCA2246T, BioRad), goat anti-VASA/DDX4 (1:200, AF2030, R&D), rabbit anti-alpha smooth muscle actin/ACTA2 (1:200; ab5694, Abcam), mouse anti-8-OHdG (1:1000, sc66036, Santa Cruz), mouse anti-CD68 (1:50, M087629-2, DAKO), sheep anti-COL1A1 (1:200, af6220, R&D Systems), goat anti-FOXL2 (1:200, ab5096, Abcam), rabbit anti-NR2F2 (1:250, ab211776, Abcam), mouse anti-PCNA (1:100, SC-56, SantaCruz), rabbit anti-Cytokeratin19/KRT19 (1:100, ab76539, Abcam).

Techniques: Preserving, Recombinant, Saline, Adhesive, TUNEL Assay, In Situ, Picogreen Assay, Single-cell Transcriptomics, Software, Microscopy

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: SH-SY5Y cells were treated with hemin (10 μM) over different time intervals to analyze time-dependent kinetics, DMSO-treated cells were used as control, and etoposide (10 µM) treatment for 1 h served as a positive control. a A neutral comet assay was performed at the indicated time points, and the mean tail moment (fold change) of 25 randomly selected hemin-treated cells was quantified and represented in a line graph. The bar graph represents the mean tail moment fold change between DMSO, etoposide, and hemin-treated for 1 h, derived from 25 randomly selected cells, the data presented as mean ± s.e.m. from three independent experiments. b SA-β-Gal staining was performed to identify senescence-positive cells, representative images, and quantitation of senescent-positive cells per field in hemin-treated cells at indicated time points, the data presented is derived from three independent experiments. Scale bar = 20 µm. Zoomed inserts on the red-bordered box represent 1.5x enlarged senescence-positive cells. c Western blot analysis of indicated DDR markers (p-53BP1-S1778, p-ATM - S1981, and p53), inflammatory marker (NF-ĸB-p65), senescence marker (p21), and heme catabolizing enzyme (HO-1) was performed on cells treated with either DMSO (lanes 1-7) or hemin (lanes 9-15) at indicated time points; untreated in lane 8. Normalized western blot band intensity (relative to representative loading control) was quantified and represented as a fold change of protein level in histograms. Total ATM and 53BP1 were used to normalize p-ATM and p-53BP1 levels, respectively, and β-actin was used to normalize other proteins. The data are presented as mean ± s.e.m. from three independent experiments. d Time kinetics of hemin-induced cellular events such as Senescence (SA- β-Gal staining), DNA damage (comet assay), and HO-1 induction (western blot) are presented as fold change. The graph on the left shows early time points, while the graph on the right includes all the time points in the study. All statistical analyses were performed by one-way ANOVA, p-values are indicated in the respective graphs.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Control, Positive Control, Neutral Comet Assay, Derivative Assay, Staining, Quantitation Assay, Western Blot, Marker, Single Cell Gel Electrophoresis

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: a A schematic representation depicting the process of differentiating neurons from iPSCs. b Western blot analysis of markers for iPSCs (OCT-4), NPSCs (SOX-2), and neurons (β-III Tubulin). c Immunofluorescence (IF) staining of iPSCs-derived neurons, showing labeling with β-III tubulin and NeuN, nuclear counterstained with DAPI. Scale bar = 10 µm. d Time kinetics of SA-β-Gal staining in iPSCs-derived neurons treated with hemin (10 µM), DMSO-treated cells were used as the control. Scale bar = 10 µm. Quantitation data presented as mean ± s.e.m. from three independent experiments. e IF analysis of p21 expression following treatment with or without hemin over time. Quantitation data presented as mean ± s.e.m. from three independent experiments. Scale bar = 20 µm. f Western blot analysis of p-ATM (S1981), NF-ĸB (p65), p21, and HO-1 in response to hemin treatment (lanes 2-6) at different time points. Quantitation data presented as mean ± s.e.m. from three independent experiments. g A schematic representation summarizing the various cellular events mediated by hemin in SH-SY5Y cells and iPSCs-derived neurons. All statistical analyses were performed by one-way ANOVA, p-values are indicated in the respective graphs.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Western Blot, Immunofluorescence, Staining, Derivative Assay, Labeling, Control, Quantitation Assay, Expressing

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: SaH-SY5Y cells were pre-treated with either KU 55933 (ATMi) (10 μM), Pifithrin-α (p53i) (10 μM) and in combination for 2 h before exposing them to hemin (10 μM). a Western blot analysis was conducted to assess the expression of p-ATM/ATM, p53, HO-1, and p21 proteins at indicated time points after hemin treatment, the band intensity was quantified and normalized using respective loading control. The quantitation data is presented as mean ± s.e.m. from three independent experiments. b SA-β-Gal staining was performed on cells treated with hemin and inhibitors (ATMi and p53i) at indicated time points, representative images, and quantitation of senescent positive cells per field in hemin-treated cells at indicated time points, the data presented is derived from three independent experiments. Scale bar = 20 µm. c DNA integrity was analyzed using LA-PCR, left panel represents an agarose gel (1%) image of the amplicon, the right panel shows the quantitation of amplified DNA using plate reader-based Picogreen assay, and represents DNA integrity (fold change) mean ± s.e.m. from three independent experiments. d A scheme illustrating the role of DDR factors ATM and p53 in hemin-mediated senescence and HO-1 induction. All statistical analyses were performed by one-way ANOVA, p-values are indicated in the respective graphs.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Western Blot, Expressing, Control, Quantitation Assay, Staining, Derivative Assay, Agarose Gel Electrophoresis, Amplification, Picogreen Assay

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: SH-SY5Y cells were pre-treated with either BMS-345541 (NF-ĸBi, 10 μM), ML385 (Nrf2i, 10 μM) or in combination for 2 h before exposure to hemin (10 μM). a Western blot analysis was conducted to assess the expression of NF-ĸB (p65), NRF2, HO-1, p21, and cleaved caspase-3 proteins at indicated time points after hemin treatment, The band intensity was quantified and normalized using a respective loading control. The quantitation data is presented as mean ± s.e.m. from three independent experiments. b SA-β-Gal staining was performed on cells after treatment with hemin and inhibitors of NF-ĸB and Nrf2 at indicated time points representative images, and quantitation of senescent positive cells per field in hemin-treated cells at indicated time points, the data presented is derived from three independent experiments. Scale bar = 20 µm. All statistical analyses were performed by one-way ANOVA, p-values are indicated in the respective graphs.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Western Blot, Expressing, Control, Quantitation Assay, Staining, Derivative Assay

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: SH-SY5Y cells were treated with hemin (10 μM) for indicated time points, and DMSO-treated cells were used as the control. a Co-staining of colorimetric SA-β-Gal staining, p21, and HO-1 by IF in cells treated for 1 h, 6 h, and 12 h with 10 μM hemin. Scale bar = 20 µm. The quantitation data is presented as mean ± s.e.m. from three independent experiments, arrow indicates senescence-positive cells. b The co-expression of HO-1 in senescence cells (SA-β-Gal/p21) was illustrated using a grouped bar graph. c IF co-staining of SA-β-Gal and HO-1 was performed in cells treated with hemin for 6 h and 12 h. Scale bar = 20 µm. The quantitation data is presented as mean ± s.e.m. from three independent experiments. d The co-expression of HO-1 in senescence cells (SA-β-Gal) was illustrated using a grouped bar graph. All statistical analyses were performed by two-sided Student’s t-test, p-values are indicated in the respective graph.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Control, Staining, Quantitation Assay, Expressing

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: a Graphical representation of the presence of hematoma and the tissue surrounding the hematoma. b Presurgical brain imaging of ICH patients, ICH-1: A non-contrast-enhanced computed tomography (CT) scan of the brain reveals a significant subcortical hemorrhage within the parietal lobe, measuring 5.5 × 6.4 cm. Associated findings include extensive vasogenic edema and a prominent mass effect. Hypertensive etiology was clinically correlated. ICH-2: Large right parenchymal hemorrhage, measuring 6.8 × 2.8 × 4.5 cm. Associated findings include moderate vasogenic edema and mild midline shift. hypertensive etiology was clinically correlated. ICH-3: Magnetic resonance imaging (MRI) of the brain was performed. The axial T2 image shows a left parenchymal hemorrhage within the left thalamocapsular region measuring 4.5 × 3.9 × 4.6 cm in the context of recurrent cavernoma. c H & E staining of ICH patient tissues with appropriate age-matched non-hemorrhagic control tissue. Scale bar = 10 µm. d DNA damage in ICH patient tissues was evaluated using the TUNEL assay compared with appropriate age-matched non-hemorrhagic controls. Scale bar = 50 µm. e IF co-staining of SA-β-Gal and NeuN (neuronal marker) in ICH patient tissue with appropriate age-matched non-hemorrhagic control, Scale bar = 20 µm. f IF co-staining of SA-β-Gal and HO-1 was conducted in ICH patients ( n = 3), including appropriate age-matched non-hemorrhagic control ( n = 3), Scale bar = 20 µm.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Imaging, Computed Tomography, Magnetic Resonance Imaging, Staining, Control, TUNEL Assay, Marker

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: a Western blot analysis was conducted with or without p21i to examine the expression of p21, p53, HO-1, and cleaved PARP proteins at the specified time points after hemin (10 µM) treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. b Western blot analysis was performed with or without HO-1 knockdown to examine the expression of HO-1, p53, and cleaved caspase-3 proteins at the indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. c Western blot analysis was carried out after transfecting with or without HO-1 expression plasmid to examine the expression of HO-1, p21, and GPX-4 proteins at the indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. d Measuring cellular lipid peroxidation in SH-SY5Y cells transfected with or without the HO-1 plasmid and treated with or without hemin using the C11 BODIPY™ 581/591 fluorescent probe. Total C11 BODIPY™ 581/591 (red), oxidized C11 BODIPY™ 581/591 (green), and Hoechst-stained nuclei (blue). Increased green fluorescence suggests higher lipid peroxidation, while the red fluorescence reflects areas of non-oxidized lipid content. Scale bar = 20 µm. Zoomed image is shown at 3x magnification. e Viability assay using CellTiter-Glo reagent showing results for control cells, HO-1 knockdown cells, and HO-1 overexpressing cells at indicated time points after hemin treatment. The quantitation data presented as mean ± s.e.m. from three independent experiments. f A schematic illustration of the sequential cellular responses to hemin exposure and its significance in counteracting ICH-associated hemin and iron toxicity. Any interference with these sequential processes, such as inhibition of transient senescence or altering the timing of HO-1 induction without corresponding co-expression with senescence, can lead to unsynchronized cellular response and cause ferroptotic and/or apoptotic cell death. All statistical analyses were performed by two-sided Student’s t-test, p-values are indicated in the respective graph.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Western Blot, Expressing, Quantitation Assay, Knockdown, Plasmid Preparation, Transfection, Staining, Fluorescence, Viability Assay, Control, Inhibition

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: iPSC-derived neurons were exposed to PEG-OAC and DEF-OAC-PEG nanoparticles 1 h after hemin (10 μM) treatment. a Representative images and quantification of SA-β-Gal positive cells per field in iPSCs-derived neurons treated with or without hemin and nanoparticles (PEG-OAC and DEF-OAC-PEG) for 24 h, Scale bar = 20 µm. The data presented is derived from three independent experiments. b Viability assay (CellTiter-Glo) of iPSC-derived neurons treated with or without hemin and nanoparticles (PEG-OAC and DEF-OAC-PEG) for 24 h, the data presented is derived from three independent experiments. c Western blot analysis to assess the expression of p-ATM (S1981), HO-1, p21, cleaved PARP, and GPX-4 proteins at indicated time points, the data presented is derived from three independent experiments. d Representative image of the lipid peroxidation assay using the C11 BODIPY™ 581/591 fluorescent probe in iPSC-derived neurons following 24-hour treatment with or without hemin and nanoparticles (PEG-OAC and DEF-OAC-PEG). Total C11 BODIPY™ 581/591 (red), oxidized C11 BODIPY™ 581/591 (green), and Hoechst-stained nuclei (blue). Increased green fluorescence suggests higher lipid peroxidation, while the red fluorescence reflects areas of non-oxidized lipid content. Scale bar = 20 µm. Zoomed image is shown at 3x magnification. All statistical analyses were performed by one-way ANOVA, p-values are indicated in the respective graphs.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Derivative Assay, Viability Assay, Western Blot, Expressing, Peroxidation Assay, Staining, Fluorescence

Journal: Communications Biology

Article Title: Hemin-induced transient senescence via DNA damage response: a neuroprotective mechanism against ferroptosis in intracerebral hemorrhage

doi: 10.1038/s42003-025-07983-3

Figure Lengend Snippet: Intracerebral hemolysis results in the release of hemin from ruptured blood cells. Initially, cells respond by activating DNA damage response (DDR) and NF-ĸB pathways, leading to a temporary onset of the senescence like phenotype. This response acts as a critical protective mechanism against the neurotoxic effects of hemin and iron following a brain hemorrhage. This is followed by the stimulation of HO-1, an enzyme responsible for breaking down heme. However, continuous exposure to hemin can cause cells to enter a state of prolonged senescence. Our study demonstrates that a bifunctional carbon nanoparticle comprising anti-oxidant + iron chelating activity (DEF-PEG-OAC) alleviates genome damage and cellular senescence.

Article Snippet: The GFP-tagged HMOX1 plasmid (pCX-HO1-2A-EGFP) was generously provided by Roberto Giovannoni (Addgene plasmid # 74672) .

Techniques: Activity Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Characterizing Adult Cochlear Supporting Cell Transcriptional Diversity Using Single-Cell RNA-Seq: Validation in the Adult Mouse and Translational Implications for the Adult Human Cochlea

doi: 10.3389/fnmol.2020.00013

Figure Lengend Snippet: Key resources.

Article Snippet: FACS-purified adult cochlear supporting cell single-cell RNA-Seq (Fluidigm C1) , This article , .

Techniques: RNAscope, In Situ, Immunohistochemistry, In Situ Hybridization, Adhesive, Sequencing, Sample Prep, Picogreen Assay, Suspension, Binding Assay, Transgenic Assay, Software

Journal: iScience

Article Title: Ex vivo removal of pro-fibrotic collagen and rescue of metabolic function in human ovarian fibrosis

doi: 10.1016/j.isci.2025.112020

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were rabbit anti-COLIV/COL4A1 (1:50, ab217147, Abcam), mouse anti-AMH (1:30, MCA2246T, BioRad), goat anti-VASA/DDX4 (1:200, AF2030, R&D), rabbit anti-alpha smooth muscle actin/ACTA2 (1:200; ab5694, Abcam), mouse anti-8-OHdG (1:1000, sc66036, Santa Cruz), mouse anti-CD68 (1:50, M087629-2, DAKO), sheep anti-COL1A1 (1:200, af6220, R&D Systems), goat anti-FOXL2 (1:200, ab5096, Abcam), rabbit anti-NR2F2 (1:250, ab211776, Abcam), mouse anti-PCNA (1:100, SC-56, SantaCruz), rabbit anti-Cytokeratin19/KRT19 (1:100, ab76539, Abcam).

Techniques: Preserving, Recombinant, Saline, Adhesive, TUNEL Assay, In Situ, Picogreen Assay, Single-cell Transcriptomics, Software, Microscopy

Journal: iScience

Article Title: Ex vivo removal of pro-fibrotic collagen and rescue of metabolic function in human ovarian fibrosis

doi: 10.1016/j.isci.2025.112020

Figure Lengend Snippet:

Article Snippet: The primary antibodies used were rabbit anti-COLIV/COL4A1 (1:50, ab217147, Abcam), mouse anti-AMH (1:30, MCA2246T, BioRad), goat anti-VASA/DDX4 (1:200, AF2030, R&D), rabbit anti-alpha smooth muscle actin/ACTA2 (1:200; ab5694, Abcam), mouse anti-8-OHdG (1:1000, sc66036, Santa Cruz), mouse anti-CD68 (1:50, M087629-2, DAKO), sheep anti-COL1A1 (1:200, af6220, R&D Systems), goat anti-FOXL2 (1:200, ab5096, Abcam), rabbit anti-NR2F2 (1:250, ab211776, Abcam), mouse anti-PCNA (1:100, SC-56, SantaCruz), rabbit anti-Cytokeratin19/KRT19 (1:100, ab76539, Abcam).

Techniques: Preserving, Recombinant, Saline, Adhesive, TUNEL Assay, In Situ, Picogreen Assay, Single-cell Transcriptomics, Software, Microscopy

Journal: Cell

Article Title: Endocrine-exocrine signaling drives obesity-associated pancreatic ductal adenocarcinoma

doi: 10.1016/j.cell.2020.03.062

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Single-cell library preparation was performed using a Chromium Single Cell 3’ Reagent Kit v3 (10X Genomics) and sequenced by Illumina HiSeq.

Techniques: Virus, Plasmid Preparation, Generated, Transplantation Assay, Recombinant, DNA Extraction, Lysis, Extraction, Blocking Assay, Western Blot, Enzyme-linked Immunosorbent Assay, Cell Viability Assay, Reverse Transcription, Bicinchoninic Acid Protein Assay, Picogreen Assay, Derivative Assay, Software