Journal: Cell Proliferation

Article Title: Establishment of a porcine pancreatic stem cell line using T‐REx ™ system‐inducible Wnt3a expression

doi: 10.1111/cpr.12188

Figure Lengend Snippet: The expression of GFP and Wnt3a was regulated by Dox in a time‐ and dose‐dependent manner. (a) The expression of GFP in PSCs was observed under fluorescence microscope after treatment with different concentrations of Dox (0, 0.1, 1, 5, 10 μg/ml) for different times (0, 24, 48 and 72 h) (bar = 200 μm). (b) The relative Wnt3a mRNA expression level in PSCs after Dox treatment for different times analyzed by QRT‐PCR. (c) The relative Wnt3a mRNA expression level in PSCs after treatment with different concentrations of Dox analysed by QRT‐PCR. (d) GFP‐positive PSCs were quantified by flow cytometric after induced by Dox. (e) The histogram of statistical result for Fig. d. (f) The Wnt3a protein expression in PSCs after Dox addition for different times detected by Western blotting. (g) The Wnt3a protein expression in the supernate of cultured PSC after Dox treatment for 72 h. (h) Immunostaining of Wnt3a and active β‐catenin in PSCs after treatment with or without Dox (bar = 100 μm).

Article Snippet: Wnt3a was amplified from mouse tail vein‐derived samples using Vazyme Blood Direct PCR Kit (Vazyme, Nanjing, China), by PCR.

Techniques: Expressing, Fluorescence, Microscopy, Quantitative RT-PCR, Western Blot, Cell Culture, Immunostaining

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: An image of a haematoxylin and eosin (HE)-stained section of HT1080 tumours showing the presence of abundant vascular channels containing red blood cells is illustrated. A magnified image of these vascular channels is depicted on the right hand side. (Original magnifications 100X and 400X respectively) B. Paraffin sections of HT1080 tumours were subjected to immuno histochemistry (IHC) analyses using antibodies to various angiogenic markers. The tumour cells were positive for PECAM, VE-Cadherin, VEGF, NRP-1, VEGF 165 and VEGFR-2 (violet colour). PECAM positive micro-vessel formation is indicated by a black arrow (top layer, right panel). C. Nuclear localization of HIF-1α in the tumour cells confirms the presence of in situ hypoxia. The sections were counterstained with hematoxylin to demarcate the nuclei (blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Staining, Immunohistochemistry, In Situ

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Confocal microscopy analyses show that the hypoxic cells exhibit up-regulation of NRP-1 ( A ) and nuclear stabilization of HIF-1α ( B ). Nuclei are demarcated by DAPI (Blue). Mean fluorescence intensity (M.F.I.) of the cells was measured by Image J software (NIH) at membrane (for NRP-1) and in the nuclear region (for HIF-1α). The M.F.I. of 30 randomly selected cells was used to calculate mean ± S.E.M. The analyses have been graphically depicted (b and d for NRP-1 and HIF-1α respectively) ** p<0.01 and *** p<0.001. C. Quantitative PCR analyses for NRP-1 and HIF-1α mRNA show 2.4 and 1.7 folds up-regulation of these genes in the cells incubated under hypoxia compared to normoxia. (N = 3; *p<.05 and ** p<.01). D. Western blot experiments performed on the cells grown under normoxia vs. hypoxia show that the protein levels of both HIF-1α (upper panel) and NRP-1 (middle panel) are up-regulated in the hypoxic cells compared to the normoxic ones (∼1.4 and 3.2 folds respectively). E. Results of quantitative PCR experiments showed that the hypoxia-induced up-regulation of NRP-1 and VEFG 165 mRNA was sensitive to the presence of chetomin in the medium, indicating that these genes are down-stream events in the HIF-1α-mediated transcription process. F. Confocal microscopy analysis shows that the hypoxia-mediated up-regulation of NRP-1 protein (Cyan, upper panel) is abrogated in the presence of chetomin in the medium (lower panel), suggesting that NRP-1 expression critically depends on the HIF-1α -mediated transcription. Nuclei are demarcated by DAPI (Blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Confocal Microscopy, Fluorescence, Software, Real-time Polymerase Chain Reaction, Incubation, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Silencing of NRP-1 expression in the HT/shNRP-1 cells was validated at transcript level by performing quantitative PCR experiments. The NRP-1 mRNA was ∼500 folds down-regulated in the shNRP-1 clone (N = 3; ***p<0.001) showing that the shRNA constructs used were effective. B. The results obtained in the PCR experiments were validated by western blot experiments. The NRP-1 expression was down-regulated in the HT/shNRP-1 cells at protein level as well. C. Quantitative PCR analyses confirmed the higher expression of NRP-1 transcript in the HT/flNRP-1 clone compared to the HT1080/Scr cells (∼2 folds, N = 3; *p<0.05). D. Western blot experiments were performed on the HT1080/Scr, HT/shNRP-1 and HT/flNRP-1 cells. Data show the presence of a 150 kDa band of the NRP-GFP fusion protein in the HT/flNRP-1 cells when the blot was probed with antibodies to NRP-1 (upper panel) and GFP (middle panel). The down-regulation of NRP-1 in the HT/shNRP-1 clone is also seen (upper panel). E. Confocal microscopy analyses show a higher expression of NRP-1 in the HT/flNRP-1 clone (upper panel) and a reduced expression of NRP-1 in HT/shNRP-1 clone (Lower panel) compared to the HT1080/Scr cells (middle panel). Membrane-localized NRP-1-GFP fusion protein is seen in the HT/flNRP-1 cells. The RFP fluorescence is seen in the nuclei of HT1080/Scr and HT/shNRP-1 cells respectively. Nuclei are demarcated by DAPI (Blue).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, shRNA, Construct, Western Blot, Confocal Microscopy, Fluorescence

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Expression of PECAM and VEGFR-2 mRNA was quantified by performing real time PCR experiments on the HT1080/WT cells that were incubated under normoxia and hypoxia – with or without chetomin. Both PECAM and VEGFR-2 mRNAs were up-regulated by hypoxia. Presence of chetomin abrogated the up-regulation of PECAM and VEGFR2 genes by hypoxia indicating that these genes are regulated by the HIF-1α-mediated transcription. B. Quantitative PCR experiments performed on the HT1080/Scr and HT/shNRP-1 cells incubated under hypoxia show that hypoxia failed to up-regulate the expression of PECAM, VEGF 165 as well as VEGFR-2 in the HT/shNRP-1 cells, indicating that the hypoxia-induced angiogenic program in the HT1080 cells is controlled by NRP-1. C. Tubule formation on matrigel by HT1080/Scr and HT/shNRP-1 cells under normoxia and hypoxia is depicted. The HT/shNRP-1 cells failed to form tubules even after priming with hypoxia (lower right hand panel), indicating that NRP-1 expression in critical for the enhanced tubule formation by the hypoxia-primed HT1080 cells. The hypoxia-primed wild type cells formed robust tubules as seen in the earlier experiments (upper right hand panel). D. HT/flNRP-1 cells form dense tubules even without the hypoxia-priming. The tubule formation by these cells started very early (2 hours) and became very dense by 6 hours (upper right hand panel). After 6 hours, the tubules collapsed as the HT/flNRP-1 cells invaded the matrigel vigorously (lower panel) forming a monolayer in the well. NRP-1 controls the tumorigenic properties of HT1080 cells. E. Matrigel-invasion property of HT1080/Scr cells was compared with that of HT/flNRP-1 and HT/shNRP-1 cells. A representative image of the invaded cells stained with crystal violet is depicted (Original magnification: 100X). Quantification of the invaded cells showed that the HT/flNRP-1 cells possessed significantly enhanced invasive property (N = 3; ** p<0.01) while the HT/shNRP-1 cells showed a significantly reduced invasive ability (N = 3; ***p<0.001). F. Anchorage-independent growth of HT1080/Scr, HT/shNRP-1 and HT/flNRP-1 was examined by performing soft agar colony assay. A representative phase contrast image of the colonies formed by these cells is illustrated (original magnification: 100X). The HT/flNRP-1 cells formed large colonies having loose migrating cells at the border (middle panel), while the HT/shNRP-1 cells formed very small compact colonies. Quantification of the colony formation shows that the number of colonies formed by the HT/flNRP-1 cells was significantly higher (**p<0.01) while that by the HT/shNRP-1 cells was significantly lower (* p<0.05) compared to the HT1080/Scr cells. Data show that NRP-1 expression is necessary for the anchorage-independent growth of HT1080 cells. Data are represented as mean ± S.E.M.

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Expressing, Real-time Polymerase Chain Reaction, Incubation, Staining, Colony Assay

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: HT1080/Scr, HT/flNRP-1 and HT/shNRP-1 cells were injected subcutaneously in the flanks of NOD/SCID mice (9 mice per set) to compare their tumorgenic potential. HT/flNRP-1 cells formed larger tumours (mouse in the middle) compared to those formed by the HT1080/Scr cells (mouse on the left). HT/shNRP-1 cells did not form tumour (mouse on right, 0/9). B. A comparative image of the tumours formed by the HT1080/Scr and the HT/flNRP-1 cells is depicted. The tumours formed by HT1080/Scr and HT/flNRP-1 cells were weighed. The mean weight of the tumours formed by the HT/flNRP-1 was significantly higher than that of the HT1080/Scr cells. (N = 9, ***p<0.001) C. The kinetics of tumour formation by the HT/flNRP-1 cells was significantly higher compared to that of HT1080/Scr cells (**p<0.01, ***p<0.001). D. HT/flNRP-1 tumor sections were subjected to IHC analyses using antibodies to various angiogenic markers like PECAM, VE-Cadherin, VEGF, VEGF 165 , NRP-1 and VEGFR-2. The sections showed a strong expression of all these markers indicating that the HT/flNRP-1 tumours are highly angiogenic (original magnification: 200X). E. Paraffin sections of the tumours formed by the HT1080/Scr and the HT/flNRP-1 were immuno-stained with an anti-PECAM antibody. The HT/flNRP-1 tumours showed a high density of PECAM-positive tube-like structures (indicated by arrows) compared to the HT1080/Scr tumours indicating a high level of angiogenesis (original magnification: 100X) F. The strong nuclear localization of HIF-1α seen in these tumours confirms a high level of in situ hypoxia (original magnification: 200X). Hypoxia-priming enhances the in vivo tumour formation. G. HT1080/Scr and HT/flNRP-1 cells – primed or not with hypoxia – were injected sub-cutaneously in the skin of NOD/SCID mice. The size of the tumours formed by the hypoxia-primed cells was significantly larger that their respective normoxic controls. Quantification of the tumour weights (N = 9) shows that hypoxia-priming significantly enhances the tumour formation by both HT1080/Scr as well as by the HT/flNRP-1 cells (Data are represented as mean± S.E.M., N = 9, ***p<.001 and **p<.01.) H. The kinetics of tumour formation by the hypoxia-primed cells at various time points was significantly higher compared to their normoxic counterparts (*p<0.05, **p<0.01, ***p<0.001).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Injection, Expressing, Staining, In Situ, In Vivo

Journal: PLoS ONE

Article Title: Vasculogenic Mimicry of HT1080 Tumour Cells In Vivo : Critical Role of HIF-1α-Neuropilin-1 Axis

doi: 10.1371/journal.pone.0050153

Figure Lengend Snippet: Quantitative PCR analyses for OCT3/4, c-Myc and KLF4 mRNA shows that hypoxia up-regulated the expression of OCT3/4 and c-Myc while down-regulates that of KLF4. B. Presence of chetomin in the incubation medium of cells incubated under hypoxia resulted in a further up-regulation of the expression of OCT3/4 and c-Myc mRNA indicting that these genes are independent of HIF-1α-mediated transcription and is under the control of a mechanism that is negatively regulated by the HIF-1α-mediated transcription. The down-regulation of KLF4 by hypoxia was however, rescued by chetomin showing that it was a HIF1-α-dependent process. C. Quantification of OCT3/4, c-Myc and KLF4 mRNA in the hypoxia-primed HT1080/Scr and HT/shNRP-1 cells shows that the expression of OCT3/4 and c-Myc is significantly higher in the hypoxia-primed HT/shNRP-1 cells compared to the hypoxia-primed HT1080/Scr cells, showing that the up-regulation of these genes by hypoxia is not only NRP-1 independent, but also gets further enhanced when NRP-1 is silenced.The suppression of KLF4 expression by hypoxia was partially rescued by silencing of NRP-1 indicating the role of HIF-1α-NRP-1 axis in its down-regulation by hypoxia. (Data in all panels are represented as mean ± S.E.M; N = 3 and *** p<0.001, ** p<0.01 and *p<0.5).

Article Snippet: A set of four unique shRNA constructs directed against various regions of human NRP-1 mRNA were purchased from Origene (HuSH 29mer shRNA constructs against human NRP-1, Origene technologies, Inc.; Rockville, MD, USA).

Techniques: Real-time Polymerase Chain Reaction, Expressing, Incubation

Journal: Biomedicines

Article Title: Apabetalone Downregulates Fibrotic, Inflammatory and Calcific Processes in Renal Mesangial Cells and Patients with Renal Impairment

doi: 10.3390/biomedicines11061663

Figure Lengend Snippet: Apabetalone blocks TGF-β1-induced HRMC activation. ( A ): HRMCs were treated with TGF-β1 ± BETi or TGFBRi for 24 h followed by gene expression analysis by real-time PCR ( n = 5). ( B ): Representative images of HRMCs treated with TGF-β1 ± apabetalone for 48 h, followed by immunofluorescence microscopy for α-SMA (green); nuclei were stained with DAPI (blue); apa = apabetalone. ( C ): Fluorescence intensity of α-SMA was quantified as percent of the image area. ( D ): Collagen gel contraction was evaluated after 4 days of treatment ( n = 6). ( E ) Collagen deposition was evaluated by picrosirius red staining after 5 days of treatment ( n = 4). Data in bar graphs are the mean ± SD. Statistical analysis by one-way ANOVA followed by Dunnett’s Multiple Comparison Test. *** p < 0.001, ** p < 0.01, NS not significant. ACTA2: α-SMA gene. α-SMA: alpha smooth muscle actin. TGF-β1: Transforming growth factor β1. Apabetalone: BD2-selective BET inhibitor. JQ1: pan-BET inhibitor. MZ1: PROTAC that directs BET proteins for degradation. TGFBRi: small molecule inhibitor of the TGF-β receptor.

Article Snippet: Collagen was stained with picrosirius red solution (Abcam # ab246832) for 60 min, followed by destaining with 0.1 N HCl.

Techniques: Activation Assay, Expressing, Real-time Polymerase Chain Reaction, Immunofluorescence, Microscopy, Staining, Fluorescence

Journal: bioRxiv

Article Title: Electrochemical Metabolic Profiling Reveals Mitochondrial Hyperactivation and Enhanced Neural Progenitor Proliferation in MLC1-Mutant Human Cortical Organoids

doi: 10.1101/2025.04.16.647364

Figure Lengend Snippet: (A) Sanger sequencing showing the homozygous c.824C>A (p.Ala275Asp) mutation in exon 10 of the MLC1 gene in patient-derived hiPSCs. (B) Karyotyping analysis confirming normal chromosomal integrity in both control and MLC1 mutant hiPSCs. (C) Representative bright-field images showing typical colony morphology of control and MLC1 mutant hiPSCs. (D) qPCR analysis showing comparable expression of pluripotency markers (OCT4, SOX2, NANOG, LIN28A) between control and MLC1 mutant lines (PBMC, n=4; Control, n=6; MLC1 mutant, n=6). (E) Immunofluorescence staining of OCT4, SOX2, NANOG, and SSEA-4 confirming pluripotency in both lines. Scale bars, 50 µm. (F) Immunostaining for PAX6 (ectoderm), SM22A (mesoderm), and FOXA2 (endoderm) in control and MLC1 mutant hiPSCs following directed differentiation. Scale bars, 20 µm. (G) Schematic showing human cortical organoid (hCO) generation from hiPSCs.(H) Western blot showing temporal expression of MLC1 protein in control and MLC1 mutant hCOs from day 15 to day 90. (I) Densitometric quantification of MLC1 protein levels normalized to β-actin, showing similar expression kinetics between groups (n=4). (J) Immunofluorescence of MLC1 and N-cadherin in day 30 hCOs. MLC1 is localized to neuroepithelial cells in both control and MLC1 mutant lines. Scale bar, 50 µm. (K) Quantification of MLC1⁺/N-cadherin⁺ immunoreactive area as a percentage of total hCO cross-sectional area, showing no significant difference between control (n=12) and MLC1 mutant hCOs (n=11). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data are presented as mean ± S.E.M.

Article Snippet: The membrane was then blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-MLC1 (1:1000, ARP35249-P050, Aviva Systems Biology), anti-glutathione peroxidase 1 (GPX1, 1:1000, LF-PA0019, Lab Frontier), anti-glutathione peroxidase 4 (GPX4, 1:1000, LF-PA0055, Lab Frontier), anti-catalase (1:1000, ab52477, Abcam), anti-superoxide dismutase 1 (SOD1, 1:500, MA1-105, Invitrogen), anti-superoxide dismutase 2 (SOD2, 1:500, sc-18503, Santa Cruz Biotechnology), OPA1 (1:1000, 612606, BD Transduction Laboratories), DRP1 (1:1000, 611112, BD Transduction Laboratories) and MFN2 (1:1000, ab56889, Abcam) antibodies.

Techniques: Sequencing, Mutagenesis, Derivative Assay, Control, Expressing, Immunofluorescence, Staining, Immunostaining, Western Blot

Journal: bioRxiv

Article Title: Electrochemical Metabolic Profiling Reveals Mitochondrial Hyperactivation and Enhanced Neural Progenitor Proliferation in MLC1-Mutant Human Cortical Organoids

doi: 10.1101/2025.04.16.647364

Figure Lengend Snippet: (A) Fabrication and EC detection of the MAGO (MAtrigel-coated GOld nanostructured) platform designed for in situ monitoring of mitochondrial activity in hCOs. (B-D) Characterization of the ITO/gold nanostructure (Control) and ITO/gold nanostructure/Matrigel (MAGO) platform using (B) FE-SEM images, (C) AFM analysis, and (D) conductive AFM (c-AFM) analysis. (E) Root mean square roughness (Rq) analysis for Control and MAGO platform groups (n=10). (F) Quantified results from the c-AFM images (D). (G) DPV graphs from MLC1 mutant organoids on Control and MAGO platform (left-panel) and the quantified DPV results presented as a bar graph (right-panel) (n=6). (H) DPV results with varying numbers of hCOs, ranging from 1 hCO to 4 hCOs (left-panel) and the quantified results presented as a bar graph (right-panel) (n=3). (I) DPV results from normal hCO (control) and MLC1 mutant hCO (left-panel) and the quantified results (n=7). (J) Scheme depicting the time-dependent monitoring of hCOs and MLC1 mutant models. (K) DPV graphs from Control (left-panel) and MLC1 mutant groups (right-panel) for 90 days. (L) Quantified DPV results presented as a line graph (n=5). (M) Cell viability tests after DPV detection presented as a line graph (n=5). (N) Representative DAPI images of control and MLC1 mutant hCOs at day 30, day 60, and day 90. Scale bar, 200 µm. (O) Mean hCO cross-sectional area of control and MLC1 mutant hCOs showing growth plateau beyond day 60, dissociating metabolic signals from physical expansion (Control: D30, n=12; D60, n=12, D90, n=10; MLC1 mutant: D30, n=12; D60, n=12, D90, n=6). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data are presented as mean ± S.E.M.

Article Snippet: The membrane was then blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-MLC1 (1:1000, ARP35249-P050, Aviva Systems Biology), anti-glutathione peroxidase 1 (GPX1, 1:1000, LF-PA0019, Lab Frontier), anti-glutathione peroxidase 4 (GPX4, 1:1000, LF-PA0055, Lab Frontier), anti-catalase (1:1000, ab52477, Abcam), anti-superoxide dismutase 1 (SOD1, 1:500, MA1-105, Invitrogen), anti-superoxide dismutase 2 (SOD2, 1:500, sc-18503, Santa Cruz Biotechnology), OPA1 (1:1000, 612606, BD Transduction Laboratories), DRP1 (1:1000, 611112, BD Transduction Laboratories) and MFN2 (1:1000, ab56889, Abcam) antibodies.

Techniques: In Situ, Activity Assay, Control, Mutagenesis

Journal: bioRxiv

Article Title: Electrochemical Metabolic Profiling Reveals Mitochondrial Hyperactivation and Enhanced Neural Progenitor Proliferation in MLC1-Mutant Human Cortical Organoids

doi: 10.1101/2025.04.16.647364

Figure Lengend Snippet: (A) Intracellular ATP levels measured under galactose-based conditions at day 60. MLC1 mutant hCOs show elevated ATP levels, which are suppressed by oligomycin, indicating enhanced OXPHOS-dependent ATP production (Control, n=6; MLC1 mutant, n=5). (B) Mitochondrial membrane potential assessed via TMRM fluorescence by flow cytometry. Basal TMRM signal is reduced in MLC1 mutant hCOs and completely abolished by CCCP (Control, n=6; MLC1 mutant, n=5). (C) Intracellular ROS levels measured using H₂DCFDA fluorescence. MLC1 mutant hCOs display elevated basal ROS levels, which are attenuated by NAC and enhanced by H₂O₂ (Control, n=6; MLC1 mutant, n=5). (D) Western blot analysis of antioxidant enzymes. GPX4 and catalase are upregulated, while SOD2 is downregulated in MLC1 mutant hCOs relative to controls. (E) Densitometric quantification of protein expression from (D), normalized to β-actin (n=18). (F) Transmission electron microscopy showing elongated mitochondrial morphology in MLC1 mutant hCOs. Scale bar, 1 µm. (G) Western blot of mitochondrial fusion/fission regulators. MFN2 and S-OPA1 are increased in MLC1 mutant hCOs, while L-OPA1 and DRP1 remain unchanged. (H) Quantification of mitochondrial dynamics markers and OPA1 S/L ratio from (G) (MFN2: Control, n=15; MLC1 mutant, n=27, OPA-1: Control, n=15; MLC1 mutant, n=23, DRP1: Control, n=12; MLC1 mutant, n=22). (I) Schematic of EC detection in control and MLC1 mutant hCOs treated with glycolytic and mitochondrial inhibitors. (J) DPV graphs of hCOs and MLC1 mutant hCOs after 24-hour exposure to various concentrations of FCCP, oligomycin, and rotenone & antimycin A. (K) Heatmap depicting electrical signals in hCOs and MLC1 mutant hCOs (n=3) following the administration of mitochondrial inhibitors, derived from DPV graph (J). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data are presented as mean ± S.E.M.

Article Snippet: The membrane was then blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-MLC1 (1:1000, ARP35249-P050, Aviva Systems Biology), anti-glutathione peroxidase 1 (GPX1, 1:1000, LF-PA0019, Lab Frontier), anti-glutathione peroxidase 4 (GPX4, 1:1000, LF-PA0055, Lab Frontier), anti-catalase (1:1000, ab52477, Abcam), anti-superoxide dismutase 1 (SOD1, 1:500, MA1-105, Invitrogen), anti-superoxide dismutase 2 (SOD2, 1:500, sc-18503, Santa Cruz Biotechnology), OPA1 (1:1000, 612606, BD Transduction Laboratories), DRP1 (1:1000, 611112, BD Transduction Laboratories) and MFN2 (1:1000, ab56889, Abcam) antibodies.

Techniques: Mutagenesis, Control, Membrane, Fluorescence, Flow Cytometry, Western Blot, Expressing, Transmission Assay, Electron Microscopy, Derivative Assay

Journal: bioRxiv

Article Title: Electrochemical Metabolic Profiling Reveals Mitochondrial Hyperactivation and Enhanced Neural Progenitor Proliferation in MLC1-Mutant Human Cortical Organoids

doi: 10.1101/2025.04.16.647364

Figure Lengend Snippet: DPV graphs of hCOs (above) and MLC1 mutant hCOs (below), and the quantified DPV results are presented as bar graphs (n=3).

Article Snippet: The membrane was then blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-MLC1 (1:1000, ARP35249-P050, Aviva Systems Biology), anti-glutathione peroxidase 1 (GPX1, 1:1000, LF-PA0019, Lab Frontier), anti-glutathione peroxidase 4 (GPX4, 1:1000, LF-PA0055, Lab Frontier), anti-catalase (1:1000, ab52477, Abcam), anti-superoxide dismutase 1 (SOD1, 1:500, MA1-105, Invitrogen), anti-superoxide dismutase 2 (SOD2, 1:500, sc-18503, Santa Cruz Biotechnology), OPA1 (1:1000, 612606, BD Transduction Laboratories), DRP1 (1:1000, 611112, BD Transduction Laboratories) and MFN2 (1:1000, ab56889, Abcam) antibodies.

Techniques: Mutagenesis

Journal: bioRxiv

Article Title: Electrochemical Metabolic Profiling Reveals Mitochondrial Hyperactivation and Enhanced Neural Progenitor Proliferation in MLC1-Mutant Human Cortical Organoids

doi: 10.1101/2025.04.16.647364

Figure Lengend Snippet: (A, B) Immunofluorescence staining of PAX6 (A) and SOX2 (B) in control and MLC1 mutant hCOs at days 30 and 60, showing enriched expression in rosette structures. Scale bars, 50 µm. (C, D) Quantification of PAX6⁺ and SOX2⁺ immunoreactive areas at days 30 and 60. MLC1 mutant hCOs show significantly expanded progenitor populations (n=12). (E) BrdU pulse-chase assay (24 h pulse on day 30, analysis on day 37) showing increased BrdU⁺ area in MLC1 mutant hCOs (n=12). Scale bar, 50 µm. (F) Schematic of CRISPR-mediated prime editing to introduce the c.824C>A (p.Ala275Asp) mutation into control hiPSCs. (G) Next-generation sequencing confirms >95% biallelic editing in two selected prime editing-derived clones. (H) Representative karyotyping result showing normal chromosomal integrity in prime-edited MLC1 mutant hiPSCs. (I) Immunostaining of OCT4, SOX2, NANOG, and SSEA-4 confirming maintenance of pluripotency in prime-edited lines. Scale bar, 50 µm. (J) Targeted deep sequencing of top 10 predicted off-target sites showing no detectable off-target editing in prime-edited clones compared to untreated controls. (K) BrdU pulse-chase assay (24 h pulse on day 30, analysis on day 37) showing increased BrdU⁺ area in prime-edited MLC1 mutant hCOs (PE-Control, n=5; PE-MLC1 mutant, n=4). Scale bar, 50 µm. (L) DPV graphs of hCOs, MLC1 mutant hCOs, PE-Control hCOs, and PE-MLC1 mutant hCOs (left-panel) and the quantified DPV results (right-panel) (n=6). (M) Cell viability tests after DPV detection are presented as a bar graph (n=3). * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001. Data are presented as mean ± S.E.M.

Article Snippet: The membrane was then blocked with 3% bovine serum albumin in Tris-buffered saline containing 0.05% Tween-20 for 1 h at room temperature and incubated overnight at 4°C with anti-MLC1 (1:1000, ARP35249-P050, Aviva Systems Biology), anti-glutathione peroxidase 1 (GPX1, 1:1000, LF-PA0019, Lab Frontier), anti-glutathione peroxidase 4 (GPX4, 1:1000, LF-PA0055, Lab Frontier), anti-catalase (1:1000, ab52477, Abcam), anti-superoxide dismutase 1 (SOD1, 1:500, MA1-105, Invitrogen), anti-superoxide dismutase 2 (SOD2, 1:500, sc-18503, Santa Cruz Biotechnology), OPA1 (1:1000, 612606, BD Transduction Laboratories), DRP1 (1:1000, 611112, BD Transduction Laboratories) and MFN2 (1:1000, ab56889, Abcam) antibodies.

Techniques: Immunofluorescence, Staining, Control, Mutagenesis, Expressing, Pulse Chase, CRISPR, Introduce, Next-Generation Sequencing, Derivative Assay, Clone Assay, Immunostaining, Sequencing

Journal: Cancers

Article Title: The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

doi: 10.3390/cancers13081957

Figure Lengend Snippet: Identification of RAD51 and MRE11 inhibitors as prexasertib-sensitizing agents in CRC-SCs. ( A ) CRC-SCs previously characterized as resistant to ATR-CHK1 abrogation (RES-CRC-SCs) were left untreated or treated for 72 h with the CHK1/2 inhibitor prexasertib (CHK1i), and/or a set of modulators of the DNA damage response (DDR) with known prexasertib-sensitizing effect (i.e., adavosertib and triapine) or with unknown impact on prexasertib CSC toxicity (i.e., B02, KU-60019, mirin, NU7026, and VE-821), as indicated. Cell proliferation and viability were assessed by CellTiter-Glo ® assay. The heatmap shows prexasertib-sensitizing effects of DDR modulators, with values corresponding to the percentage of viable cells upon normalization on control conditions. Data are means of three independent experiments, with values reported in . The heatmap and clusterization were generated with Python. ( B , C ) Cell proliferation/viability (assessed by CellTiter-Glo ® assay) of distinct RES-CRC-SCs left untreated or exposed for 96 h to CHK1i alone or in combination with RAD51i (B02) ( B ) or MRE11i (mirin) ( C ), as indicated. Results are means±SEM and individual data points of six (RAD51i-treated #19RES), five (RAD51i-treated #1RES), four (MRE11i-treated #1RES), or three (MRE11i-treated #19RES) independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA and Bonferroni or Dunnett’s T3 post-hoc test) as reported. ( D , E ) Cell proliferation/viability (evaluated by CellTiter-Glo ® assay) of representative CRC-SCs sensitive to CHK1i (SENS-CRC-SCs) ( D ) or intrinsically resistant to CHK1i (innRES-CRC-SCs) ( E ) left untreated or subjected for 96 h to CHK1i alone or in combination with MRE11i or RAD51i, as indicated. Results are means ± SEM and individual data points of four (MRE11i-treated SENS-CRC-SCs and #innRES) or three (RAD511i-treated SENS-CRC-SCs) independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA and Bonferroni or Dunnett’s T3 post-hoc test) as reported. .

Article Snippet: Membranes were incubated with primary antibodies directed against MRE11 (1:1000; #sc-22767; RRID:AB_2145247 and #sc-135992; RRID:AB_2145244) from Santa Cruz Biotechnology (Dallas, TX, USA), PARP1 (#9542) from Cell Signaling Technology, RAD51 (1:1000; #sc-398587; RRID: AB_2756353 and #sc-377467) from Santa Cruz Biotechnology, RPA32 (#A303-874A) from Bethyl Laboratories (Montgomery, TX, USA) and pRPA32 (S4/S8) (#A300-245A) from Bethyl Laboratories, as well as with antibodies recognizing β-actin (1:2000; #A5441; RRID:AB_476744), cofilin (#3318, Cell Signaling Technology), nucleolin (1:1000; #14574; Cell Signaling Technology; RRID:AB_2798519), or β-tubulin (#T4026).

Techniques: Glo Assay, Control, Generated

Journal: Cancers

Article Title: The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

doi: 10.3390/cancers13081957

Figure Lengend Snippet: Combined inhibition of CHK1 and MRE11 or CHK1 and RAD51 induces replication stress in prexasertib-resistant CRC-SCs. ( A ) Western-blot analysis of representative RES-CRC-SCs left untreated or administrated for 24 h with prexasertib (CHK1i), either alone or in combination with MRE11i (mirin) or RAD51i (B02), and then stained with antibodies recognizing phospho(p)RPA32 (S4/S8) and RPA32 (as markers of replication stress, RS) and nucleolin (to ensure equal lane loading). One representative western-blot and the quantification of the ratio pRPA32/nucleolin are shown (see also ). Results are expressed as means±SEM and individual data points of five independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal-Wallis test and Dunn’s post-hoc test) compared to untreated conditions. ( B ) Flow cytometry analysis in representative RES-CRC-SCs left untreated or treated with CHK1i, either alone or combined with MRE11i or RAD51i, and then stained with a DNA intercalant (DAPI) together with an anti-γH2AX antibody. Cell cycle profiles and quantitative data (means ± SEM; six independent experiments at 24 h and seven independent experiments at 48 h) are reported. In cell cycle profiles, cells positive for γH2AX in S-phase are in green. Numbers indicate the percentage of corresponding events. In the histograms, the percentage of γH2AX + cells in all cell cycle phases (total) are in dark color, while the percentage of γH2AX + cells in S-phase are in pale color. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal-Wallis test and Dunn’s post-hoc test), as indicated (for γH2AX + cells in all cell cycle phases). # p < 0.05, ## p < 0.01, ### p < 0.001 (Kruskal-Wallis test and Dunn’s post-hoc test), as indicated (for γH2AX + cells in S-phase). ( C ) Immunofluorescence analysis in representative RES-CRC-SCs left untreated or exposed for 24 h to CHK1i, either alone or in combination with MRE11i or RAD51i, and then strained with an antibody recognizing γH2AX. Representative images and quantification of percentages of γH2AX + cells presenting “focal”, “partially diffuse” or “pan-nuclear” γH2AX positivity are shown. For more information about the category of γH2AX positivity, see Materials and Methods. Data are expressed as means±SEM and individual data points of five independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001 (Kruskal-Wallis test and Dunn’s post-hoc test) compared to untreated conditions (for all γH2AX + cells); # p < 0.05, ## p < 0.01, ### p < 0.001 (Kruskal-Wallis test and Dunn’s post-hoc test) compared to untreated conditions (for each distinct category of γH2AX positivity, depicted with the indicated color code). Dose range in ( A – C ): 100 nM CHK1i, 20 µM MRE11i for #19RES or 30 µM MRE11i for #1RES, 7.5 µM RAD51i; a.u., arbitrary units. .

Article Snippet: Membranes were incubated with primary antibodies directed against MRE11 (1:1000; #sc-22767; RRID:AB_2145247 and #sc-135992; RRID:AB_2145244) from Santa Cruz Biotechnology (Dallas, TX, USA), PARP1 (#9542) from Cell Signaling Technology, RAD51 (1:1000; #sc-398587; RRID: AB_2756353 and #sc-377467) from Santa Cruz Biotechnology, RPA32 (#A303-874A) from Bethyl Laboratories (Montgomery, TX, USA) and pRPA32 (S4/S8) (#A300-245A) from Bethyl Laboratories, as well as with antibodies recognizing β-actin (1:2000; #A5441; RRID:AB_476744), cofilin (#3318, Cell Signaling Technology), nucleolin (1:1000; #14574; Cell Signaling Technology; RRID:AB_2798519), or β-tubulin (#T4026).

Techniques: Inhibition, Western Blot, Staining, Flow Cytometry, Immunofluorescence

Journal: Cancers

Article Title: The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

doi: 10.3390/cancers13081957

Figure Lengend Snippet: Combined inhibition of MRE11 and CHK1 or of RAD51 and CHK1 causes an accumulation of premature and damaged mitoses in CRC-SCs resistant to prexasertib. ( A – D ) Cytofluorimetric assessment of cell cycle profiles ( A , B ) or the levels of phospho(p)H3 (S10) ( A , C ) and/or pH3 and γH2AX ( D ) in representative RES-CRC-SC left untreated or treated for 24 h with prexasertib (CHK1i) alone or in combination with MRE11i (mirin) or RAD51i (B02), and then stained with DAPI and the appropriates antibodies. Cell cycle profiles and quantitative data (means ± SEM from six independent experiments) are reported. In ( A ), cells positive for pH3 in S-phase and G 2 /M-phase are in orange and red, respectively. Numbers indicate the percentage of corresponding events. In ( C ), the percentage of premature mitosis corresponds to the percentage of pH3 + cells with a DNA content lower than 4 n among all pH3 + cells, while the percentage of normal mitoses corresponds to the percentage of pH3 + cells with a 4 n DNA content among all cells. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA and Bonferroni or Dunnett’s T3 post-hoc test in ( B ), in ( C ) for the analysis of the percentages of pH3 + cells and of premature mitoses, and in ( D ); Kruskal-Wallis test and Dunn’s post-hoc test in ( C ), for the analysis of the percentages of mitoses), as indicated. ( E ) Immunofluorescence detection of DNA damage in mitosis in RES-CRC-SCs left untreated or exposed for 24 h to CHK1i, alone or in combination with MRE11i or RAD51i, and then strained with DAPI and an antibody recognizing γH2AX. The panel shows representative images of (pro)metaphases (as demonstrated by the classical chromosome condensation in gray scale images) with DNA damage foci. Green numbers refer to numbers of analyzed mitoses (n) and the percentages of γH2AX + mitoses pooled from five independent experiments. Dose range in ( A – E ): 100 nM CHK1i, 20 µM MRE11i for #19RES or 30 µM MRE11i for #1RES, 7.5 µM RAD51i; a.u., arbitrary units.

Article Snippet: Membranes were incubated with primary antibodies directed against MRE11 (1:1000; #sc-22767; RRID:AB_2145247 and #sc-135992; RRID:AB_2145244) from Santa Cruz Biotechnology (Dallas, TX, USA), PARP1 (#9542) from Cell Signaling Technology, RAD51 (1:1000; #sc-398587; RRID: AB_2756353 and #sc-377467) from Santa Cruz Biotechnology, RPA32 (#A303-874A) from Bethyl Laboratories (Montgomery, TX, USA) and pRPA32 (S4/S8) (#A300-245A) from Bethyl Laboratories, as well as with antibodies recognizing β-actin (1:2000; #A5441; RRID:AB_476744), cofilin (#3318, Cell Signaling Technology), nucleolin (1:1000; #14574; Cell Signaling Technology; RRID:AB_2798519), or β-tubulin (#T4026).

Techniques: Inhibition, Staining, Immunofluorescence

Journal: Cancers

Article Title: The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

doi: 10.3390/cancers13081957

Figure Lengend Snippet: The inhibition of MRE11 or RAD51 sensitizes CRC-SCs to prexasertib by inducing a caspase-dependent mitotic catastrophe. ( A , B ) Western-blot analysis of MRE11 and RAD51 levels in SENS-CRC-SCs ( A ) and RES-CRC-SCs ( A , B ) left untreated or administrated with prexasertib (CHK1i), alone ( A , B ) or in combination with MRE11i (mirin) or RAD51i (B02) ( B ) (as indicated), and then stained with the appropriates antibodies. In ( B ), two independent experiments for #1RES ( 1 and 2 ) are shown. Cofilin, nucleolin and β-tubulin were used to ensure equal lane loading. Representative western-blots are reported. Quantifications are shown in . ( C , D ) Immunofluorescence- and flow cytometry-mediated detection of the activation of caspase 3 (CASP3A) in RES-CRC-SCs upon treatment for 24 h ( C ) or 48 h ( D ) with CHK1i, MRE11i, and/or RAD51i as indicated, followed by co-staining with the DNA intercalant Hoechst ( C ) or DAPI ( D ) and an anti-CASP3a antibody. In ( C ), representative images and data quantification are shown. In ( D ), cell cycle profiles (left) and quantification of the percentage of CASP3A + cells among all cells (center) and the relative percentage of CASP3A + cells in G 1 -, S-, G 2 /M-phase (right) are illustrated. In cell cycle profiles, positivity for CASP3A in G 1 -, S-, G 2 /M-phase is depicted in pale blue, violet and dark blue, respectively. Numbers indicate the percentages of corresponding events. In the histograms, results are expressed as means ± SEM from three or seven independent experiments in ( C , D ), respectively. * p < 0.05, ** p < 0.01, *** p < 0.001 (one-way ANOVA and Bonferroni post-hoc test) compared to untreated conditions. ( E ) Western-blot analysis in RES-CRC-SCs treated for 48 h as indicated, using an antibody directed against PARP1, also recognizing the cleaved (c) form. β-Actin was used to monitor equal lane loading. Representative western-blots are shown (see also ). Quantification of data, expressed as means ± SEM, and individual data points are from three independent experiments. cPARP1, cleaved PARP1. Dose range in A – E : 100 nM CHK1i, 20 µM MRE11i for #19RES or 30 µM MRE11i for #1RES, 15 µM Q-VD-Oph, 7.5 µM RAD51i; a.u., arbitrary units. .

Article Snippet: Membranes were incubated with primary antibodies directed against MRE11 (1:1000; #sc-22767; RRID:AB_2145247 and #sc-135992; RRID:AB_2145244) from Santa Cruz Biotechnology (Dallas, TX, USA), PARP1 (#9542) from Cell Signaling Technology, RAD51 (1:1000; #sc-398587; RRID: AB_2756353 and #sc-377467) from Santa Cruz Biotechnology, RPA32 (#A303-874A) from Bethyl Laboratories (Montgomery, TX, USA) and pRPA32 (S4/S8) (#A300-245A) from Bethyl Laboratories, as well as with antibodies recognizing β-actin (1:2000; #A5441; RRID:AB_476744), cofilin (#3318, Cell Signaling Technology), nucleolin (1:1000; #14574; Cell Signaling Technology; RRID:AB_2798519), or β-tubulin (#T4026).

Techniques: Inhibition, Western Blot, Staining, Immunofluorescence, Flow Cytometry, Activation Assay

Journal: Cancers

Article Title: The Targeting of MRE11 or RAD51 Sensitizes Colorectal Cancer Stem Cells to CHK1 Inhibition

doi: 10.3390/cancers13081957

Figure Lengend Snippet: The cooperation between MRE11+CHK1 and RAD51+CHK1 is essential for the survival of CRC-SCs resistant to prexasertib. ( A ) Live cell microscopy assessment of apoptosis induction in representative RES-CRC-SCs left untreated or exposed for 48 h to prexasertib (CHK1i), MRE11i (mirin) and/or RAD51i (B02) as indicated, and then incubated for 10 min with SYTOX (which incorporates only dead cells) and the DNA dye Hoechst. SYTOX incorporation (as a parameter of regulated cell death activation) was evaluated by live fluorescence microscopy and image analysis. Nuclear fluorescence was used to discriminate spheres (area higher than 1000 pixels) from single cells or small cell aggregates (area lower than 1000 pixels) on the basis of the threshold indicated with a red arrow point (1000 pixels). Spheres were further classified in small spheres (area comprised between 1000 and 3000 pixels) and big spheres (area > 3000 pixels) on the basis of the threshold indicated with a yellow arrow point (approximately 3000 pixels). Green intensity means (i.e., SYTOX incorporation) were quantified in such spheres. Data, expressed as ratios of SYTOX/Hoechst intensity, are a pool of two independent experiments performed on distinct RES-CRC-SCs, and are shown as box-plots with means and individual data points. In the box-plot on the right, spheres are divided in small and big spheres. * p < 0.05, *** p < 0.001 (Kruskal-Wallis ANOVA and Dunn’s post-hoc test). ( B ) Live cell videomicroscopy analysis of one representative RES-CRC-SCs (#19RES) grown as 3D tumorspheres, left untreated or exposed to CHK1i, either alone or in combination with MRE11i or RAD51i. Images were taken every 20 min for up to 67 h (see Materials and Methods). Representative frames of the fate of one sphere per condition are shown, with numbers referring to the time passed from the beginning of the recording. Expulsion of one or more dead/inert cells or of one or more dead/inert cell aggregates are depicted with a red arrow point ( Exp_not viable ), while sphere disaggregation and/or death with a dark arrow point ( Death ). The fate of all spheres analyzed (at least 50 spheres per condition) are represented on the right using the indicated color code. In such “sphere fate profile”, each single sphere is depicted by a hyphen, with the first and last spheres analyzed also illustrated by a number. The following events are included: (i) expulsion of one or more viable cells or cell aggregates ( Exp_viable ; in green), (ii) sphere fusion ( Fusion ; in orange), (iii) expulsion of one or more cells or cell aggregates with an apoptotic or inert morphology ( Exp_not viable ; in red), (iv) sphere disaggregation and/or death ( Death ; in dark). The growth of each sphere was determined by measuring the area of the sphere at the beginning and at the end of the recording, and then calculating the ratio of the latter on the former. We considered as growing spheres only those with a growth ratio higher than 1.3. Growing and not-growing spheres are respectively colored in blue and grey. Numbers indicate the total number of spheres for each condition, counted in two separate videos. In the histogram in the panel, results are expressed as percentages of abnormal spheres (comprising spheres not growing and/or disaggregated/dead spheres) on the top and of spheres undergoing multiple (i.e., more than three) rounds of expulsion of inert/non-viable cells or cell aggregates on the bottom. For more information about the experiments, categories, and exclusion criteria see Materials and Methods. See also Supplementary Videos. Dose range in A–D: 100 nM CHK1i, 20 µM MRE11i for #19RES or 30 µM MRE11i for #1RES, 7.5 µM RAD51i; a.u., arbitrary units. .

Article Snippet: Membranes were incubated with primary antibodies directed against MRE11 (1:1000; #sc-22767; RRID:AB_2145247 and #sc-135992; RRID:AB_2145244) from Santa Cruz Biotechnology (Dallas, TX, USA), PARP1 (#9542) from Cell Signaling Technology, RAD51 (1:1000; #sc-398587; RRID: AB_2756353 and #sc-377467) from Santa Cruz Biotechnology, RPA32 (#A303-874A) from Bethyl Laboratories (Montgomery, TX, USA) and pRPA32 (S4/S8) (#A300-245A) from Bethyl Laboratories, as well as with antibodies recognizing β-actin (1:2000; #A5441; RRID:AB_476744), cofilin (#3318, Cell Signaling Technology), nucleolin (1:1000; #14574; Cell Signaling Technology; RRID:AB_2798519), or β-tubulin (#T4026).

Techniques: Microscopy, Incubation, Activation Assay, Fluorescence