Journal: bioRxiv

Article Title: HAUSP Stabilizes SOX2 through Deubiquitination to Maintain Self-renewal and Tumorigenic Potential of Glioma Stem Cells

doi: 10.1101/2021.06.09.447550

Figure Lengend Snippet: (A) Immunoblot analyses of differentiated cell markers TUJ1 (a neuronal marker) and GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and harvested for immunoblot analyses with specific antibodies against TUJ1, GFAP, HAUSP, and GAPDH (loading control). (B) Quantitative PCR (RT-PCR) analyses of TUJ1 and GFAP mRNA levels in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for the indicated days, and harvested for RT-PCR analyses. Student t test was used to assess the significance. ***, p<0.001. ( C and D ) Immunofluorescent staining of TUJ1 (a neuronal marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against TUJ1 (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of TUJ1-positive cells were shown (D). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001. ( E and F ) Immunofluorescent staining of GFAP (an astrocyte marker) in GSCs expressing shHAUSP or shNT (control). GSCs (T4121) were transduced with shHAUSP or shNT through lentiviral infection for 48 hours, then cultured in the medium with reduced growth factors for indicated days, and immunostained with a specific antibody against GFAP (in green) and counterstained with DAPI to indicate nuclei (in blue). Scale bar, 30 µm. Quantifications of GFAP-positive cells were shown (F). Student t test was used to assess the significance. ns, no significant difference; ***, p<0.001.

Article Snippet: Specific antibodies against HAUSP (Bethyl, A300-033A), SOX2 (Bethyl, A201-741A; or Santa Cruz, SC-365964), OLIG2 (R&D System, AF2418), GFAP (Biolegend, 644702), TUJ1 (Biolegend, 801201), MAP2 (Biolegend, 801801), GAPDH (R&D System, MAB5718), HA (Sigma, 11867423001), α-Tubulin (Sigma, T6199), Flag (Sigma, F1804), Ubiquitin (Biolegend, 646302), Cleaved Caspase 3 (Cell Signaling, 9661), Cleaved PARP (Cell Signaling, 5625), and anti-Flag-agarose (Sigma, A2220) were used for immunoblot and/or immunoprecipitation.

Techniques: Western Blot, Marker, Expressing, Control, Transduction, Infection, Cell Culture, Real-time Polymerase Chain Reaction, Reverse Transcription Polymerase Chain Reaction, Staining

Journal: International Journal of Molecular Sciences

Article Title: ARPC2 Promotes Pulmonary Fibrosis by Regulating MRTFA Activity Independent of the Canonical ARP2/3 Complex

doi: 10.3390/ijms27062729

Figure Lengend Snippet: ARPC2 knockdown inhibits TGF-β1 induced fibrotic response in MRC-5 cell. MRC-5 cells were infected with lentiviruses expressing shRNA targeting ARPC2 or ACTR2 mRNA, followed by 72-h incubation under serum-starved conditions with TGF-β1 to evaluate fibrotic attenuation. ( a ) Western blot analysis using primary antibodies against of ACTA2, COL4A1, ARPC2, ACTR2, and GAPDH. Representative bands are shown in the left panel. Quantification of fibrosis markers (right panel) is normalized to the GAPDH and compared to TGF-β1-treated group (100%). Mean ± SD ( n = 3; ** p < 0.01). ( b ) RNA levels of ACTA2 , COL4A1 , ARPC2 and ACTR2 were normalized to the GAPDH and compared to TGF-β1-treated group (100%). Mean ± SD ( n = 3; * p < 0.05; ** p < 0.01). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Primary antibody incubations with anti-MRTFA (Proteintech, Rosemont, IL, USA, 21166-1-AP) and GAPDH (Bio-Rad, Hercules, CA, USA, MCA4740) were performed overnight at 4 °C, followed by three washes in 0.1% TBS-T. Incubations with fluorochrome-labeled secondary antibodies were performed in Antibody diluent reagent solution (Invitrogen, Waltham, MA, USA, 003218) for 1 h at room temperature.

Techniques: Knockdown, Infection, Expressing, shRNA, Incubation, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: ARPC2 Promotes Pulmonary Fibrosis by Regulating MRTFA Activity Independent of the Canonical ARP2/3 Complex

doi: 10.3390/ijms27062729

Figure Lengend Snippet: Ectopic overexpression of ARPC2 induces fibrotic response in MRC-5 cells. MRC-5 cells were infected with adenoviruses expressing EGFP-fused ARPC2 or ACTR2 and incubated for 72 h under serum-starved conditions to assess fibrotic induction. ( a ) Western blot analysis using primary antibodies against of EGFP, ACTA2, COL4A1, GAPDH, ARPC2, and ACTR2. Representative bands are shown in the left panel. Quantification of fibrosis markers (right panel) is normalized to GAPDH and compared to the control group (100%). Mean ± SD ( n = 4; * p < 0.05; ** p < 0.01). ( b ) RNA levels of ACTA2 , COL4A1 , ARPC2 and ACTR2 were normalized to the GAPDH and compared to the control group (100%). Mean ± SD ( n = 4; * p < 0.05; ** p < 0.01). Statistical significance was determined by one-way ANOVA followed by Dunnett’s multiple comparisons test.

Article Snippet: Primary antibody incubations with anti-MRTFA (Proteintech, Rosemont, IL, USA, 21166-1-AP) and GAPDH (Bio-Rad, Hercules, CA, USA, MCA4740) were performed overnight at 4 °C, followed by three washes in 0.1% TBS-T. Incubations with fluorochrome-labeled secondary antibodies were performed in Antibody diluent reagent solution (Invitrogen, Waltham, MA, USA, 003218) for 1 h at room temperature.

Techniques: Over Expression, Infection, Expressing, Incubation, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: ARPC2 Promotes Pulmonary Fibrosis by Regulating MRTFA Activity Independent of the Canonical ARP2/3 Complex

doi: 10.3390/ijms27062729

Figure Lengend Snippet: Transcriptomic analysis reveals a shared fibrotic regulon between ARPC2 and MRTFA. Transcriptomic analysis was performed in the MRC-5 cells by comparing (i) pre- and post- TGF-β1 treatment groups (control; n = 8 per group) and (ii) lentiviral transduction and TGF-β1 treatment condition ( n = 4 per group). All DEGs were filtered using cut-off values of absolute log 2 fold change > 1 and FDR < 0.01. ( a ) Volcano plot displaying differential gene expression between TGF-β1-treated and unstimulated control cells. Significant DEGs are highlighted in red (upregulated) and blue (downregulated). The top 5 most significantly altered genes and pro-fibrotic genes ( ARPC2 , FN1 , COL1A1 and COL4A1 ) are labeled. ( b ) Venn diagram illustrating the overlap of DEGs between ARPC2- and MRTFA-knockdown groups, categorized by their response to TGF-β1 (upregulated or downregulated). Gene counts for each category are indicated. ( c ) Heatmap depicting the relative expression levels of key fibrotic response genes across all experimental groups. Pre- and post-TGF-β1 treatment groups were indicated by dash line. ( d ) Validation of gene expression profiles from ( c ) using RT-qPCR. Dots represent individual biological replicates. Mean ± SD ( n = 4; * p < 0.05; ** p < 0.01; compared to TGF-β1 treated control). Representative bands of Western blot analysis using primary antibodies against of ARPC2, ACTR2, MRTFA, and GAPDH are shown in the right panel. ( e ) Gene Ontology (GO) enrichment analysis of biological processes associated with the genes commonly downregulated by both ARPC2 and MRTFA depletion.

Article Snippet: Primary antibody incubations with anti-MRTFA (Proteintech, Rosemont, IL, USA, 21166-1-AP) and GAPDH (Bio-Rad, Hercules, CA, USA, MCA4740) were performed overnight at 4 °C, followed by three washes in 0.1% TBS-T. Incubations with fluorochrome-labeled secondary antibodies were performed in Antibody diluent reagent solution (Invitrogen, Waltham, MA, USA, 003218) for 1 h at room temperature.

Techniques: Control, Transduction, Gene Expression, Labeling, Knockdown, Expressing, Biomarker Discovery, Quantitative RT-PCR, Western Blot

Journal: Molecular Neurobiology

Article Title: Comparative Analysis of Red Onion-Derived Exosome-Like Nanovesicles and Extract Reveals Sustained Immunomodulatory Effects in LPS/IFN-γ-Stimulated Microglia

doi: 10.1007/s12035-026-05820-0

Figure Lengend Snippet: Anti-inflammatory and antioxidant properties of RO-ELNs and extract against ATP + LPS/IFN-γ stimulated pro-inflammatory mediators in HMC3 cells. RO-ELNs with a concentration of 1 × 10 9 particles/mL were pretreated for 20 or 6 h before acute or prolonged neuroinflammation models. Extract with a concentration of 125 µg/mL was incubated for only 6 h for both acute and prolonged neuroinflammation models. Western blot and quantitative analyses show the ability of RO-ELNs and extract in inhibiting P-NF-κB ( A , B ) and COX-2 ( C , D ) expression levels in HMC3 cells at different time points 4 and 48 h of neuroinflammation. GAPDH was used as a housekeeping protein. The antioxidative properties of RO-ELNs and extract ( E ) and their ability to decrease IL-6, TNF-α secretion levels ( F , G ) at 4 and 48 h of neuroinflammation. Data are expressed as mean ± standard ( n ≥ 3): * P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001. As a result of the initial reversed loading sequence for COX-2, lanes were rearranged digitally to align with the experimental grouping presented in the paper. All lanes originated from the same membrane and exposure. A separation dash marks the realignment. Uncropped original membranes can be found in Supplementary Figure

Article Snippet: 3033), and GAPDH (Boster, M00227-7) antibodies overnight at 4 °C.

Techniques: Concentration Assay, Incubation, Western Blot, Expressing, Sequencing, Membrane