Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: LPS induces an inflammatory response in mouse mammary glands. ( A , B ) Anatomical images of LPS-induced mammary gland tissue damage. ( C , D ) H&E staining and IHC staining for IL-1β, TNF-α, and IL-6. MA: mammary alveolus; MEC: mammary epithelial cell; NEUT: neutrophils; RBC: red blood cell. ( E , F ) Relative protein and mRNA expression levels of IL-1β, TNF-α, and IL-6 in mouse mammary tissue (n = 3); the IHC micrographs share identical magnification parameters. A representative scale bar is depicted on the left of panel C. Statistical significance was determined by comparison with the control group (* p < 0.05; ** p < 0.01). Note: CM, clinical mastitis.

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Staining, Immunohistochemistry, Expressing, Comparison, Control

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: LPS-induced inflammation model in MAC-T cells. ( A ) Cell viability of MAC-T cells following 24 h of stimulation with different concentrations of LPS. ( B , C ) Relative protein and mRNA expression levels of IL-1β, TNF-α, and IL-6 in MAC-T cells after 24 h of stimulation with varying LPS concentrations (n = 3). Statistical significance was determined by comparison with the control group (* p < 0.05; ** p < 0.01; ns, not significant).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Expressing, Comparison, Control

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: The inhibition of ARPC3/ARPC4 regulates inflammatory factor expression. ( A ) Western blot analysis of the relative protein expression levels of IL-1β, TNF-α, IL-6, and IL-8 following CK666 treatment. ( B ) qRT-PCR analysis of the relative mRNA expression levels of IL-1β , TNF-α , IL-6 , and IL-8 after CK666 treatment (n = 3). Statistical significance was determined by comparison with the LPS + DMSO group (** p < 0.01).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Comparison

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis.

doi: 10.3390/biom15040538

Figure Lengend Snippet: Figure 5. The effect of LPS treatment on the expression of apoptosis-related factors in MAC-T cells. (A–C) Relative mRNA (A) and protein (B,C) expression levels of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in MAC-T cells following LPS treatment. (D–F) Relative mRNA (D) and protein (E,F) expression levels of Caspase3, Caspase7, Caspase8, Bax, and Bcl-2 in mouse mammary tissue following LPS treatment (n = 3). Statistical significance was determined by comparison with the control group (* p < 0.05; ** p < 0.01). Note: CM, clinical mastitis.

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 ◦C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Expressing, Comparison, Control

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis.

doi: 10.3390/biom15040538

Figure Lengend Snippet: Figure 7. Effect of ARPC3/ARPC4 inhibition on HSP70 expression and cell apoptosis. (A–C) Relative protein and mRNA expression levels of ARPC3/ARPC4, HSP70, Caspase3, and Caspase7 in LPS- induced MAC-T cells following CK666 treatment. (D) Flow cytometry analysis of apoptosis rates in cells treated with CK666 and LPS. (E) Localization of ARPC3/ARPC4 expression in MAC-T cells (scale: 120 µm) (n = 3). All micrographs share identical magnification parameters. A representative scale bar (120 µm) is depicted in the higher left corner of panel E. Statistical significance was determined by comparison with the LPS + DMSO group ( ** p < 0.01).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 ◦C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Inhibition, Expressing, Flow Cytometry, Comparison

Journal: bioRxiv

Article Title: ARPC5 Isoforms Drive Distinct Arp2/3-dependant Actin Remodeling Events in CD4 T Cells

doi: 10.1101/2022.01.25.477674

Figure Lengend Snippet: (A) Representative immunoblots shows induction of phospho levels of DNA damage sensor CHK-1 in JNLA.GFP cells upon replication stress induction by Aphidicolin (15µM, APH) for 3h. Membranes were first probed with phospho specific antibodies, followed by stripping and re-probing with antibodies against total protein and GAPDH. Immunoblots are representative of three independent experiments. (B) Single cell tracking of 10 cells per condition (denoted by different colors for NTC, C5 or C5L KO) for the entire timeframe of 5h post pre-treatment with APH, shows the APH mediated NFA kinetics in KO and control JNLA cells. (C) shows relative comparison (Fold change, FC) of cells forming either NFA or AR, respectively, in Control and KO JNLA cells upon two different modes of T cell activation i.e., activation with PMA+Ionomycin (P/I) or on anti CD3/28 coated coverslips. Bars represent mean values from three independent experiments and error bars were calculated from mean±SD of 3 independent experiments where at least 30 cells were analyzed per condition per experiment for NFA quantification and more than 100 cells per condition per experiment were analyzed for AR quantification. (D) Blocking of the calcium signalling pathway downstream of Calmodulin, using inhibitors STO609, KN93 and KN62, respectively on JNLA cells, followed by induction of replication stress with APH does not impair the replication stress mediated NFA bursts. 30 cells/ condition were analyzed. Statistical significance was calculated using One-way ANOVA (multiple comparison) or Welch’s t-test according to the experimental setup. * P ≤ 0.0332, ** P ≤ 0.0021, ***P ≤ 0 . 0002, ****P ≤ 0 . 000021 and ns: not significant.

Article Snippet: Other antibodies used were mouse-anti-ARP3, 1:10,000 (cloneFMS338, SIGMA), mouse-anti-GAPDH, 1:2500 (G9, Santa Cruz), mouse anti-p16-ARC, 1:500 (#305011, Synaptic systems & sc-166760, SCBT), rabbit anti-ARPC5L, 1:1000 (GTX120725 GeneTex and 22025-1-AP Proteintech), rabbit anti-ARPC1A, 1:500 (#HPA004334, Sigma), mouse anti-ARPC1B, 1:500 (SCBT), rabbit anti-ARPC2, 1:1000 (EPR8533 Abcam), mouse anti-ARPC3, 1:500 (#HPA006550, Sigma Aldrich), mouse anti-ARPC4, 1:500 (#NBP1-69003, Novus Biologicals), mouse anti-mCherry, 1:1000 for WB and 1:500 for IF (NBP1-96752), rabbit anti-mCherry, 1:1000 for WB and 1:500 for IF (ab167453), rabbit anti-pTyr, 1:100 (#sc18182, SCBT), rabbit anti-pSLP76, 1:1000 (#ab75829, Abcam), HRP-coupled secondary rabbit or mouse antibodies for immunoblotting were obtained from Jackson Immuno Research was used at a dilution of 1:5000 for all samples.

Techniques: Western Blot, Stripping Membranes, Single Cell Tracking, Control, Comparison, Activation Assay, Blocking Assay

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: LPS induces an inflammatory response in mouse mammary glands. ( A , B ) Anatomical images of LPS-induced mammary gland tissue damage. ( C , D ) H&E staining and IHC staining for IL-1β, TNF-α, and IL-6. MA: mammary alveolus; MEC: mammary epithelial cell; NEUT: neutrophils; RBC: red blood cell. ( E , F ) Relative protein and mRNA expression levels of IL-1β, TNF-α, and IL-6 in mouse mammary tissue (n = 3); the IHC micrographs share identical magnification parameters. A representative scale bar is depicted on the left of panel C. Statistical significance was determined by comparison with the control group (* p < 0.05; ** p < 0.01). Note: CM, clinical mastitis.

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Staining, Immunohistochemistry, Expressing, Comparison, Control

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: LPS-induced inflammation model in MAC-T cells. ( A ) Cell viability of MAC-T cells following 24 h of stimulation with different concentrations of LPS. ( B , C ) Relative protein and mRNA expression levels of IL-1β, TNF-α, and IL-6 in MAC-T cells after 24 h of stimulation with varying LPS concentrations (n = 3). Statistical significance was determined by comparison with the control group (* p < 0.05; ** p < 0.01; ns, not significant).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Expressing, Comparison, Control

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: The JC-1 fluorescent probe was used to detect the effect of inhibiting ARPC3/ARPC4 on the mitochondrial membrane potential of MAC-T cells (scale: 180 μm). All micrographs share identical magnification parameters. A representative scale bar (180 μm) is depicted in the higher left corner of the panel. Statistical significance was determined by comparison with the DMSO group (* p < 0.05; ** p < 0.01).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Membrane, Comparison

Journal: Biomolecules

Article Title: Targeted Regulation of HSP70 by the ARP2/3 Complex in Mammary Epithelial Cells and Its Impact on Host Cell Apoptosis

doi: 10.3390/biom15040538

Figure Lengend Snippet: The inhibition of ARPC3/ARPC4 regulates inflammatory factor expression. ( A ) Western blot analysis of the relative protein expression levels of IL-1β, TNF-α, IL-6, and IL-8 following CK666 treatment. ( B ) qRT-PCR analysis of the relative mRNA expression levels of IL-1β , TNF-α , IL-6 , and IL-8 after CK666 treatment (n = 3). Statistical significance was determined by comparison with the LPS + DMSO group (** p < 0.01).

Article Snippet: The membranes were blocked with rapid Sealing Solution (P0222, Beyotime, Shanghai, China) for 10–20 min at room temperature and incubated 10–12 h at 4 °C with primary antibodies against ARPC3, ARPC4, Bax, Bcl-2, HSPA1A, HSPA1L, Caspase7, IL-1β, TNF-α, IL-6, β-actin (Proteintech, Wuhan, China), IL-8 (Affinity Biosciences, Cincinnati, OH, USA), Caspase3, and Caspase8 (Bioss, Beijing, China).

Techniques: Inhibition, Expressing, Western Blot, Quantitative RT-PCR, Comparison