Journal: Immunity, Inflammation and Disease

Article Title: Human umbilical cord mesenchymal stem cells improve disease characterization of Sjogren's syndrome in NOD mice through regulation of gut microbiota and Treg/Th17 cellular immunity

doi: 10.1002/iid3.1139

Figure Lengend Snippet: Umbilical cord mesenchymal stem cells (UC‐MSCs) and UC‐MSCs and derived exosomes (UC‐MSCs‐exo) improved disease characterization of Sjogren's syndrome (SS) in Nonobese Diabetic (NOD) mice by affecting co‐cultured T cells. (A) Saliva flow. (B) The levels of Ro/SSA antibody and α‐Fodrin immunoglobulin A were detected by enzyme‐linked immunosorbent assay (ELISA). (C) Pathological changes of the submandibular gland and intestine were observed by hematoxylin‐eosin staining. Scale bar: 100 μm (up), 25 μm (down). (D) The ratio of Treg/Th17 cells in the spleen was determined by flow cytometry. (E) The levels of interferon‐gamma (IFN‐γ), interleukin (IL)‐2, IL‐6, IL‐10, IL‐17, Foxp3, lipopolysaccharide, tumor necrosis factor‐alpha, and transforming growth factor‐β1 were analyzed by ELISA. * p < .05 versus Model, # p < .05 versus UC‐MSCs, and p < .05 versus UC‐MSCs‐exo.

Article Snippet: The levels of IFN‐γ (KE10001; Proteintech), IL‐2 (CSB‐E04627m; CUSABIO), IL‐6 (KE10007; Proteintech), IL‐10 (CSB‐E04594m; CUSABIO), IL‐17 (KE10020; Proteintech), prostaglandin E2 (PGE2, YJ028719; MLBIO), transforming growth factor‐beta1 (TGF‐β1, KE10005; Proteintech), SS‐related antigen A (Ro/SSA) antibody (69‐20401; MSKBIO), α‐Fodrin immunoglobulin A (IgA) (ml001942; MLBIO), Foxp3 (YJ037859; MLBIO), lipopolysaccharide (LPS, CSB‐E13066m; CUSABIO), and TNF‐α (CSB‐E04741m; CUSABIO) were determined by ELISA kits.

Techniques: Derivative Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Staining, Flow Cytometry

Journal: The Journal of Biological Chemistry

Article Title: A surge of cytosolic calcium dysregulates lysosomal function and impairs autophagy flux during cupric chloride–induced neuronal death

doi: 10.1016/j.jbc.2023.105479

Figure Lengend Snippet: Cytosolic Ca 2+ -dependent impairment of autophagy flux in MN9D cells following CuCl 2 treatment. A , immunoblot analyses were performed using anti-CB300 antibody using cell lysates obtained from stable MN9D/Neo and MN9D/CaBP cells. Anti-GAPDH antibody was utilized as a loading control. B – O , MN9D/Neo or MN9D/CaBP cells were treated with or without 250 μM CuCl 2 for 15 h. B , cells were stained with 3 μM Fluo-3 ( green ). Representative confocal images are provided. Nuclei were counterstained with Hoechst 33258 ( blue ). Merged images are shown to the right . Scale bar represents 20 μm. C , MTT reduction assay was performed to assess cell viability, which was expressed as a percentage over untreated matching control (value = 1). Data are shown as the mean ± S.D. of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001. D , immunoblot analyses were performed using anti-LC3, anti-fodrin, or anti-p62 antibodies. The calpain-cleaved band of fodrin (c-fodrin) was detected by an anti-fodrin antibody. Relative intensities of LC3-II ( E ) and p62 ( F ) signals were measured using ImageJ software, normalized by the intensity of GAPDH signal, and expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗∗ p < 0.001; NS, not significant. G , immunocytochemical analyses were performed using anti-LC3 ( green ) and anti-p62 ( red ), and nuclei were counterstained with Hoechst 33258 ( blue ). Representative confocal images are provided. Merged images are shown to the right . Scale bar represents 10 μm. The number ( H ) and area ( I ) of LC3 and area of p62 ( J ) puncta per cell were quantified using ImageJ software. Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. ∗∗ p < 0.01; ∗∗∗ p < 0.001. K , cellular lysates were subjected to immunoblot analyses using the indicated antibodies. After normalization against the intensity of total protein, relative intensities of the phosphorylated forms of mTOR ( L ), p-AMPK ( M ), p-Akt ( N ), and p-p70S6K ( O ) are expressed as fold change relative to untreated control (value = 1). Data are shown as the mean ± S.D of three independent experiments. Two-way ANOVA followed by Tukey’s post hoc test was performed. (( N ) ANOVA p value is 0.229215) ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001; NS, not significant.

Article Snippet: The following primary antibodies were used: rabbit anti-LC3 antibody (Cell Signaling Technology, 2775), mouse anti-fodrin antibody (ENZO Life Sciences, BML-FG6090), guinea pig anti-p62/SQSTM1 antibody (Progen, GP62-C), rabbit anti-p-mTOR antibody (Cell Signaling Technology, 2971), rabbit anti-mTOR antibody (Cell Signaling Technology, 2972), rabbit anti-p-p70S6K antibody (Cell Signaling Technology, 2708), rabbit anti-p70S6K antibody (Cell Signaling Technology, 9234), rabbit anti-p-AMPK antibody (Cell Signaling Technology, 2535), rabbit anti-AMPK antibody (Cell Signaling Technology, 2603), rabbit anti-p-Akt antibody (Cell Signaling Technology, 4060), rabbit anti-Akt antibody (Cell Signaling Technology, 4685), rat anti-LAMP1 antibody (Developmental Studies Hybridoma Bank, 1D4B), rabbit anti-cathepsin D antibody (Santa Cruz, sc-10725), mouse anti-NeuN antibody (Sigma Aldrich, MAB377), mouse anti-Calbindin D28K antibody (Swant, 300) and rabbit anti-GAPDH antibody (Bethyl, A300–641A).

Techniques: Western Blot, Staining, MTT Reduction Assay, Software

Journal: Frontiers in Cellular Neuroscience

Article Title: Prevention of noise-induced hearing loss by calpain inhibitor MDL-28170 is associated with upregulation of PI3K/Akt survival signaling pathway

doi: 10.3389/fncel.2023.1199656

Figure Lengend Snippet: MDL-28170 treatment inhibits noise-induced α-fodrin activation in the cochlear tissues. (A) Representative immunoblots from whole cochlear homogenates revealed 240 kDa α-fodrin and the cleaved 150 kDa fragment 3 h after noise exposure. GAPDH (37 kDa) was used as the sample loading control. (B,C) Semi-quantifications of each band density show that the ratio of cleaved to full α-fodrin increased with noise exposure and MDL treatment prevented such an increase (B) , while the cleaved band density divided by GAPDH was not different among the three groups (C) . Data are presented as means + SD; n = 3 in each group with one mouse (two cochleae) per sample, * p < 0.05. (D) Representative images were taken from the basal turn of cochlear epithelia at the time point 3 h after noise exposure. MDL treatment alone showed similar immunolabeling intensity for cleaved α-fodrin (red) in OHCs as the DMSO-treated group. Noise exposure significantly increased cleaved α-fodrin in OHCs, whereas MDL treatment prevented such effects. Phalloidin (green) was a counterstain for visualization of OHCs. The enlarged OHCs allow for better visualization of the immunolabeling. Scale bar = 10 μm. (E) Confocal images used to compare fluorescence intensity by semi-quantification of immunolabeling for cleaved α-fodrin in OHCs were acquired under the same settings and the same processing enhancement for the captured images was used. The person analyzing images was blind to the treatment groups. It confirmed a significant increase after noise exposure, whereas MDL treatment prevented such effects. Data are presented as means + SD; the n is indicated in the bar for each condition with use of one cochlea per mouse. ** p < 0.01, **** p < 0.0001.

Article Snippet: The specimens were first permeabilized in 2% Triton X-100 solution and then blocked with 10% normal goat serum for 30 min each step at room temperature, followed by incubation with primary antibodies: polyclonal rabbit anti-Calpain I (Abcam, Cambridge, UK, #ab39170, 1:50), polyclonal rabbit anti-Calpain II (Abcam #ab39165, 1:50), cleaved α-fodrin (Cell Signaling Technology, Danvers, MA, USA, #2121, 1:50), monoclonal rabbit anti-PI3K, p85α (Millipore, Burlington, MA, USA, #04-403, 1:50), monoclonal rabbit anti-p-Akt (Ser473) (Cell Signaling Technology #4060, 1:50) at 4°C for overnights (24–48 h).

Techniques: Activation Assay, Western Blot, Control, Immunolabeling, Fluorescence