Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 5. Skeletal muscle autophagy is dysregulated in mKO animals during atrophy. (A) Representative western blots of LC3-I, LC3-II, and SQSTM1 in TA muscles of WT and mKO mice treated with Sal or Col under fed and 48 h fast conditions, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (B-C) Graphical summaries of LC3-II and SQSTM1 flux in WT and mKO animals under fed and 48 h fast conditions. Data are expressed as protein content relative to WT fed (n = 12–16). (D) Representative image from mKO muscle of double-membrane autophagic vacuole (yellow arrow). Scale bar: 200 nm. (E) Graphical summary of total number of autophagic vacuoles in WT and mKO mice treated with Sal or Col under fed and 48 h fast conditions. (F) Heatmap of macroautophagy genes in TA muscle with enrichment unique to carm1 mKO after fasting. Data are expressed relative to WT fed (n = 3). (G) typical western blots of phosphorylated autophagy-related 16 (p-ATG16L1 [Ser278]), total ATG16L1, phosphorylated (p)-MTOR (mechanistic target of rapamycin kinase; Ser2448), total MTOR, p-ULK1 (unc-51 like autophagy activating kinase 1; Ser555), p-ULK1 (Ser757), total ULK1, TFEB (transcription factor EB), SKP2 (S-phase kinase associated protein 2), BECN1, LAMP1

Article Snippet: Antibodies against LC3 (1:1,000; Cell Signaling Technology, 4108S), SQSTM1/p62 (1:1,000; Sigma-Aldrich, P0067), phosphorylated ATG16L1 (Ser278, 1:1,000; Abcam, ab195242), ATG16L1 (1:1,000; Abcam, ab188642), phosphorylated MTOR (Ser2448; 1:1,000; Cell Signaling Technology, 2971S), MTOR (1:1,000; Cell Signaling Technology, 2972S), phosphorylated ULK1 (Ser555; 1:1,000; Cell Signaling Technology, 5869S), phosphorylated ULK1 (Ser757; 1:1,000; Cell Signaling Technology, 14202S), ULK1 (1:1,000; Cell Signaling Technology, 8054S), TFEB (1:1,000; Cell Signaling Technology, 4240S), SKP2 (1:1,000; Cell Signaling Technology, 4313S), BECN1 (1:1,000; Cell Signaling Technology, 3738S), LAMP1 (1:1,000; Abcam, ab24170), LAMP2 (1:1,000; Abcam, ab13524), and CTSD/cathepsin D (1:1,000; Cell Signaling Technology, 2284S) were used to evaluate autophagy.

Techniques: Western Blot, Muscles, Staining, Membrane

Journal: Autophagy

Article Title: CARM1 drives mitophagy and autophagy flux during fasting-induced skeletal muscle atrophy.

doi: 10.1080/15548627.2023.2288528

Figure Lengend Snippet: Figure 7. The impact of fasting on human skeletal muscle fiber size, CARM1 biology, and atrophy- and autophagy-related signaling. (A) Representative images of H&E-stained vastus lateralis QUAD muscle cross sections from healthy male participants collected 1 h postprandial (fed) and following 48 h of fasting (fast). Scale bar: 50 μm. (B) graphical summary of the average myofiber CSA of QUAD muscles from adult humans in response to fasting. Data are expressed as CSA relative to fed (n = 9). (C) distribution of CSA (μm2) in QUAD of fed (solid line) and fast (dashed line) human myofibers (n = 9). (D) heatmap visualizing the average mRNA expression of PRMT family members in QUAD muscle of adult humans during fed and 40 h fast conditions. Microarray data extracted from GSE28016 (n = 7). (E) Representative western blots of CARM1, asymmetric arginine dimethylated CARM1 substrates, SMARCC1 me2a (Arg1064), total SMARCC1, PABPC1 me2a (Arg455/Arg460), total PABPC1, PRKN, BNIP3, LC3-I, LC3-II, p-ATG16L1 (Ser278), total ATG16L1, p-ULK1 (Ser757), and total ULK1 in QUAD muscles from healthy humans in response to 48 h of fasting, accompanied by a typical Ponceau stain. Molecular masses (kDa) are shown at the right of blots. (F-N) graphical summaries of CARM1, CARM1 substrate,

Article Snippet: Antibodies against LC3 (1:1,000; Cell Signaling Technology, 4108S), SQSTM1/p62 (1:1,000; Sigma-Aldrich, P0067), phosphorylated ATG16L1 (Ser278, 1:1,000; Abcam, ab195242), ATG16L1 (1:1,000; Abcam, ab188642), phosphorylated MTOR (Ser2448; 1:1,000; Cell Signaling Technology, 2971S), MTOR (1:1,000; Cell Signaling Technology, 2972S), phosphorylated ULK1 (Ser555; 1:1,000; Cell Signaling Technology, 5869S), phosphorylated ULK1 (Ser757; 1:1,000; Cell Signaling Technology, 14202S), ULK1 (1:1,000; Cell Signaling Technology, 8054S), TFEB (1:1,000; Cell Signaling Technology, 4240S), SKP2 (1:1,000; Cell Signaling Technology, 4313S), BECN1 (1:1,000; Cell Signaling Technology, 3738S), LAMP1 (1:1,000; Abcam, ab24170), LAMP2 (1:1,000; Abcam, ab13524), and CTSD/cathepsin D (1:1,000; Cell Signaling Technology, 2284S) were used to evaluate autophagy.

Techniques: Staining, Muscles, Expressing, Microarray, Western Blot

Journal: International Journal of Biological Sciences

Article Title: Low-intensity pulsed ultrasound promotes skeletal muscle regeneration via modulating the inflammatory immune microenvironment

doi: 10.7150/ijbs.79685

Figure Lengend Snippet: LIPUS regulated macrophage polarization through the WNT pathway in LPS-induced M1 macrophages. (A-B) The changes in protein expression of FZD5 and β-catenin in LPS-stimulated RAW 264.7 cells were exposed and recorded over two times of LIPUS treatment. Relative protein expression of β-catenin was calculated after normalized with Lamin B1. (n=4). *P < 0.05. Relative protein expression of FZD5 was calculated after normalized with GAPDH. (n=4). **P < 0.01. (C) Immunofluorescence staining of FZD5(green) expressed on the cell membrane and Phalloidin (red) of macrophage in different groups after treatment with LIPUS. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The relative fluorescence intensity of FZD5 was compared among different groups. (n=4). *P < 0.05. ***P < 0.001. (D)Images showed Immunofluorescence staining of β-catenin(green) expressed in the nucleus, and Phalloidin (red) of macrophages in different groups after treatment with LIPUS. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The nuclear translocation percentage for β-catenin was compared among different groups. (n=4) *P < 0.05 ***P < 0.001 ****P<0.0001. (E-F) Immunofluorescence staining of β-catenin(green) expressed in the nucleus, and Phalloidin (red) of macrophage in different groups after treating different combinations of UltrasoundH, XAV-939, and QS11. The nuclei were dyed with Dapi (blue). Scale bar=25μm. The nuclear translocation percentage for β-catenin was compared among different groups. (n=4) *P < 0.05 **P < 0.01 ***P<0.001. No significant changes were found when LIPUS and XAV-939 were combined. (G) The gene expression level of CD86, iNOS, ARG1, and CD206 in different groups after treating different combinations of LIPUS, ultrasoundH, XAV-939, and QS11 were detected by qPCR. (n=3)

Article Snippet: Phalloidin , Abcam , ab176757 , Mouse mAb , 1:1000(IF) , .

Techniques: Expressing, Immunofluorescence, Staining, Membrane, Fluorescence, Translocation Assay, Gene Expression

Journal: Redox Biology

Article Title: Tobacco toxins induce osteoporosis through ferroptosis

doi: 10.1016/j.redox.2023.102922

Figure Lengend Snippet: ROS and mitochondrial damage activated AMPK and furtherly promoted CSE-induced ferritinophagy in rBMSCs a. GSEA analysis presented gene expression in mTOR signaling pathway. b. WB showing expression level of p-mTOR, mTOR, p-ULK1, ULK1, p-AMPK and AMPK in rBMSCs treated with 4.5 % CSE for 0, 15, 30, 45 and 60 min. c. WB showing expression level of Ferritin, NCOA4, LC3-II/I and p62 in rBMSCs treated with 4.5 % CSE for 0,15,30,45 and 60 min. d. WB showing expression level of TFRC in rBMSCs treated with 4.5 % CSE for 0,15,30,45 and 60 min and with 0,1.5,3,4.5,6 % CSE for 1h. e. Representative microscopy images of DCFH-DA staining in rBMSCs treated with 4.5 % CSE for 1h, scale bar: 100 μm. f. WB showing expression level of AMPK, Ferritin and LC3-II/I in rBMSCs after 4.5 % CSE treatment for 12h following AMPK knockdown. g. CCK-8 assay and C11-BODIPY 581/591 analysis of rBMSCs after 4.5 % CSE treatment for 12h following AMPK knockdown. h. WB showing expression level of p-ULK1, ULK1, p-AMPK and AMPK in rBMSCs treated with 4.5 % CSE for 0,1,3,6,9,12h. i. Representative TEM images of mitochondria in rBMSC treated with 4.5 % CSE for 3h. Yellow arrows showed swollen mitochondria with severely disrupted cristae. j. Representative fluorescence confocal images of mROS with MitoSox dye in rBMSCs treated with 4.5 % CSE for 3h. Scale bar = 25 μm. k. Flow cytometry of mitochondrial membrane potential labeled by TMRE in rBMSCs treated with 4.5 % CSE for 3h and 12h (n = 3). l. ATP production detection of rBMSCs following 4.5 % CSE treatment for 3h and 12h (n = 3). m. CCK-8 assay (m) and C11-BODIPY 581/591 analysis (n) of rBMSCs treated with 4.5 % CSE for 12h combined with NAC (2 mM) or MitoQ (5 μM) (n = 3). o. Statistical analysis of mean fluorescence intensity (MFI) of TMRE-labeled rBMSCs following 4.5 % CSE treatment for 12h combined with NAC (2 mM) (n = 3) p. WB showing expression level of Slc7a11, GPx4, p62, LC3-II/I, Ferritin, p-AMPK, AMPK, p-ULK1 and ULK1 in rBMSCs treated with 4.5 % CSE combined with NAC (2 mM). q. Schematic diagram illustrated exogenous ROS and iron in CSE firstly induce AMPK-regulated autophagic dependent Fenton reaction, which attacked mitochondria furtherly boost endogenous ROS and aggravated ferroptotic death. *P < 0.05, **P < 0.01, ***P < 0.001 versus control. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The primary antibodies used in this study were listed below: GPx4 (ab125066, 1:1000), Ferritin (ab183781, 1:1000) were obtained from Abcam; LC3A/B (12741, 1:1000), p62 (23214, 1:1000), p62 (5114, 1:1000), ATG5 (12994, 1:1000), Phospho-mTOR (Ser2448) (5536, 1:1000), MTOR (2983; 1:1,000), phospho-PRKAA/AMPK (Thr172) (2535, 1:1000), PRKAA/AMPK (5832; 1:1000), phospho-ULK1 (Ser555; 5869; 1:1,000), ULK1 (8054, 1:1000) were purchased from Cell Signaling Technology; NCOA4 (sc-373739; 1:150) and Ferritin heavy chain (sc-376594; 1:150) were purchased from Santa Cruz Biotechnology; β-actin (66009-1-Ig, 1:3000), mouse secondary antibodies (SA00001-1, 1:6000) and rabbit secondary antibodies (SA00001-2, 1:6000) were obtained from Proteintech; Slc7a11 (A2413, 1:1000) were purchased from ABclonal.

Techniques: Gene Expression, Expressing, Microscopy, Staining, Knockdown, CCK-8 Assay, Fluorescence, Flow Cytometry, Membrane, Labeling, Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 1. Impaired hMOG- induced EAE in Aicda2/2 mice is restored by a passive transfer of anti-MOG IgG1 Abs. Clinical scores (A–D) and weight loss (C and D) of animals were measured daily fol- lowing immunization until date of harvest. (A) Separately caged WT (n = 25) and Aicda2/2 (n = 26) mice or (B) littermates from Aicda+/2 3 Aicda+/2 parents (WT n = 5; Aicda2/2 n = 5) were immunized with hMOG1–120. (C) Following im- munization, Aicda2/2 mice (n = 5) were treated with 200 mg of anti- MOG IgG1 Ab (clone 818c5) or an isotype control (n = 5) at days 4 and 11 postimmunization. (D) Following immunization, WT mice (n = 5) were treated with 200 mg of anti- MOG IgG1 Ab (clone 818c5) or an isotype control (n = 4) at days 4 and 11 postimmunization. Arrows indi- cate i.v. injections of 818c5 Ab or an isotype control. (A) Represents data pooled over three separate experi- ments. (B–D) Data shown are from a representative experiment from at least two experiments with similar outcomes. Means and SEM are shown. Statistical significance was determined by two-way ANOVA (*p . 0.05, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Control

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 2. WT and Aicda2/2 mice exhibit clinically similar EAE in response to rMOG. (A) Separately caged WT (n = 9) and Aicda2/2 (n = 10) mice were immunized with rMOG1–120, and clinical scores were measured daily until day 26 postimmunization. Data show a representative experiment from two separate experiments with similar outcomes. (B) Titers of anti-rMOG IgM (WT n = 8; Aicda2/2 n = 10) and IgG1 (WT n = 6; Aicda2/2 n = 10) Abs in serum were determined by ELISA. Corresponding dilution curves used to calculate titers can be found in Supplemental Fig. 1A. Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mouse were subjected to H&E staining with a representative example for each genotype shown in (C). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (D). Coronal sections of formalin-fixed paraffin-embedded thoracic spinal cords from each WT (n = 4) and Aicda2/2 (n = 5) mice were subjected to LFB staining with a representative example for each genotype shown in (E). Subsequent blinded quantification using one coronal thoracic section for each animal is shown in (F). Please refer to Materials and Methods for quantification details. Arrows indicate examples of cellular accumulation (C) or demyelination (E) throughout the white matter area of the spinal cord. Means and SEM (A) or means and SD (B, D, and F) are displayed. Statistical significance was determined by using a two-way ANOVA (A) or a Mann–Whitney U test (B, D, and F) (*p . 0.05, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Enzyme-linked Immunosorbent Assay, Staining, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 5. Ung2/2 mice exhibit an impaired class-switch autoantibody response in response to hMOG. (A) Serum from WT (n = 5; Ung+/2 n = 8) and Ung2/2 (n = 10) mice was diluted to a starting concentration of 0.2 mg/ml anti-MOG IgG1 and added to plates coated with hMOG, followed by washes with increasing concentrations of a stripping buffer (NH4SCN) to generate an affinity index. (B) Serum was measured for relative titers of anti-MOG IgM (WT n = 4; Ung+/2 n = 2; Ung2/2 n = 12) and anti-hMOG IgG1 (WT n = 3; Ung+/2 n = 11; Ung2/2 n = 17). (C) Supernatants from brain homogenates were measured for total IgM (WT n = 5; Ung+/2 n = 8; Ung2/2 n = 10) and anti-hMOG IgG1 (WT n = 6; Ung+/2 n = 8; Ung2/2 n = 10). (D) Supernatants from spinal cord homogenates (WT n = 4; Ung+/2 n = 4; Ung2/2 n = 5) were measured for total IgM and anti-hMOG IgG1. Statistical significance was determined by Mann–Whitney U test (*p . 0.05, **p . 0.01, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Concentration Assay, Stripping Membranes, MANN-WHITNEY

Journal: Journal of immunology (Baltimore, Md. : 1950)

Article Title: Isotype-Switched Autoantibodies Are Necessary To Facilitate Central Nervous System Autoimmune Disease in Aicda -/- and Ung -/- Mice.

doi: 10.4049/jimmunol.1700729

Figure Lengend Snippet: FIGURE 6. Class-switched autoantibodies detected in the serum of WTand Ung2/2 mice recognize conformational epitopes of MOG. HEK293 cells transfected with MOG-GFP were overlaid with serum from WT (n = 8), Aicda2/2 (n = 6), or Ung2/2 (n = 11) mice, and IgM (A) or IgG1 (B) Abs were detected by secondary Abs tagged with Alexa Fluor 555 (AF555). DAPI staining for nuclei and merged RGB color images are shown for reference. (C and D) GFP+AF555+DAPI+ cellular events were counted and represented as a ratio of total GFP+DAPI+ cells. Images were captured at original magnification 320. Statistical significance was de- termined by Mann–Whitney U test (**p . 0.01, ***p . 0.001).

Article Snippet: DAPI and Alexa Fluor 555 Rat Anti-Mouse IgG1 or IgM (SouthernBiotech) were used to detect nuclei and Abs, respectively.

Techniques: Transfection, Staining, MANN-WHITNEY

Journal: bioRxiv

Article Title: Photoactivated SOPP3 enables APEX2-mediated proximity labeling with high spatio-temporal resolution in live cells

doi: 10.1101/2024.06.23.595995

Figure Lengend Snippet: a Schematic of APEX2+SOPP3 mediated photoactivated-proximal labeling (photo-PL). B, Biotin. b Photosensitizer screening for APEX2 activation. Streptavidin blot showed photo-PL efficiency. anti-V5 and anti-HA indicated expression level of APEX2 and photosensitizers, respectively. Anti-α-tubulin, internal loading control for western blot. c , d Schematic ( c ) and construct designs ( d ) of APEX2+SOPP3 mediated PL to map proteome on ER-Mito contact sites. BP, Biotin Phenol. e Evaluation on the efficiency of photo-PL at ER-Mito contact sites. Anti-HA and anti-V5 indicated expression level of SOPP3-ERM and OMM-APEX2 respectively. f Left: confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on ER-Mito contact sites. Outer membrane of mitochondria (OMM)-localized APEX2 and ER membrane (ERM)-localized SOPP3 were visualized by anti-V5 and anti-HA antibody. Biotinylation signals on the contact sites were visualized by Alexa Fluor 555-conjugated streptavidin. Illumination time, 5 seconds. Scale bar, 9 μm. Right: the ‘Surface’ tool in Imaris software was used to create a 3D rendering from each channel of confocal images of boxed region. Contact area algorithm was further performed to determine the interface (yellow) between ER (green) and Mito (cyan). Scale bar, 0.8 μm. g Validation on MAMs proteins from streptavidin-enriched PL samples. h , i Schematic and construct designs ( h ) and evaluation on photo-PL efficiency on cell-cell contact sites via western blotting analysis ( i ). Anti-V5 and anti-HA indicated expression level of APEX2-TM and SOPP3-TM respectively. j Confocal fluorescence imaging of APEX2+SOPP3 mediated photo-PL on cell-cell contact sites, stained with Alexa Fluore 555-conjugated streptavidin. Scale bar, 2 μm. Zoomed image from boxed region showed biotinylation labeling precisely on cell-cell contact sites. Scale bar, 1 μm. Illumination time, 10 seconds. k Schematic and construct design of chimeric APEX2-SOPP3-mediated photo-PL in ERM. l Evaluation on the efficiency of photo-PL mediated by chimeric APEX2-SOPP3 targeted to ERM via western blotting analysis. Anti-HA/Flag/V5 indicated expression level of chimeric APEX2-SOPP3-ERM, SOPP3-ERM and OMM-APEX2 respectively. m Confocal fluorescence imaging of photo-PL via APEX2-SOPP3 targeted to ERM. Chimeric APEX2-SOPP3-ERM and biotinylation signals was visualized by anti-HA antibody and Alexa Fluor 555-conjugated streptavidin respectively. Anti-Flag blot indicated expression level of chimeric APEX-SOPP3 targeted to various subcellular compartments. Illumination time, 5 seconds. Scale bar, 10 μm. n , o Schematic and construct designs ( n ) and evaluation on chimeric APEX2-SOPP3 on cell-cell contact sites via western blotting analysis ( o ). Anti-HA indicated expression level of chimeric APEX2-SOPP3-TM. p Confocal fluorescence imaging of chimeric APEX2-SOPP3 mediated photo-PL on cell surface. Chimeric APEX2-SOPP3 was visualized by anti-HA antibody. Biotinylation signals were visualized by Alexa Fluor 555-conjugated streptavidin. Scale bar, 8 μm. Illumination time, 5 seconds. q Flow cytometry analysis on p (30,000 cells per condition). After photo-PL, cells were stained with Alexa Fluor 555-conjugated streptavidin, followed with flow cytometry gating for quantification of biotinylation labeling on the surface of APEX2-SOPP3-TM cells (anti-HA positive, q up) and neighboring WT cells (anti-HA negative, q down). Blue light-activated SOPP3 showed more efficient to facilitate APEX2-mediated proximity labeling compared to that of 1 mM H 2 O 2 treatment. Illumination time, 10 seconds.

Article Snippet: The following antibodies were used: mouse anti-V5 (Abclonal, AE017), rabbit anti-HA (Cell signaling technology, 3724S), Goat-Alexa Fluore 647-conjugated anti-mouse (Invitrogen, A32728), Goat-Alexa Fluore 488-conjugated anti-rabbit (Invitrogen, A11034) and Alexa Fluore 555-conjugated streptavidin (Invitrogen, S32355).

Techniques: Labeling, Activation Assay, Expressing, Control, Western Blot, Construct, Fluorescence, Imaging, Membrane, Software, Biomarker Discovery, Staining, Flow Cytometry

Journal: iScience

Article Title: Bidirectional signals generated by Siglec-7 and its crucial ligand tri-sialylated T to escape of cancer cells from immune surveillance

doi: 10.1016/j.isci.2024.111139

Figure Lengend Snippet:

Article Snippet: Anti-rabbit IgG Fab2 Alexa 555 , Cell Signaling Technology , Cat# 4413, RRID: AB_10694110.

Techniques: Blocking Assay, Plasmid Preparation, Purification, Control, Staining, Recombinant, Membrane, Mutagenesis, Cytotoxicity Assay, Cloning, Expressing, Sequencing, Software