Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 5 ISG15 upregulated by PAPR12, increasing ISGylation of MFN1/2 and then attenuating the ubiquitylation and SUMOylation to inhibit PINK1/Parkin-dependent mitophagy. a Immunoblotting (IB) analysis of ubiquitylation and ISG15 in PHCs following transfection with KD-ISG15 or KD-NC. b Protein quantification of (a) using ImageJ. n = 3 per group. c IB analysis of ubiquitylation of mitochondrial proteins. Mitochondria were isolated from KD-NC and KD-ISG15 PHCs transfected with a HA-ubiquitin construct. d Protein quantification of (d) using ImageJ. n = 3 per group. e IB analysis of SUMO2/3 and ubiquitin with SUMO2/3-conjugated proteins in KD-NC and KD-ISG15 PHCs immunoprecipitated using SUMO2/3 affinity beads. f IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3 and ISG15 in KD-NC and KD-ISG15 PHCs treated with or without MG-132 after immunoprecipitation of MFN1/2. The molecular marker of IB plots of ubiquitin, SUMO2/3, and ISG15 conjugates with MFN1/2 is 70 kD and above when considering the molecular weight of MFN1/2 at 85 kD. g Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (f) using ImageJ after immunoprecipitation of MFN1/2. n = 3 per group. h IB analysis of MFN1/2, MFN1, MFN2, ubiquitin, SUMO2/3, and ISG15 in PHCs following either knockdown or overexpression of PARP12 after immunoprecipitation of MFN1/2. i Quantification of MFN1/2, ubiquitin, SUMO2/3, and ISG15 of (h) using ImageJ. n = 3 per group. j MitoTracker Red and LysoTracker Green staining of PHCs was observed by confocal microscopy. Scale bars: 10 µm. k Western blot analysis of PARP12, ISG15, LC3B, p62, MFN1, MFN2, PINK1, Parkin, COL2A1, aggrecan, MMP13, RUNX2, Bcl2, Bax, and NLRP3 inflammasome activity in PHCs coinfected with PARP12 KD-03 shRNA and ISG15-OE adenovirus. n = 3 per group. l ROS staining with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. m JC-1 staining in PHCs following transfection with PARP12 knockdown and ISG15 overexpression. Scale bars: 100 µm. Data are presented as the mean ± SD. Paired t-test (b, d, i) and one-way analysis of variance followed by Tukey’s multiple comparison test (g) were used for statistical analysis. *P < 0.05, **P < 0.01, and ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Western Blot, Transfection, Isolation, Ubiquitin Proteomics, Construct, Immunoprecipitation, Marker, Molecular Weight, Knockdown, Over Expression, Staining, Confocal Microscopy, Activity Assay, shRNA, Comparison

Journal: Bone research

Article Title: IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating ubiquitylation and SUMOylation of MFN1/2.

doi: 10.1038/s41413-024-00363-3

Figure Lengend Snippet: Fig. 8 PARP12 modulates osteoarthritis (OA) pathogenesis in monosodium iodoacetate (MIA)-treated rats. a Experimental diagram of the MIA OA rat model treated with XAV-939 or PARP12 overexpression (OE) adenovirus. Rats were evaluated at age of 10 weeks. n = 6 per group. b 3D reconstruction images of micro-CT scanning of the knees of rats treated with XAV-939 or PARP12-OE adenovirus. n = 5 per group. c–f Analysis of BV/TV, BS/TV, trabecular thickness, and trabecular numbers. n = 5 per group. g Western blot analysis of PARP12, COL2A1, aggrecan, MMP13, RUNX2, Bcl2/Bax, LC3B, p62, MFN1, MFN2, PINK1, Parkin and NLRP3 inflammasome activity in chondrocytes of rats treated with XAV-939 or PARP12 overexpression (OE) adenovirus. n = 3 per group. h Representative images of Safranin O and IHC staining of PARP12, COL2A1 and MMP13. Scale bars: 250 µm (first row) and 50 µm (second row). i Quantification of macroscopic score based on staining results in (h). n = 3 per group. j–l Quantification of PARP12, COL2A1, and MMP13 positive chondrocytes based on staining results in (h). n = 3 per group. m–o ROS staining, ATP level and JC-1 staining in chondrocytes of rats treated with XAV-939 or PARP12 OE adenovirus. p Schematic representation of the mechanism by which IRF1-mediated upregulation of PARP12 promotes cartilage degradation by inhibiting PINK1/Parkin dependent mitophagy through ISG15 attenuating the ubiquitylation and SUMOylation of MFN1/2. Data are presented as the mean ± SD. Paired t-test (c–f, j–l, n) and non-parametric Mann-Whitney U test (i) were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001

Article Snippet: According to the manufacturer’s instructions of SUMOylation 2/3 affinity beads (BK162, Cytoskeleton, Inc.), SUMO2/3-conjugated proteins in chondrocytes lysates were immunoprecipitated.

Techniques: Over Expression, Micro-CT, Western Blot, Activity Assay, Immunohistochemistry, Staining, MANN-WHITNEY

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: The SH2 domain of STAT5A is required for efficient binding to Src kinases. (A) Domain structure of fluorescently labeled STAT5A-eYFP. (B) Subcellular localization of STAT5A-eYFP in the absence or presence of Epo. HeLa T-REx HA-EpoR cells stably transfected with STAT5A-eYFP were stimulated with 1 U/ml Epo for 30 min and the localization of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (C) Subcellular localization of STAT5A-eYFP (upper panel), STAT5A R618Q -eYFP (middle panel) and STAT3-eYFP (lower panel) was investigated in the presence of vSrc-dsRed. HeLa T-REx vSrc-dsRed cells were treated with 5 ng/ml doxycycline and transfected with the indicated constructs and the distribution of fluorescently labeled fusion proteins was analyzed after 24 h by confocal microscopy. Scale bars: 20 μm. (D) Quantification of the relative subcellular distribution of eYFP-labeled STAT3 and STAT5A constructs in HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP (B) and HeLa T-REx vSrc-dsRed cells transfected with STAT5A-eYFP, STAT5A R618Q -eYFP or STAT3-eYFP (C) . The expression of the HA-EpoR and vSrc-dsRed was induced with 5 ng/ml doxycycline for 24 hours. Mean fluorescence intensities (MFI) of the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. The data shown are means ± SD of n = 30 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005. n.s. = not significant. (E + F) HeLa T-REx FRT cells were co-transfected with plasmids coding for STAT5A-eYFP or STAT5A R618Q -eYFP and vSrc-dsRed or Hck-dsRed. Fluorescently labeled STAT5 was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed or Hck-dsRed 24 h after transfection. The expression and phosphorylation of STAT5A and vSrc/Hck proteins was analyzed in the whole cellular lysates (WCL) using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Labeling, Stable Transfection, Transfection, Confocal Microscopy, Construct, Expressing, Fluorescence, Software, Immunoprecipitation, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: STAT5A binds to the phosphorylated activation loop of SFK. (A) Domain structure of vSrc-dsRed. Selected amino acids are highlighted. A multiple sequence alignment of the activation loop of SFK is shown. Autophosphorylation site is highlighted (red). (*) conserved amino acids, (:) similar properties . Bold characters highlight peptide sequence used for precipitation. (B + C) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with the indicated vSrc-dsRed variants. Phosphorylation was analyzed 24 h after transfection using antibodies against pY 416 -Src, Src, pY 694/699 -STAT5A/B and STAT5A. CTRL = untransfected cells. (D) Quantification of relative subcellular distribution of STAT5A in HeLa T-REx FRT stably expressing STAT5A-eYFP and the indicated vSrc-dsRed mutants. Mean fluorescence intensity (MFI) of eYFP-fluorescence in the cytoplasm and nucleus were determined using the Zen 2012 software and changes in the ratio between the compartments were plotted. Data show means ± SD of n = 10 cells and were statistically evaluated by Student’s t -test. ***p < 0.0005, **p < 0.005, *p < 0.05, n.s. = not significant. (E) HeLa T-REx FRT cells stably expressing STAT5A-eYFP were transfected with vSrc K295N -dsRed or vSrc Y416F -dsRed. Subcellular distribution of STAT5A-eYFP was analyzed 24 h after transfection by confocal microscopy. Scale bars: 20 μm. (F + G) HeLa T-REx FRT cells were co-transfected with plasmids coding for vSrc-dsRed (Hck-dsRed), vSrc K295N -dsRed (Hck K269N -dsRed) or vSrc Y416F -dsRed (Hck Y390F -dsRed) and STAT5A-eYFP. STAT5-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of vSrc-dsRed (Hck-dsRed) 24 h after transfection. Expression and phosphorylation of STAT5A and vSrc proteins was analyzed in the WCL using antibodies against pY 416 -Src, Src, Hck, pY 694/699 -STAT5A/B and GFP. (s) short exposure, (l) long exposure. (H) HeLa T-REx FRT cells expressing STAT5A-eYFP or STAT5A R618Q -eYFP were lysed and incubated with a Src-peptide containing tyrosine- or phosphotyrosine 416. Precipitates and WCL were analyzed by immunoblotting using a GFP-specific antibody.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Activation Assay, Sequencing, Stable Transfection, Expressing, Transfection, Fluorescence, Software, Confocal Microscopy, Immunoprecipitation, Western Blot, Incubation

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: SFK-mediated cytoplasmic localization of STAT5A is dominant over BCR-ABL induced nuclear accumulation. (A) HeLa T-REx BCR-ABL cells were transiently transfected with STAT5A-eYFP and either treated with 5 ng/ml doxycycline for 24 h to induce BCR-ABL expression (lower panel) or left untreated (upper panel). Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using a cABL-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. The subcellular distribution of STAT5A-eYFP was analyzed by confocal microscopy. Scale bars: 20 μm. (B) The subcellular distribution of STAT5A-eYFP was investigated in the presence of vSrc-dsRed (upper panel), vSrc K295N -dsRed (middle panel) or vSrc Y416F -dsRed (lower panel) in HeLa T-REx BCR-ABL cells that were treated with 5 ng/ml doxycycline for 24 h. Fixation was performed with methanol. Fixed cells were stained for BCR-ABL using an Abl-specific primary antibody and a secondary antibody conjugated to Alexa Fluor-405. Scale bars: 20 μm. (C) HeLa T-REx BCR-ABL cells were co-transfected with vSrc-dsRed, or the respective kinase activity affecting mutants vSrc K295N -dsRed or vSrc Y416F -dsRed and STAT5A-eYFP. The cells were either treated with 5 ng/ml doxycycline for 24 h (lanes 1–3) to induce the expression of BCR-ABL or left untreated (lane 4). Protein expression and phosphorylation in the cellular extracts was investigated by immunoblotting with antibodies against pY 412 -cABL, cABL, pY 694/699 -STAT5A/B, STAT5A, pY 416 -Src and Src. α-Tubulin served as a loading control.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Transfection, Expressing, Staining, Confocal Microscopy, Activity Assay, Western Blot

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Binding of STAT5A to Src kinases interferes with dimerization. (A) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The cells were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the HA-tagged EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP were transfected with STAT5A-FLAG. The expression of vSrc-dsRed was induced for 8 h with 5 ng/ml doxycycline or the cells were left untreated. STAT5A-eYFP was immunoprecipitated from cell lysates using a GFP antibody and analyzed by immunoblotting for the presence of STAT5A-FLAG. The expression and phosphorylation of STAT5A-eYFP and STAT5A-FLAG was analyzed in the WCL using antibodies against pY 694/699 -STAT5A/B, GFP and the FLAG-tag. (B) HeLa T-REx HA-EpoR cells stably expressing STAT5A-eYFP were treated with 5 ng/ml doxyxcyline for 24 h to induce the expression of the human EpoR and stimulated with 5 U/ml Epo for 30 minutes or left untreated. HeLa T-REx vSrc-dsRed cells stably expressing STAT5A-eYFP or a STAT5A S710F -eYFP were treated with 5 ng/ml doxyxcline for 8 h or the cells were left untreated. Cellular extracts were prepared under native conditions and STAT5A-eYFP dimers were separated from monomers by blue native PAGE electrophoresis (NP). STAT5A-eYFP dimer complexes were measured by the detection of the eYFP fluorescence. The cellular extracts were subjected to immunoblotting using antibodies against pY 694/699 -STAT5A/B, STAT5A, Src and the HA-tag of the EpoR. (C) Confocal microscopy analysis of HeLa T-REx FRT cells co-expressing vSrc-dsRed together with STAT5A S710F -eYFP (upper panel), a serine phosphorylation mimicking mutant STAT5A S710D -eYFP (middle panel) or a serine phosphorylation deficient mutant STAT5A S710A -eYFP (lower panel). Methanol fixation was performed 24 h after transfection. Scale bars: 20 μm.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Binding Assay, Stable Transfection, Expressing, Transfection, Immunoprecipitation, Western Blot, FLAG-tag, Blue Native PAGE, Electrophoresis, Fluorescence, Confocal Microscopy, Mutagenesis

Journal: Cell Communication and Signaling : CCS

Article Title: Src family kinases interfere with dimerization of STAT5A through a phosphotyrosine-SH2 domain interaction

doi: 10.1186/s12964-014-0081-7

Figure Lengend Snippet: Activated SFK interfere with dimerization and nuclear translocation of pSTAT5A in BCR-ABL expressing cells. Left scheme: Classical activation of the JAK2-STAT5A signaling pathway downstream of the EpoR. Right scheme: BCR-ABL directly phosphorylates STAT5A Y694 resulting in STAT5A dimerization, nuclear accumulation and finally target gene expression . In the presence of BCR-ABL, a predominantly cytoplasmic localization of pSTAT5A is achieved (i) upon binding to the scaffolding adaptor Gab2 resulting in pro-survival signaling through PI3K/Akt activation and (ii) through binding of the STAT5A SH2 domain to the phosphorylated activation loop of SFK, a mechanism that interferes with STAT5A dimerization and subsequent nuclear accumulation. Constitutively active STAT5A S710F escapes the SFK-mediated cytoplasmic retention. Flashes indicate phosphorylation events.

Article Snippet: Anti-pY 694/699 -STAT5A/B (#9351), anti-pY 416 -Src (#2101), anti-pY 412 -Abl (#2865), anti-Hsp70 (#4872, Cell Signaling, Beverly, USA), anti-STAT5A (clone #5073, rabbit polyclonal antiserum was kindly provided by Richard Moriggl, Ludwig Boltzmann Institute for Cancer Research (LBI-CR), Vienna, Austria), anti-GFP (600-103-215, Rockland, Gilbertsville, USA), anti-FLAG (F3165), anti-α-tubulin (T5168, Sigma, St. Louis, USA), anti-GAPDH (sc-32233), anti-Abl (sc-131), anti-Hck (sc-72), anti-cSrc (sc-19, Santa Cruz Biotechnology, Santa Cruz, USA), anti-vSrc (MABS193, Millipore, Billerica, MA, USA) and anti-HA (MMS-101R, Covance, Princeton, New Jersey, USA) antibodies were used for immunoblotting.

Techniques: Translocation Assay, Expressing, Activation Assay, Binding Assay, Scaffolding

Journal: Journal of Nanobiotechnology

Article Title: Momordica charantia L.-derived exosome-like nanovesicles stabilize p62 expression to ameliorate doxorubicin cardiotoxicity

doi: 10.1186/s12951-024-02705-z

Figure Lengend Snippet: MC-ELNs prevent p62 from DOX-induced ubiquitination degradation in cardiomyocytes. H9c2 cells were pre-treated with 10 µg/mL MC-ELNs for 6 h, and then cultured with or without 1 µM doxorubicin (DOX) for another 24 h. Western blot images ( A ) and quantitative analysis ( B ) on the p62 protein levels. ( C ) The p62 mRNA levels in H9c2 cells treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. ( D ) H9c2 cells were treated with PBS or 1 µM DOX in the presence or absence of 2 µM Lactacystin (LACTA) for 24 h. ( E ) H9c2 cells were incubated in the absence or presence of MC-ELNs (10 µg/mL) in combination with the protein synthesis inhibitor cycloheximide (CHX, 10 µg/mL) for 2–4 h. Western blot analysis of the expressions of p62. ( F ) Immunoprecipitation assay (IP) and western blot analysis of cell lysate derived from PBS- or DOX-treated H9c2 cells in the absence or presence of 10 µg/mL of MC-ELNs (DOX, DOX + MC-ELNs). IP was performed with an antibody against p62. The blot was probed with antibodies against p62, Ub, K48-Ub, and K63-Ub. β-actin was employed as a loading control. Western blot analysis on the identified E3 ligase enzyme bound with p62 including Parkin ( G ), TRIM21 ( H ), and TRAF6 ( I ) in H9c2 cells treated with 1 µM DOX in the absence or presence of 10 µg/mL MC-ELNs. All data are presented as mean ± SD ( n = 3 experiments per group for A - E , G - I ; n = 1 experiment per group for F ). Comparisons among more than two groups were performed by ordinary one-way analysis of variance (ANOVA) followed by the Tukey’s multiple comparisons test

Article Snippet: The primary antibodies included Cleaved caspase3 (9664S, CST), Caspase3 (9665S, CST), Cleaved caspase7 (8438S, CST), Caspase7 (12827T, CST), Cleaved PARP (9545, CST), PARP (9532S, CST), γ-H2A.X (ab2893, Abcam), Cyclin D1 (2978S, CST), Cyclin E1 (20808S, CST), Cyclin B1 (4138S, CST), Nrf2 (SAB4501984, Sigma), HO-1 (10701-1AP, proteintech), SQSTM1/p62 (ab109012, Abcam), Keap1 (8047S, CST), Ubiquitin (ab140601, Abcam), K48-Ub (ab140601, Abcam), K63-Ub (ab179434, Abcam), TRIM21 (ab207728, Abcam), TRAF6 (HY-P80919, MCE), Parkin (ab77924, Abcam), PCNA (ab29, Abcam), LC3B (NB100-2220, NOVUS), NQO1 (67240-1-lg, proteintech), Catalase (21260-1-AP, proteintech), SOD2 (24127-1-AP, proteintech), GPX4 (ab125066, Abcam), β-actin (HRP-600008, proteintech).

Techniques: Ubiquitin Proteomics, Cell Culture, Western Blot, Incubation, Immunoprecipitation, Derivative Assay, Control

Journal: International Journal of Molecular Sciences

Article Title: DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

doi: 10.3390/ijms22115675

Figure Lengend Snippet: Inhibiting ERK1/2 signaling decreased the effect of DHA against oxidative stress. AML12 cells were pre-treated with DHA (50 μM) and TUG891 (10 μM) for 12 h, and then treated with H 2 O 2 (400 μM) for another 2 h. ( A ) The protein expression of ERK1/2, P-ERK1/2, AMPK, and P-AMPK was determined by Western-blotting. ( B ) The protein expression of ERK1/2 and P-ERK1/2 in AML12 cells after treated with U0126. ( C ) Cells were pre-treated with U0126 (10 μM) for 2 h, and then treated with DHA (50 μM) for 12h followed with H 2 O 2 (400 μM) 2h challenge. Cellular ROS was stained with DCFH-DA fluorescent probe and detected by Flow cytometry. ( D ) The cell viability of AML12 cells. All data were expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Article Snippet: Therefore, we inhibited the expression of ERK1/2 with the specific inhibitor U0126 (HY-12031A, MCE, Shanghai, China).

Techniques: Expressing, Western Blot, Staining, Flow Cytometry

Journal: International Journal of Molecular Sciences

Article Title: DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

doi: 10.3390/ijms22115675

Figure Lengend Snippet: The inhibition of ERK1/2 signaling blocked DHA-mediated mitophagy. Cells were pre-treated with U0126 (10 μM) for 2 h, and then treated with DHA (50 μM) for 12 h followed with H 2 O 2 (400 μM) 2h challenge. ( A ) The protein expression of Beclin1, P62/SOSTM1, LC3B, PINK, and Parkin was determined by Western-blotting. ( B ) The immunofluorescence detected the expression of PINK in AML12 cells. ( C ) The lysosomes in AML12 cells were stained with LysoSensor™ and detected by using confocal microscopy after treatment. All data are expressed as mean ± SEM of three independent experiments. Significant differences between each group are shown with different letters (e.g., a, b, and c mean a statistically significant differences between each other, p < 0.05).

Article Snippet: Therefore, we inhibited the expression of ERK1/2 with the specific inhibitor U0126 (HY-12031A, MCE, Shanghai, China).

Techniques: Inhibition, Expressing, Western Blot, Immunofluorescence, Staining, Confocal Microscopy

Journal: International Journal of Molecular Sciences

Article Title: DHA Protects Hepatocytes from Oxidative Injury through GPR120/ERK-Mediated Mitophagy

doi: 10.3390/ijms22115675

Figure Lengend Snippet: The schematic representation shows that DHA attenuates H 2 O 2 -induced hepatocyte injury through ERK1/2-dependent mitophagy. Oxidative stress would induce hepatocyte apoptosis by impairing mitochondrial function. DHA and TUG891 (a agonist of GPR120) supplementation activates GPR120/ERK1/2 signaling pathway which regulates PINK1/Parkin-mediated mitophagy. The up-regulation of protective mitophagy level promotes mitochondrial renewal and maintains mitochondrial homeostasis during oxidative injury. However, inhibiting the activation of ERK1/2 and autophagy by selective inhibitor U0126 or 3-MA, respectively, could block the protective effect of DHA.

Article Snippet: Therefore, we inhibited the expression of ERK1/2 with the specific inhibitor U0126 (HY-12031A, MCE, Shanghai, China).

Techniques: Activation Assay, Blocking Assay

Journal: Journal of Nanobiotechnology

Article Title: Inverse-designed plasmonic biosensors with LSPR-SPP-wood anomaly coupling enhanced for biomolecular analysis

doi: 10.1186/s12951-025-03930-w

Figure Lengend Snippet: High-throughput affinity sensing platform for ADC drug development. ( a ) The association process of incubating PD-1 on the chip to detect Sintilimab. ( b ) The long-time dissociation process of PD-1 with Sintilimab. ( c ) Dynamic real-time and fitting curves of the association and dissociation phases of the interaction between PD-1 and Sintilimab. ( d ) Schematic diagram of the high-throughput antibody affinity detection protocol, with each group, repeated three times in the three samples tested, and the concentration of A-H in each column decreasing incrementally. ( e ) Schematic diagram of the main events in the antibody drug fishing and affinity detection process. ( f-h ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the unoptimized sensor. ( i-k ) Dynamic real-time and fitting curves of the association and dissociation phases for Polatuzumab with CD79B, Adalimumab with TNF-α, and Sacituzumab govitecan with TROP2, detected using the optimized IDMM-SPR sensor

Article Snippet: Bovine serum albumin (BSA), Phosphate-buffered saline (PBST), 11-mercaptoundecanoic acid (MUA), 1-ethyl3-(dimethylaminopropyl) carbodiimide (EDC), N-hydroxysuccinimide (NHS), Dimethyl Sulfoxide (DMSO), Fish gelatin,, ethanolamine, and phosphate-buffered saline (PBS) buffer, Polyethylene glycol 2000 (PEG) were purchased from Sigma-Aldrich; Protein A, IgG, CRP, and CRP ab, hTGFBR1 were purchased from monoclonal anti-CRP capture and detection antibodies were purchased from Beijing Sino Biological, Inc. (Beijing, China); Sintilimab, Adalimumab, Polatuzumab, Sacituzumab, PD-1, TNF-α, CD79B, TROP2, Quercetin were purchased from MedChemExpress.

Techniques: High Throughput Screening Assay, Concentration Assay

Journal: International journal of oral science

Article Title: WNT7A promotes tumorigenesis of head and neck squamous cell carcinoma via activating FZD7/JAK1/STAT3 signaling.

doi: 10.1038/s41368-024-00279-y

Figure Lengend Snippet: Fig. 4 WNT7A upregulated expression of STAT3 target genes in HNSCC cells. a Heatmap of differentially expressed genes based on RNA- seq data. The top 30 deregulated genes were selected for further analysis. b, c Validation of the promote proliferation, self-renewal, and anti- apoptosis correlated genes by real-time RT-PCR. d Design of ChIP-qPCR primers by using Cistrome Data Browser. e ChIP-qPCR analysis was performed to examine the enrichment of STAT4, HCAR2, and BIRC3 after overexpression of WNT7A. The immunoprecipitation of the STAT3 was compared to that of the IgG antibody and IRF1 was a positive control in the analysis. Data shown as mean ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.000 1

Article Snippet: . . Huang et al. 9 International Journal of Oral Science (2024) 16:7 Chromatin immunoprecipitation (ChIP-qPCR) ChIP assays were conducted using an anti-STAT3 antibody (Table S4 shows the antibodies used for ChIP) and Protein G Magnetic Beads (HY-K0204, MCE, USA) to pull down DNA fragments bound to STAT3.

Techniques: Expressing, RNA Sequencing, Biomarker Discovery, Quantitative RT-PCR, ChIP-qPCR, Over Expression, Immunoprecipitation, Positive Control

Journal: International journal of oral science

Article Title: WNT7A promotes tumorigenesis of head and neck squamous cell carcinoma via activating FZD7/JAK1/STAT3 signaling.

doi: 10.1038/s41368-024-00279-y

Figure Lengend Snippet: Fig. 6 WNT7A may activate STAT3 signaling pathway through FZD7/JAK1. a Western blot analysis demonstrated that overexpression of WNT7A in HN30 and HN6 cells leaded to increased Tyr1034/1035 phosphorylation of JAK1, while total protein levels of JAK1 remain unchanged. b Protein network prediction using the STRING database identified FZD5 and FZD7 as potential receptor candidates for WNT7A. c Analysis of single-cell sequencing data (GSE103322) revealed higher enrichment of FZD7 in cancer cells. d, e Co-immunoprecipitation results confirm interactions between WNT7A, FZD7, and JAK1. f Co-localization assay by staining FZD7 (red), JAK1 (green), and nucleus (DAPI)

Article Snippet: . . Huang et al. 9 International Journal of Oral Science (2024) 16:7 Chromatin immunoprecipitation (ChIP-qPCR) ChIP assays were conducted using an anti-STAT3 antibody (Table S4 shows the antibodies used for ChIP) and Protein G Magnetic Beads (HY-K0204, MCE, USA) to pull down DNA fragments bound to STAT3.

Techniques: Western Blot, Over Expression, Phospho-proteomics, Single Cell, Sequencing, Immunoprecipitation, Staining

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: Downregulation of PDIA3 inhibits gastric cancer cell growth through cell cycle regulation.

doi: 10.1016/j.biopha.2024.116336

Figure Lengend Snippet: Fig. 8. STAT3 was associated with PDIA3-mediated cyclin G1 regulation. (A) The protein expressions of STAT3, phosphor-STAT3 (Tyr705) and cyclin G1 after downregulation of PDIA3 by sgRNA-2. (B) The expression of cyclin G1 after treatment of a STAT3 inhibitor cryptotanshinone in TMK1 and AGS. (C) The expression of cyclin G1 after siSTAT3 in TMK1 and AGS.

Article Snippet: The inhibitor for STAT3, cryptotanshinone, was purchased from MedChemExpress LLC. (Shanghai, China).

Techniques: Expressing