Journal: Journal of pharmaceutical analysis

Article Title: Monoclonal antibody targeting mu-opioid receptor attenuates morphine tolerance via enhancing morphine-induced receptor endocytosis.

doi: 10.1016/j.jpha.2023.06.008

Figure Lengend Snippet: Fig. 4. Effects of 3A5C7 monoclonal antibody (mAb) on mu-opioid receptor (MOR) recycling from cytoplasm to cell membrane. (A) Immunofluorescence staining of HEK293T-MOR cells to demonstrate the recycling of MOR. (B) Immunofluorescence staining of SH-SY5Y cells to demonstrate the recycling of MOR. (C, D) Flow cytometry demonstrating the recycling of MOR in HEK293T-MOR cells (C) and corresponding quantification as percentages of control cell surface MOR without any treatment (D). (E, F) Flow cytometry demonstrating the recycling of MOR in SH-SY5Y cells (E) and corresponding quantification as percentages of control cell surface MOR without any treatment (F). HEK293T-MOR or SH-SY5Y cells were exposed to morphine (10 mM) and 3A5C7 (10 mg/mL) for 1 h to induce internalization which were washed out thereafter. MOR recycling was monitored at 0 min, 15 min, 30 min, and 1 h after washout. (G) Western blot showed the MOR recycling from cytoplasm to cell membrane. HEK293T-MOR or SH-SY5Y cells were treated as indicated and cell membrane and plasma proteins were extracted for assays. (H) Immunofluorescence staining showed the relationship between internalized MOR and lysosome associated membrane protein-1 (LAMP-1), the marker of lysosome. Cells were treated with morphine and 3A5C7 mAb for 1 h at 37 C before assay. Naþ-Kþ adenosine triphosphatase (ATPase) a1 subunit was used as internal reference of cell membrane and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as internal reference of cytoplasm. Student's t tests were used for statistical analysis. Data were presented as mean ± standard error of mean (n ¼ 3 independent experiments). *P < 0.05, **P < 0.01, and ****P < 0.0001 versus recycle 0 min in Figs. 4D and F. DAPI: 40,6-diamidino-2-phenylindole dihydrochloride; NC: negative blank control.

Article Snippet: The mouse monoclonal anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody (60004-1-Ig), rabbit polyclonal anti-lysosome associated membrane protein-1 (LAMP-1) antibody (21997-1-AP), rabbit polyclonal antiGRK2 antibody (13990-1-AP), and rabbit polyclonal anti-barrestin2 antibody (10171-1-AP) were purchased from Proteintech (Chicago, IL, USA).

Techniques: Membrane, Staining, Flow Cytometry, Control, Western Blot, Clinical Proteomics, Marker

Journal: Cellular and molecular life sciences : CMLS

Article Title: The impact of VPS35 D620N mutation on alternative autophagy and its reversal by estrogen in Parkinson's disease.

doi: 10.1007/s00018-024-05123-4

Figure Lengend Snippet: Fig. 4 WASH1 is involved in Rab9 subcellular localization, but estrogen has no effect on WASH1-VPS35 colocalization. A Rab9 and LAMP1 localiza- tion in WASH1-knockdown cells compared to that in control cells. B Colocalization analysis of Rab9 and LAMP1. (n = 3 independent experiments; 30 cells in each group.) The data were analyzed using Student’s t test. C HeLa cells transiently expressing the mCherry- EGFP-Rab9 protein under WASH1 knockdown conditions compared to control cells. D The signal intensity ratio of EGFP/mCherry of HeLa cells in C. (n = 3 independent experi- ments; 30 cells in each group.) The data were analyzed using Student’s t test. E VPS35 and WASH1 localization in HeLa cells transiently transfected with VPS35 (WT or D620N)-Venus. Arrows represent colocalization of VPS35 and WASH1. F Colo- calization analysis of VPS35 and WASH1. (n = 3 independent experiments; 30 cells in each group.) The data were analyzed using Kruskal–Wallis test, fol- lowed by multiple comparisons through the Bonferroni method. * p < 0.05, ** p < 0.01, and *** p < 0.001. The bar graph represents the mean + SD. Scale bars = 10 μm

Article Snippet: Primary antibodies against the following proteins were used for these analyses: MAP2 (chicken, Abcam), TH (mouse, Millipore), cleaved caspase-3 (rabbit, Cell Signaling), LAMP1 (mouse, Santa Cruz), Rab9 (rabbit, Abcam), and α-synuclein (rabbit, Cell Signaling), GPR30 (rabbit, #107748, GeneTex), WASH1 (mouse, SAB4200552, Sigma-Aldrich).

Techniques: Knockdown, Control, Expressing, Transfection

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 2. Inhibition of miR-29b-1-5p ameliorates lipopolysaccharide (LPS)-induced septic myocardial injury in mice (A) Mice received a single intraperitoneal injection of 25 mg/kg LPS (lethal dose) or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. An equal amount of saline was injected into the control group. n=20. Mice received a single intraperitoneal injection of 10 mg/kg LPS or a tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving LPS injection). (B) Twenty-four hours after LPS injection, echocardiography tests were performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via hematoxylin and eosin (HE) staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) α-Smooth muscle actin (α-SMA) expression in mouse myocardial tissues was determined via immunohistochemistry (IHC) staining. Scale bar: 20 μm. (I‒L) Tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and cardiac troponin I (cTnI) levels in mouse serum were determined by enzyme-linked immunosorbent assay (ELISA). (M) The miR-29b-1-5p expression level in mouse myocardial tissue was determined by real-time quantitative reverse transcription PCR (qRT-PCR). n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs. the LPS+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Saline, Control, Staining, Expressing, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Reverse Transcription, Quantitative RT-PCR, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 3. Inhibition of miR-29b-1-5p ameliorates CLP-induced septic myocardial injury in mice (A) Mice were subjected to CLP surgery or tail vein injection of 80 mg/kg miR-29b-1-5p/NC antagomir for 3 consecutive days (24 h later receiving CLP surgery), after which the survival conditions were assessed for 96 h, and survival rate curves were generated. n=20. A sham operation was performed on the control group. n=20. (B) Twenty- four hours after CLP surgery, echocardiography was performed. (C,D) Mice were euthanized, and histological analysis of myocardial tissue injury was performed via HE staining and Masson’s trichrome staining. Scale bar: 50 μm. (F,G) The cardiac injury score and fibrotic area were assessed via HE and Masson’s trichrome staining, respectively. (E,H) The protein levels and distribution of α-SMA in mouse myocardial tissues were examined using IHC staining. Scale bar: 20 μm. (I‒L) TNF-α, IL-1β, IL-6 and cTnI levels in mouse serum were determined by ELISA. (M) The miR-29b- 1-5p expression level in mouse myocardial tissue was determined by qRT-PCR. n=6. (N) The protein levels of TLR2, TLR4, TFEB, LAMP1 and LC3II in mouse myocardial tissues were determined by western blot analysis. n=3. **P<0.01 vs the control group; #P<0.05 and ##P<0.01 vs the CLP+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Injection, Generated, Control, Staining, Immunohistochemistry, Enzyme-linked Immunosorbent Assay, Expressing, Quantitative RT-PCR, Western Blot

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 4. Inhibition of miR-29b-1-5p ameliorates LPS-induced cardiomyocyte dysfunction in mice in vitro (A) Twenty-four hours after transfecting HL-1 cells with the NC or miR-29b-1-5p antagomir, LPS (1 μg/mL) stimulation was applied for another 24 h, and miR-29b-1-5p expression was validated in each group by qRT-PCR. (B) After LPS treatment for 0, 24, 48, or 72 h, MTT assay was performed to examine the viability of the HL-1 cells. (C) Flow cytometry was used to evaluate apoptosis in HL-1 cells. (D‒F) TNF-α, IL-1β, and IL-6 levels in HL-1 cell supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (G‒I) Western blot analysis was used to measure Bax, Bcl-2, cleaved caspase-3, pro- caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II protein levels in HL-1 cells. n=3. **P<0.01 vs the control group; ##P<0.01 vs the LPS+NC antagomir group.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, In Vitro, Expressing, Quantitative RT-PCR, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot, Control

Journal: Acta biochimica et biophysica Sinica

Article Title: miR-29b-1-5p exacerbates myocardial injury induced by sepsis in a mouse model by targeting TERF2.

doi: 10.3724/abbs.2024020

Figure Lengend Snippet: Figure 6. Silencing of TERF2 partially reverses the ameliorative effect of miR-29b-1-5p inhibition on LPS-induced cardiomyocyte apoptosis and inflammation (A) HL-1 cells were transfected with sh-TERF2 or sh-NC vector. Forty-eight hours later, HL-1 cells were collected to detect the protein expression of TERF2. (B‒I) HL-1 cells were transfected with sh-TERF2 or the miR-29b-1-5p antagomir for 24 h, followed by stimulation with LPS. MTT assay was performed to examine HL-1 cell viability (B). Flow cytometry was performed to evaluate HL-1 cell apoptosis (C). ELISA was also conducted to evaluate TNF-α, IL-1β, and IL-6 levels in HL-1 cells (D‒F). Western blot analysis was conducted to determine the protein levels of TERF2, cleaved PARP, cleaved caspase-3, pro-caspase-3, TLR2, TLR4, TFEB, LAMP1 and LC3II in HL-1 cells (G–I). **P<0.01 vs LPS+NC antagomir+ sh-NC; #P<0.05, ##P<0.01 vs LPS+miR-29b-1-5p antagomir+sh-TERF2.

Article Snippet: Jiang et al. Acta Biochim Biophys Sin 2024 4°C with the following antibodies: anti-TERF2 (1:1000; 66893-1-Ig; Proteintech, Wuhan, China), anti-Bax (1:1000; ab32503; Abcam), anti-Bcl-2 (1:2000; ab182858; Abcam), anti-cleaved caspase-3 (1:5000; ab214430; Abcam), anti-Pro-caspase-3 (1:10,000; ab32499; Abcam), anti-cleaved PARP (1:1000; ab32064; Abcam), anti-TLR2 (1:1000; DF7002; Affinity Bioscience, Beijing, China), anti-TLR4 (1:1000; 19811-1-AP; Proteintech), anti-TFEB (1:1000; 13372-1-AP; Proteintech), anti-LAMP1(1:1000; 67300-1-Ig; Proteintech), anti-LC3II (1:1000; 14600-1-AP; Proteintech), anti-β-actin (1:1000; ab8227; Abcam), and anti-GAPDH (1:1000, AF7021; Affinity Bioscience), followed by incubation with HPR-conjugated goat anti-mouse or rabbit IgG secondary antibody (1:2000; SA00001-2 or SA00001-1; Proteintech) for 2 h at room temperature.

Techniques: Inhibition, Transfection, Plasmid Preparation, Expressing, MTT Assay, Flow Cytometry, Enzyme-linked Immunosorbent Assay, Western Blot

Journal: bioRxiv

Article Title: FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy

doi: 10.1101/2024.05.28.596287

Figure Lengend Snippet: Hearts were collected from 8-month mice 8 weeks post-TAC or sham surgery and processed for protein quantification. Protein from (A) sham only, (B) TAC only, (C) WT only, and (D) Ficd -/- only mice were run on 15% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for LC3 and GAPDH (loading control) levels (n=6-7 mice per genotype per condition). Quantification of LC3-I, LC3-II, and GAPDH from (E) sham only, (F) TAC only, (G) WT only, (H) Ficd -/- only gels. Protein from (I) sham only and (J) TAC only mice were run on 10% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for CtsL, CtsB, CtsD, LAMP1, and GAPDH (loading control) levels (n=6-7 mice per genotype per condition). Quantification of (K) CtsL, (L), CtsB, (M) CtsD, and (N) LAMP1 levels relative to GAPDH controls in sham only mice. Quantification of (O) CtsL, (P), CtsB, (Q) CtsD, and (R) LAMP1 levels relative to GAPDH controls in TAC only mice. Indicated P values were calculated using unpaired t tests.

Article Snippet: The following antibodies were used for western blotting and immunofluorescence studies: Alpha-actinin (D6F6)(Cell Signaling, 6487T, Rabbit mAb), BiP (Proteintech, 665741IG, mouse mAb), CHOP (D46F1)(Cell Signaling, 5554S, Rabbit mAb), Cathepsin B (D1C7Y)(Cell Signaling, 31718S, Rabbit mAb), Cathepsin D (E179)(Cell Signaling, 69854S, Rabbit mAb), Cathepsin L (Santa Cruz Biotechnology, sc-32320, mouse mAb), FAM134B (E8Y9R)(Cell Signaling, 83414S, Rabbit mAb), GAPDH (Proteintech, 600041IG, mouse mAb), HSC70 (B-6)(Santa Cruz Biotechnology, sc-7298, mAb), LAMP1 (E6N3R)(Cell Signaling, 46843S, Rabbit mAb), LC3A/B (D3U4C)(Cell Signaling, 12741S, Rabbit mAb), Thr-AMPylation (Biointron, 17G6, mAb), Secondary 647 Fluorescent antibody (ThermoFisher, A32728).

Techniques:

Journal: bioRxiv

Article Title: FICD deficiency protects mice from hypertrophy-induced heart failure via BiP-mediated activation of the UPR ER and ER-phagy

doi: 10.1101/2024.05.28.596287

Figure Lengend Snippet: (A) Primary WT and Ficd -/- cardiomyocytes were isolated from 6-8 mice hearts per condition and plated in 24-well dishes for 48 hours at 37°C and 5% CO2. Cardiomyocytes were treated with Tm (10μg/mL) or Tg (3μM) for 10 hours and supplemented with bafilomycin (100nM) for four hours before being collected for protein quantification via western blotting. Cells were run on 15% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for LC3, FAM134B, and HSC70 (loading control) levels. Levels of (B) LC3-II/HSC70, (C) LC3-II/LC3-I, and (D) FAM134B/HSC70 were quantified from control, ER-stressed, and bafilomycin treated cardiomyocytes. (E) Primary WT and Ficd -/- cardiomyocytes were isolated from 6-8 mice hearts per condition and plated in 6-well dishes for 48 hours at 37°C 5% CO2. Cardiomyocytes were treated with Tm (10μg/mL) or Tg (3μM) for 12 hours before being collected for protein quantification via western blotting. Cells were run on 10% Tris-Glycine SDS acrylamide gels, blocked with 5% BSA, and probed for CtsL, CtsB, CtsD, LAMP1, and HSC70 (loading control) levels. Levels of (F) CtsL/HSC70, (H) CtsB/LC3-I, (I) CtsD/HSC70, and (J) LAMP1/HSC70 were quantified from control and ER-stressed cardiomyocytes. (G) CtsL activity from 50μg of isolated WT and Ficd -/- cardiomyocytes from non-treated and CtsL inhibitor treated cells. (K) Transmission Electron Microscopy images of ER whorls from isolated WT and Ficd -/- cardiomyocytes. (Scale bar, 200nm). (L) Co-immunoprecipitation of FAM134B-BiP from control, Tg-treated, and FICD(E324G) transfected WT and Ficd -/- cardiomyocytes. FAM134B antibody was incubated with 150μg of protein from each cardiomyocyte condition and immunoprecipitated with Protein A Magnetic agarose beads. 10% input and the IP-ed proteins were run on 10% Tris-Glycine acrylamide gels, blocked with 5% BSA, and probed for Thr-AMPylation, BiP, FAM134B, and HSC70 (loading control). (M) Quantification of three replicate co-immunoprecipitations of BiP/FAM134B from control, Tg, and FICD(E324G) transfected WT and Ficd -/- cardiomyocytes. Indicated P values were calculated using unpaired t tests. 2-way ANOVA tests were performed in parallel to confirm significance of results.

Article Snippet: The following antibodies were used for western blotting and immunofluorescence studies: Alpha-actinin (D6F6)(Cell Signaling, 6487T, Rabbit mAb), BiP (Proteintech, 665741IG, mouse mAb), CHOP (D46F1)(Cell Signaling, 5554S, Rabbit mAb), Cathepsin B (D1C7Y)(Cell Signaling, 31718S, Rabbit mAb), Cathepsin D (E179)(Cell Signaling, 69854S, Rabbit mAb), Cathepsin L (Santa Cruz Biotechnology, sc-32320, mouse mAb), FAM134B (E8Y9R)(Cell Signaling, 83414S, Rabbit mAb), GAPDH (Proteintech, 600041IG, mouse mAb), HSC70 (B-6)(Santa Cruz Biotechnology, sc-7298, mAb), LAMP1 (E6N3R)(Cell Signaling, 46843S, Rabbit mAb), LC3A/B (D3U4C)(Cell Signaling, 12741S, Rabbit mAb), Thr-AMPylation (Biointron, 17G6, mAb), Secondary 647 Fluorescent antibody (ThermoFisher, A32728).

Techniques: Isolation, Western Blot, Activity Assay, Transmission Assay, Electron Microscopy, Immunoprecipitation, Transfection, Incubation