Journal: Cells

Article Title: Identification of the Cysteine Protease Legumain as a Potential Chronic Hypoxia-Specific Multiple Myeloma Target Gene

doi: 10.3390/cells11020292

Figure Lengend Snippet: CRISPR/Cas9-based depletion of LGMN in MM cells confers enhanced growth disadvantage under chronic hypoxia: ( A ) Left: Competition growth assay of RPMI-8226 LGMN KO cells (sgRNA1, 3, 4 and 1 + 4) using NTC as a negative control and c-myc KO as a positive control in hypoxia (1% O 2 ). The percentage of GFP-positive cells measured via flow cytometry is normalized to NTC and day 0. Error bars indicate mean ± SEM of three technical replicates. Right: Representative Western blot of LGMN KO in RPMI-8226 cells used in the competition assay. Loading control: Nucleolin. ( B ) Left: Cumulative growth assay of RPMI-8226 LGMN KO (sgRNA5, 8) and NTC cells in normoxia (21% O 2 , blue colors) and hypoxia (1% O 2 , red colors). Error bars indicate mean ± SEM of three technical replicates. Right: Representative Western blot of LGMN KO in RPMI-8226 cells used in the cumulative growth assay. Loading control: Vinculin. ( C ) Viability assay in RPMI-8226 cells of 3 independent experiments after treatment with LGMN inhibitor 10t for 48 h under normoxia and hypoxia. Viability in [%] measured by NAD(P)H production. IC 50 is represented by black dashed line by blue dotted line in the graph and hypoxia by red dotted line. Error bars indicate the mean ± SEM of three biological replicates. ( D ) Apoptosis assay. Annexin V-PE-positive (apoptotic) RPMI-8226 cells [% of total cells] four and six days post transduction with sgRNA NTC, sgRNA LGMN(5) and sgRNA LGMN(8) in normoxic conditions. Bar graphs represent the mean ± SD of two independent experiments. ( E ) Rescue experiment using a pro-LGMN WT construct (LGMN Wt SIHW) in RPMI-8226 NTC and LGMN KO (sgRNA5) cells under normoxic conditions. Bar graphs represent the cumulative, relative cell number after 12 days of 3 independent experiments. Two-way-ANOVA with Bonferroni’s post-hoc test. *, p < 0.05; ***, p < 0.001. Values of p > 0.05 were considered not significant (ns). Abbreviations: p.t. = post transduction.

Article Snippet: A mouse monoclonal antibody against Vinculin (#ab18058) was purchased from Abcam (Cambridge, UK).

Techniques: CRISPR, Growth Assay, Negative Control, Positive Control, Flow Cytometry, Western Blot, Competitive Binding Assay, Control, Viability Assay, Apoptosis Assay, Transduction, Construct

Journal:

Article Title: The Shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets

doi: 10.1002/dvdy.21942

Figure Lengend Snippet: (A) Schematic depicting the sections of embryo imaged for B through G. (B) Lateral line placode, (C) Proctodeum, (D) Cement gland (phalloidin) and (E) Otic vesicle. green is α-tubulin, red is propidium iodide in panel B, C, E. green is phalloidin in panel D. (F–G) Fluorescence images of in situ hybridization against Shroom3, DIC: differential interference contrast image, α-tubulin: image stained with α-tubulin antibody, NBT/BCIP: NBT/BCIT precipitate following in situ hybridization by Shroom3 probe. It was detected by confocal microscopy (F) otic vesicle (G) lateral line placode. Scale bar = 50 µm.

Article Snippet: Monoclonal anti-α-tubulin antibody (1:300 dilution, DM1A, Sigma) and rabbit polyclonal anti-γ-tubulin antibodies (1:300 dilution, Abcam) were used for primary antibodies and Alex Fluor-488 goat anti-mouse or anti-rabbit IgG was used for secondary antibody (Invitrogen, 1:300 dilution).

Techniques: Fluorescence, In Situ Hybridization, Staining, Confocal Microscopy

Journal:

Article Title: The Shroom family proteins play broad roles in the morphogenesis of thickened epithelial sheets

doi: 10.1002/dvdy.21942

Figure Lengend Snippet: (A–C) Ectopic Shroom2 induces cell heightening in naïve epithelial cells. (A) Control (B) Shroom2 expressing cell (green: γ-tubulin, red: myc-Shroom2) (C) Graph of apicobasal cell height of control and Shroom2 expressing cells (mean ± s.e.m; ctl, n=16;Shroom2, n=15). (D–H) Ectopic Shroom2 expression induces cell heightening in epidermal cells. (D) Control, (E) Shroom2 expressing and (F) Shroom3 expressing epidermis (green: α-tubulin, red: myc-tagged Shroom2 or 3). (G) Graph of apicobasal cell height in Shroom2 expressing epidermal cells (mean ± s.e.m: ctl, n=25;Shroom2, n=25). (H) Graph of apicobasal cell height in Shroom3 expressing epidermal cells (mean ± s.e.m: ctl, n=39;Shroom3, n=40). Scale bar = 20 µm. Yellow arrow bars are showing the way to measure the cell height for control cells (neighboring cells) and pink arrow bars are showing that for Shroom expressing cells. (I–L) Loss of function assay for Shroom2 in neural plate. Transverse cross-section view of neural plate (green: α-tubulin, red: propidium iodide). (I) One side-Shroom2 mismatched morpholino (MM) injected embryo. (J) One side-Shroom2 morpholino (MO) injected embryo. The high magnification views of control side cells (K) and Shroom2 MO injected side cells (L). Scale bar = 50 µm.

Article Snippet: Monoclonal anti-α-tubulin antibody (1:300 dilution, DM1A, Sigma) and rabbit polyclonal anti-γ-tubulin antibodies (1:300 dilution, Abcam) were used for primary antibodies and Alex Fluor-488 goat anti-mouse or anti-rabbit IgG was used for secondary antibody (Invitrogen, 1:300 dilution).

Techniques: Control, Expressing, Functional Assay, Injection

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were transfected with either control mimic (con) or miR-30b mimic (30b) and levels of TGFβ1 and TGFβ2 mRNA were assessed by qRT-PCR. Expression levels relative to control mimic transfected cells and normalized to β-actin expression are presented as the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 expression. * P < 0.05, ** P < 0.01 as determined by unpaired Student’s t -test. (B) Cells were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of TGFβ2 protein levels by western blot. β-actin was used as endogenous control. (C) ELISAs for TGFβ1 and TGFβ2 were performed with 24 hour conditioned supernates from HUVECs transfected with 20 nM of either control or miR-30b mimic. Data represents the mean ± SEM (n = 2). Overexpression of miR-30b significantly increases TGFβ2 secretion into cell culture supernate. * P = 0.044 as determined by unpaired Student’s t -test. (D) HUVECs were transfected with 20 nM of either control mimic (control) or miR-30b mimic (miR-30b) and protein lysates were collected after 48 hours for assessment of Smad2 phosphorylation by western blot.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Transfection, Control, Quantitative RT-PCR, Expressing, Over Expression, Western Blot, Cell Culture, Phospho-proteomics

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) JDP2 mRNA expression was assessed in HUVECs transfected with miR-30b mimic (20 nM) as compared to control by qRT-PCR. Data represents the mean ± SEM (n = 3) normalized to β-actin as endogenous control. * P = 0.016 as determined by unpaired Student’s t -test. (B) HUVEC were transfected with 50 nM of either control siRNA or ATF2 siRNA 1 or 2 and RNA was isolated at 48 hours post transfection. Levels of ATF2 and TGFβ2 mRNA were assessed by qRT-PCR with β-actin as endogenous control. Data presented is mean ± SEM (n = 2). Statistically significant decreases in ATF2 and TGFβ2 expression were seen in ATF2 siRNA treated cells as compared to control siRNA treated cells. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by unpaired Student’s t -tests for each ATF2 siRNA compared to control siRNA. (C) Cells transfected with 5 nM of either control siRNA or ATF2 siRNA 1 were seeded onto growth factor reduced BME and the formation of capillary-like cord structures and number of loops was assessed after 24 hours. (D) A statistically significant increase in cord formation was observed in cells depleted of ATF2 through siRNA. Data represents the mean ± SEM (n = 2). * P = 0.041 as determined by unpaired Student’s t -test. (E) HUVECs were co-transfected with miRNA mimic (20 nM) and ATF2 siRNA 1 or 2 (50 nM) in the combinations displayed and cell lysates were collected at 48 hours post transfection and assessed for TGFβ2 mRNA expression. Data presented is mean ± SEM (n = 2). * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA with post hoc analysis. (F) Cells were transfected as in (E) using miRNA mimic (20 nM) and ATF2 siRNA 1 (5 nM) and serum starved overnight in MCDB 131 with 0.5% FBS prior to protein expression analysis by western blot. Data is representative of expression levels observed in two independently performed experiments.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Transfection, Control, Quantitative RT-PCR, Isolation, Western Blot

Journal: PLoS ONE

Article Title: MicroRNA-30b controls endothelial cell capillary morphogenesis through regulation of transforming growth factor beta 2

doi: 10.1371/journal.pone.0185619

Figure Lengend Snippet: (A) HUVECs were serum starved overnight in MCDB 131 with 0.5% FBS and stimulated with VEGF (50 ng/ml) in the presence or absence of Avastin (1 μg/ml) for 24 hours. Data represents the mean ± SEM (n = 2) for expression of TGFβ1 and TGFβ2 assessed by qRT-PCR relative to β-actin endogenous control. * P < 0.05, ** P < 0.01, *** P < 0.001 as determined by ANOVA. (B) HUVECs were treated with 5 ng/ml of TGFβ2 for 3 days prior to seeding onto growth factor reduced BME for assessment of capillary-like cord formation after 24 hours. (C) A significant decrease in cord formation is observed in the TGFβ2 treated group. Data represents the mean ± SEM (n = 2). ** P = 0.0072 as determined by unpaired Student’s t -test. (D) HUVECs transfected with 1 nM control or miR-30b mimic were treated 4 hours post transfection with 0.8 μg/ml anti-TGFβ2 neutralizing antibody or rabbit IgG. Media was refreshed after 24 hours, again with rabbit IgG or anti-TGFβ2 antibody and cells were seeded onto growth factor reduced BME 24 hours later (ie. 48 hours post transfection) in media containing rabbit IgG or anti-TGFβ2 antibody. (E) Data represents the mean ± SEM (n = 3) of the number of capillary-like cord structures or number of loops formed after 24 hours on BME. * P < 0.05, ns denotes not significant as determined by ANOVA with post hoc analysis.

Article Snippet: Primary antibodies used were: TGFβ2 (V, SC-90), ATF-2 (C-19, SC-187), and phospho-ATF-2 (F-1, SC-8398) from Santa Cruz Biotechnology (Santa Cruz, CA), phospho-Smad2 (S465/467) from Cell Signaling Technology (3101; Danvers, MA), Smad2 from Invitrogen (511300; Carlsbad, CA), β-Actin (clone AC-74) from Sigma-Aldrich (A5316; St. Louis, MO), anti-TGFβ2 neutralizing antibody (AB-12-NA) and Normal Rabbit IgG (AB-105-C) from R&D Systems (Minneapolis, MN).

Techniques: Expressing, Quantitative RT-PCR, Control, Transfection

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) Chemical structure of DOTA-cycratide. (B) Inhibition of 64Cu-cycratide binding to integrin αvβ6 on BxPC-3 cells by cycratide, DOTA-cycratide, and linear-pep. Data are shown as mean ± SD, n = 4. (C) Binding of 68Ga-cycratide to BxPC-3 with or without blocking of cold cycratide or linear-pep. %AD/106 cells = percentage of total added dose per million cells. Data are shown as mean ± SD, n = 4. (D) Metabolic stability of 68Ga-cycratide in blood and urine of BALB/c mice (data are representative of 3 independent experiments). **P < 0.01.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Inhibition, Binding Assay, Blocking Assay

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET imaging of orthotopic pancreatic cancer lesions in nude mice at 0.5 h after injection of 68Ga-cycratide without or with blocking dose of cold cycratide. Tumors are indicated by arrows. (B) Hematoxylin and eosin (H&E) staining of tumor tissues harvested from orthotopic tumor model. (C) Left, immunofluorescence staining of integrin αvβ6 from tumor tissues harvested from orthotopic tumor model. Right, negative control with secondary antibody only.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Imaging, Injection, Blocking Assay, Staining, Immunofluorescence, Negative Control

Journal: Journal of Nuclear Medicine

Article Title: Clinical Translation of a 68 Ga-Labeled Integrin α v β 6 –Targeting Cyclic Radiotracer for PET Imaging of Pancreatic Cancer

doi: 10.2967/jnumed.119.237347

Figure Lengend Snippet: (A) PET/CT images of female patient with suspected pancreatic cancer. Images were obtained at 1 h after intravenous administration of 68Ga-cycratide or 18F-FDG. Tumors are indicated by arrows. (B) Immunohistochemical (IHC) staining for integrin αvβ6 in tumor sample from same patient as in A. (C) Contrast-enhanced CT (CECT) image and PET/CT images of male patient with pancreatic cancer 7 mo after surgery at 1 h after administration of 68Ga-cycratide or 18F-FDG, as well as CECT image of same patient 3 mo later (10 mo after surgery). Occupancy lesions in CT are indicated by arrows.

Article Snippet: After endogenous peroxidase activity had been abolished using 0.3% hydrogen peroxide and antigen had been retrieved by microwave, tumor tissues were incubated with rabbit antihuman integrin α v β 6 antibody (bs-5791r; Bioss) overnight at 4°C.

Techniques: Positron Emission Tomography-Computed Tomography, Immunohistochemical staining, Immunohistochemistry

Journal: Animal Biotechnology

Article Title: Effect of Aeromonas hydrophila infection on leptin receptor overlapping transcript expression in Rana amurensis

doi: 10.1080/10495398.2024.2410742

Figure Lengend Snippet: Relative LepROT expression levels in different tissues of Rana amurensis infected (treatment group) or not (control group) with Aeromonas hydrophila . (A–H) LepROT transcripts in different tissues of Rana amurensis challenged with Aeromonas hydrophila. Each infected Rana amurensis was injected intraperitoneally with 1 mL bacterial suspension (1.5 × 10 7 CFU/mL), whereas the control group was injected with the same volume of sterile tryptone liquid medium. (A) The heart. (B) The liver. (C) The spleen. (D) The lung. (E) The kidney. (F) The skin. (G) The muscular. (H) The stomach. The samples were collected at different time points after Aeromonas hydrophila challenge (x-axis). β-actin was used as an internal control. Error bars represent one standard error. *, **, indicate a significant difference between the treatment and control group at p < 0.05 and p < 0.01, respectively.

Article Snippet: Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene fluoride (PVDF) membranes, where they were blocked with blocking buffer for 15 min (The above reagents are purchased from Beyotime Biotechnology, Jiangsu, China) and subsequently stained with specific primary antibodies of nuclear factor κB p65 subunit (NF-κB p65)(1/5000), (bs-0465R, rabbit, Bioss Antibodies, Beijing, China) and β-actin (1/5000), (bsm-33036M, mouse, Bioss Antibodies, Beijing, China) overnight at 4 °C.

Techniques: Expressing, Infection, Control, Injection, Suspension, Sterility

Journal: Animal Biotechnology

Article Title: Effect of Aeromonas hydrophila infection on leptin receptor overlapping transcript expression in Rana amurensis

doi: 10.1080/10495398.2024.2410742

Figure Lengend Snippet: Relative nuclear factor-kappa B (NF-кB), IκB kinase (IKKα, and IKKβ) expression levels in different tissues of Rana amurensis infected (treatment group) or not (control group) with Aeromonas hydrophila . (A–H) NF-кB, IKKα and IKKβ transcripts in different tissues of Rana amurensis challenged with Aeromonas hydrophila . Each infected Rana amurensis was injected intraperitoneally with 1 mL bacterial suspension (1.5 × 10 7 CFU/mL), whereas the control group was injected with the same volume of sterile tryptone liquid medium. (A) The heart. (B) The liver. (C) The spleen. (D) The lung. (E) The kidney. (F) The skin. (G) The muscular. (H) The stomach. The samples were collected at different time points after Aeromonas hydrophila challenge (x-axis). β-actin was used as an internal control. Error bars represent one standard error. *, **, indicate a significant difference between the treatment and control group at p < 0.05 and p < 0.01, respectively.

Article Snippet: Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene fluoride (PVDF) membranes, where they were blocked with blocking buffer for 15 min (The above reagents are purchased from Beyotime Biotechnology, Jiangsu, China) and subsequently stained with specific primary antibodies of nuclear factor κB p65 subunit (NF-κB p65)(1/5000), (bs-0465R, rabbit, Bioss Antibodies, Beijing, China) and β-actin (1/5000), (bsm-33036M, mouse, Bioss Antibodies, Beijing, China) overnight at 4 °C.

Techniques: Expressing, Infection, Control, Injection, Suspension, Sterility

Journal: Animal Biotechnology

Article Title: Effect of Aeromonas hydrophila infection on leptin receptor overlapping transcript expression in Rana amurensis

doi: 10.1080/10495398.2024.2410742

Figure Lengend Snippet: Western blotting tissue sections obtained from Rana amurensis infected (treatment group) or not (control group) with Aeromonas hydrophila ( Ah ). Liver tissues were collected, and the protein levels of NF-κB p65 and β-actin were analyzed by Western blotting. LB, 24h and 72h respectively represent the protein expression levels of NF-κB p65 and β-actin in the liver tissue of Rana amurensis infected with Ah.

Article Snippet: Protein samples were separated by 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and were transferred onto polyvinylidene fluoride (PVDF) membranes, where they were blocked with blocking buffer for 15 min (The above reagents are purchased from Beyotime Biotechnology, Jiangsu, China) and subsequently stained with specific primary antibodies of nuclear factor κB p65 subunit (NF-κB p65)(1/5000), (bs-0465R, rabbit, Bioss Antibodies, Beijing, China) and β-actin (1/5000), (bsm-33036M, mouse, Bioss Antibodies, Beijing, China) overnight at 4 °C.

Techniques: Western Blot, Infection, Control, Expressing

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 1: Comparison on the down-regulation of Moutan Cortex from different regions on protein expression levels of ICAM-1,TGF-β1 and FN. HBZY-1 mesangial cells were treated with 200 μg/mL AGEs in the presence or absence of Moutan Cortex (MC) extract of 200 μg/mL. Aminoguanidine of 10 μM was used as the positive control while BSA (200 μg/mL) as blank control. (A) Western blotting was performed to compare the protein expression levels. (B–D) The grayscale scan results of ICAM-1, TGF-β1 and FN. a, Control; b, 200 μg/mL AGEs; c, Positive control aminoguanidine group; “d-m” represent MC from Anhui, Guizhou, Zhejiang, Henan, Hunan, Hebei, Sichuan, Chongqing, Shandong, Gansu. Data are expressed as means ± SD, n = 3. ###P < 0.001 vs. BSA group; *P < 0.05, **P < 0.01 and ***P < 0.001 vs. AGEs group; $P < 0.05, $$P < 0.01 and $$$P < 0.001 vs. MC from Anhui group.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Comparison, Expressing, Positive Control, Control, Western Blot

Journal: Oncotarget

Article Title: Structural composition of components of geoherb Moutan Cortex contributes to anti-diabetic nephropathy activity

doi: 10.18632/oncotarget.23771

Figure Lengend Snippet: Figure 2: Effect of Moutan Cortex from different regions on ICAM-1 and TGF-β1 protein expression levels in kidney of DN rats. After being treated with STZ and/or MC extract of 5g/kg or , immunohistochemistry was conducted to evaluate the expression levels of ICAM-1 (A) and TGF-β1 (B) of renal tissues. “a” represents normal control; “b” represents model group (DN rats); “c” represents Positive control 0.1 g/kg AG; “d-m” represent Gansu, Chongqing, Shangdong, Sichuan, Zhejiang, Anhui, Hunan, Guizhou, Hebei, Henan.

Article Snippet: Rabbit anti-mouse FN, TGF-β1 and ICAM-1 monoclonal antibodies were offered by Boster Biological Engineering Co., Ltd. (Wuhan, China).

Techniques: Expressing, Immunohistochemistry, Control, Positive Control