Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A – D ) ECFC characterization and CD44 sorting and KD. ( A ) Representative images of confluent ECFC colonies taken at 5× (top) and 10× (bottom) original magnification. Scale bar: 500 μm (top), 200 μm (bottom). ( B ) Immunophenotypic characterization of ECFCs. Representative flow cytometry histograms of ECFCs demonstrated positive expression of CD13, CD31, CD105, and HLA-ABC and negative expression of hematopoietic markers CD14 and CD45, mesenchymal stem cell marker CD90, as well as HLA-DR (right-shifted, black-filled curves in comparison with gray-filled curves representing the appropriate isotype controls; n = 3 replicates). ( C ) Representative gating strategy to sort CD44 hi and CD44 lo ECFCs using FACS. ( D ) Flow cytometric analysis of CD44 in ECFCs-shCD44 (black-filled curves) and ECFCs-scrRNA (gray-filled curves) following lentivirus-mediated transduction of ECFCs with shCD44. ( E – H ) EV isolation protocol, yield, morphology, and immunophenotype. ( E ) Schematic of UF-SEC-UF protocol. ( F ) UF-SEC-UF obtained a significantly higher EV yield than differential UC. Two-tailed Student’s t test; n = 4 EV isolations. Error bars represent SEM. ( G ) TEM of EV samples isolated via differential UC (left) or UF-SEC-UF (right). Differential UC samples demonstrated aggregation of macromolecules (red arrows) and EVs (yellow arrow). UF-SEC-UF produced EV samples devoid of contaminating aggregates. Scale bars: 0.2 μm. ( H ) Representative magnetic bead–assisted flow cytometry histograms of EVs hi and EVs lo . Both populations positively expressed tetraspanins CD9, CD63, and CD81, as well as endothelial marker CD31 (right-shifted, black filled curves compared with gray-filled curves of negative control samples; n = 3 replicates). * P < 0.05. MWCO, molecular weight cutoff.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Flow Cytometry, Expressing, Marker, Comparison, Transduction, Isolation, Two Tailed Test, Produced, Negative Control, Molecular Weight

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A and B ) CD44 hi ECFCs rescued OIR mice. ( A ) Representative images and ( B ) quantification of NV (red) and VO (yellow) of retinal flat mounts from OIR mice. One-way ANOVA with Tukey’s; n = 7 retinas for CD44 hi ECFCs, n = 7 retinas for CD44 lo ECFCs, n = 5 retinas for HUVECs. ( C and D ) EVs hi rescued OIR mice. ( C ) Representative images and ( D ) quantification of retinal flat mounts. Inserts in A and C depict the original unquantified images; scale bars: 1 mm. Additional controls included EVs lo , HUVEC EVs, nonconditioned ECFC and HUVEC media subjected to UF-SEC-UF (XFM UF-SEC-UF and M200 UF-SEC-UF, respectively), and EVs hi sample depleted of vesicles via overnight UC (EVs hi depleted). Data in D are represented as a box-and-whisker plot where the top and bottom of the box represent mean of the upper and lower quartiles, horizontal line within the box represents the mean, and bars outside the box represent the min and max data points. One-way ANOVA with Tukey’s; n = 100 retinas for EVs hi , n = 102 retinas for EVs lo , n = 10 retinas for HUVEC EVs, n = 11 retinas for XFM UF-SEC-UF, n = 11 retinas for M200 UF-SEC-UF, n = 12 retinas for EVs hi depleted. ( E ) Pharmacologic exosome inhibition of CD44 hi ECFCs via GW4869 (20 μM) in DMSO (+GW4869, n = 12 retinas) attenuated the effects of CD44 hi ECFCs compared with cells incubated with DMSO alone (-GW4869, n = 12 retinas). Two-tailed Student’s t test. ( F and G ) ECFCs-shCD44 and their EVs failed to rescue OIR mice. Quantification of NV and VO in mice injected with ( F ) ECFCs-scrRNA ( n = 10 retinas) versus ECFCs-shCD44 ( n = 11 retinas) and ( G ) EVs from ECFCs-scrRNA ( n = 10 retinas) versus EVs from ECFCs-shCD44 ( n = 11 retinas). Two-tailed Student’s t test. ( H ) Dose-response curve of OIR mice injected with EVs hi . Mice were treated with a starting dose of 1.25 × 10 6 particles/0.5 μL/eye and serial 10-fold dilutions. Kruskal-Wallis test with Dunn’s multiple-comparison test; n = 6–9 retinas per group. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Whisker Assay, Inhibition, Incubation, Two Tailed Test, Injection, Comparison

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: DICER1 KD attenuated rescue effects of CD44 hi ECFCs in A and B and their EVs in C and D in OIR mice. ( A ) Representative images and ( B ) quantification of NV and VO in retinal flat mounts from OIR mice demonstrated that CD44 hi ECFCs-shDICER1 failed to rescue NV relative to mice treated with scrRNA-transfected control cells (CD44 hi ECFCs-scrRNA). ( C ) Representative images and ( D ) quantification of retinal flat mounts demonstrated that EVs from CD44 hi ECFCs-shDICER1 failed to rescue both NV and VO relative to mice treated with EVs from CD44 hi ECFCs-scrRNA EVs. Two-tailed Student’s t test; n = 10 retinas for CD44 hi ECFCs-scrRNA, n = 9 retinas for CD44 hi ECFCs-shDICER1, n = 28 retinas for CD44 hi ECFCs-scrRNA EVs, n = 18 retinas for CD44 hi ECFCs-DICER1 EVs. Error bars represent SEM. * P < 0.05, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: Transfection, Control, Two Tailed Test

Journal: JCI Insight

Article Title: Bioactive extracellular vesicles from a subset of endothelial progenitor cells rescue retinal ischemia and neurodegeneration

doi: 10.1172/jci.insight.155928

Figure Lengend Snippet: ( A ) Heatmap of differentially expressed ( q < 0.05) miRs on small RNA sequencing of EVs hi (H, n = 2) and EVs lo (L, n = 3). ( B ) Injection of miR mimics upregulated in EVs hi in A rescued NV (miR-7-5p, miR-26a-3p miR-30a-5p, miR-216a-3p, miR-381-3p, miR-503-5p) and VO (miR-30a-5p, miR-216a-3p, miR-503-5p) compared to scrmiR-injected controls. n = 12–16 retinas for miR mimics, n = 72 retinas for scrmiR. ( C ) Combinatorial injection of miR mimics rescued OIR mice dose-dependently. “Candidate miRs” miR-7-5p, miR-23a-3p, miR-216a-3p, and miR-503-5p were upregulated on small RNA sequencing in A , validated on RT-qPCR, and functional in rescuing OIR mice in B . Combination injection of the 2 most effective miR mimics (miR-216a-3p and miR-503-5p) in B and, separately, all candidate miR mimics rescued OIR mice. n = 9–14 retinas for miR-216a-3p and miR-503-5p, n = 11–16 retinas for all candidate miRs, n = 24 retinas for scrmiR. ( D ) EVs from CD44 hi ECFCs with KD expression of individual or combinatorial miRs no longer rescued OIR mice. Multiple lines of ECFCs were generated with KD expression of both miR-216a-3p and miR-503-5p, all candidate miRs together, and each candidate miR individually. EVs from miR-23a-3p KD CD44 hi ECFCs failed to rescue VO; EVs from miR-503-5p KD CD44 hi ECFCs failed to rescue NV; and EVs from CD44 hi ECFCs with KD expression of all candidate miRs failed to rescue both NV and VO in OIR mice. n = 11–20 retinas for individual miR KD, n = 7 retinas for miR-216a-3p and miR-503-5p KD, n = 12 for all candidate miR KD, n = 18 for scrmiR KD, n = 20 for PBS. One-way ANOVA with Tukey’s analysis. Error bars represent SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: CD44 expression was knocked down in ECFCs using green fluorescent protein (GFP) lentiviral shRNA clones to CD44 (CMV-Neo, GenTarget Inc., clones TRCN0000308110 and TRCN0000296190) to generate ECFCs-shCD44.

Techniques: RNA Sequencing Assay, Injection, Quantitative RT-PCR, Functional Assay, Expressing, Generated

Journal: Journal for Immunotherapy of Cancer

Article Title: Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

doi: 10.1136/jitc-2019-000195

Figure Lengend Snippet: VPA and vorinostat enhance trastuzumab-mediated ADCP. SKBR3 cells were treated with VPA or SAHA, for 24 hours, following the ADCP/ADCC assay as described in and measured by FCM. (A) ADCP | VPA (B) ADCP | vorinostat. Results are illustrated by scatter plots. Circles, boxes and triangles illustrate individual measured values. % ADCP is depicted on the ordinate, different treatments and corresponding concentrations are indicated on the abscissa. Mean ADCP % is illustrated by a long horizontal line ±SD by short horizontal lines connected with a vertical line. Differences were calculated by repeated-measure one-way ANOVA and Fisher LSD post hoc test. **p<0.01; ***p<0.001; ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cell-mediated phagocytosis; ANOVA, analysis of variance; FCM, flow cytometry; SAHA, suberanilohydroxamic acid; Tras, trastuzumab; VPA, valproic acid.

Article Snippet: Vorinostat (Zolinza), also known as suberanilohydroxamic acid (SAHA; Selleckchem, Munich, GER) was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc.).

Techniques: ADCC Assay, Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

doi: 10.1136/jitc-2019-000195

Figure Lengend Snippet: Valproic acid and vorinostat enhance trastuzumab-independent cytotoxicity. SKBR3 cells were treated with VPA or SAHA, for 24 hours, following the ADCP/ADCC as described in and measured by FCM. (A) ADCP | valproic acid (B) ADCP | vorinostat. Results are illustrated by scatter plots. Circles, boxes and triangles illustrate individual measured values. % ADCC is depicted on the ordinate, different treatments and corresponding concentrations are indicated on the abscissa. Mean % ADCC is illustrated by a long horizontal line ±SD by short horizontal lines connected with a vertical line. Differences were calculated by repeated-measure one-way ANOVA and Fisher LSD post hoc test. **p<0.01; ***p<0.001; ADCC, antibody-dependent cell-mediated cytotoxicity; ADCP, antibody-dependent cell-mediated phagocytosis; ANOVA, analysis of variance; FCM, flow cytometry; LSD, least significantdifference; SAHA, suberanilohydroxamic acid; VPA, valproic acid.

Article Snippet: Vorinostat (Zolinza), also known as suberanilohydroxamic acid (SAHA; Selleckchem, Munich, GER) was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc.).

Techniques: Flow Cytometry

Journal: Journal for Immunotherapy of Cancer

Article Title: Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

doi: 10.1136/jitc-2019-000195

Figure Lengend Snippet: VPA increases expression of the activating FcγRIIA on monocytes. SKBR3 cells were treated with VPA or SAHA, for 24 hours, without CFSE labeling of SKBR3 cells and without trastuzumab. (A) FcγRI, (B) FcγRIIA, (C) FcγRIIB and (D) FcγRIII expressions were investigated on monocytes (CD14+) after the 2.5-hour incubation time of the ADCP/ADCC assay and measured by FCM. Results are illustrated by scatter plots. Circles, boxes and triangles illustrate individual measured values. Each FcγR MFI is depicted on the ordinate, different treatments and corresponding concentrations are indicated on the abscissa. Mean MFI for each marker is illustrated by a long horizontal line ±SD by short horizontal lines connected with a vertical line. Corresponding overlay histograms are illustrated. Differences were calculated by repeated-measures one-way ANOVA and Fisher LSD post hoc test. **p<0.01; CFSE, carboxyfluorescein diacetate succinimidyl ester; FcγR, Fc gamma receptor; FI, fluorescence intensity; MFI, mean fluorescence intensity; SAHA, suberanilohydroxamic acid; VPA, valproic acid.

Article Snippet: Vorinostat (Zolinza), also known as suberanilohydroxamic acid (SAHA; Selleckchem, Munich, GER) was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc.).

Techniques: Expressing, Labeling, Incubation, ADCC Assay, Marker, Fluorescence

Journal: Journal for Immunotherapy of Cancer

Article Title: Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

doi: 10.1136/jitc-2019-000195

Figure Lengend Snippet: VPA downregulates the anti-apoptotic protein MCL1 in tumor cells. SKBR3 cells were treated with VPA or SAHA, for 24 hours. (A) MCL1 expression determined by FCM. corresponding overlay histograms are illustrated. (B) MCL1 expression after MCL1 knockdown. MCL1 expression levels were determined by FCM. Corresponding overlay histograms are illustrated. (C) % ADCP after MCL1 knockdown with trastuzumab in SKBR3 cells. ADCP/ADCC assay was performed as described in and measured by FCM. Results are illustrated by scatter plots. Circles, boxes and triangles illustrate individual measured values. MCL1 MFI and % ADCP are depicted on the ordinate, different treatments and corresponding concentrations are indicated on the abscissa. Mean MCL1 MFI and mean % ADCP are illustrated by a long horizontal line ±SD by short horizontal lines connected with a vertical line. Differences were calculated by a one-way ANOVA and Fisher LSD post hoc test and unpaired Student t-test. *p<0.05; **p<0.01; ****p<0.0001; ADCP, antibody-dependent cell-mediated phagocytosis; ANOVA, analysis of variance; FCM, flow cytometry; FI, fluorescence intensity; LSD, least significantdifference; MCL1, myeloid leukemia cell differentiation protein 1; MFI, mean fluorescence intensity; SAHA, suberanilohydroxamic acid; SCR: scrambled; siRNA, small interfering RNA, Tras: trastuzumab; VPA, valproic acid.

Article Snippet: Vorinostat (Zolinza), also known as suberanilohydroxamic acid (SAHA; Selleckchem, Munich, GER) was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc.).

Techniques: Expressing, Knockdown, ADCC Assay, Flow Cytometry, Fluorescence, Cell Differentiation, Small Interfering RNA

Journal: Journal for Immunotherapy of Cancer

Article Title: Histone deacetylase inhibitors valproic acid and vorinostat enhance trastuzumab-mediated antibody-dependent cell-mediated phagocytosis

doi: 10.1136/jitc-2019-000195

Figure Lengend Snippet: VPA and vorinostat induce an ICD and downregulates anti-phagocytic CD47 in tumor cells. SKBR3 cells were treated with VPA or SAHA, for 24 hours. (A, B) Calreticulin (CALR) and (C, D) CD47 expression levels were determined by FCM. Results are illustrated by scatter plots. Circles, boxes and triangles illustrate individual measured values. MCL1 and CD47 MFI are depicted on the ordinate, different treatments and corresponding concentrations are indicated on the abscissa. Mean CALR and CD47 MFI are illustrated by a long horizontal line ±SD by short horizontal lines connected with a vertical line. Corresponding overlay histograms are illustrated. Differences were calculated by a one-way ANOVA and Fisher’s LSD post hoc test. **p<0.01; ****p<0.0001; ANOVA, one-way analysisof variance; CALR, calreticulin; CD47, clusters of differentiation 47; FCM, flow cytometry; FI, fluorescence intensity; ICD, immunogenic cell death; LSD, least significantdifference; MCL1, myeloid leukaemia cell differentiation protein1; MFI, mean fluorescenceintensity; SAHA, suberanilohydroxamic acid; VPA, valproic acid.

Article Snippet: Vorinostat (Zolinza), also known as suberanilohydroxamic acid (SAHA; Selleckchem, Munich, GER) was dissolved in dimethyl sulfoxide (DMSO; Thermo Fisher Scientific Inc.).

Techniques: Expressing, Flow Cytometry, Fluorescence, Cell Differentiation

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 1. MARCKS KD impairs dedifferentiated VSMC migration. Human CASMCs were transfected with 100 nM of MARCKS siRNA (KD) or nontargeting, control siRNA (Control). A, At 72 hours post-siRNA treatment, protein expression of MARCKS was decreased by 93%3% compared to cells treated with control (nontargeting) siRNA. B, MARCKS KD significantly attenuated VSMC migration in the wound-healing scratch assay. C, Cell migration was determined by both reduction of the width of the wound. D, The density of cells within the wound. *P<0.05. Scale bars=500 lm. E, Starved CASMCs were stimulated with PDGF (20 ng/mL for 10 minutes), fixed, and stained with anti-cortactin (green) and anti-MARCKS (red) antibodies. White triangles denote the nucleus which stains for neither cortactin nor MARCKS. Lamellipodia, white arrows, formed around the periphery of the cell in the control group, which were seldom detected after MARCKS KD. White arrow heads denote cell nucleus. Scale bars=10 lm; shown are representative images of 3 independent experiments. F, These morphological changes are best appreciated at higher magnification. MARCKS and cortactin colocalized in lamellipodia (white arrows). Scale bar=10 lm. G, Significantly more cells formed lamellipodia in repsonse to PDGF stimulation in the control group compared to the MARCKS KD group. At least 40 cells were counted in each group per experiment. Data are presented as the mean and SE of 3 independent experiments. CASMCs indicates coronary artery smooth muscle cells; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell. *P<0.001.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Migration, Transfection, Control, Expressing, Wound Healing Assay, Staining, Knockdown, Derivative Assay, Small Interfering RNA

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 2. MARCKS KD inhibits small GTPase Rac1 and Cdc42 activation and blocks downstream signaling transduction in dedifferentiated VSMCs. A, Human CASMCs were treated with control (Control) siRNA or MARCKS siRNA (MARCKS KD). B, MARCKS siRNA reduced MARCKS protein expression by 94.7%3.1%. *P<0.0001. MARCKS KD did not affect the basal expression of small GTPase Rac1 or Cdc42, or the GTPase effector, PAK1. C, Rac1 and Cdc42 activation was detected in the presence (+PDGF) or absence (PDGF) of PDGF (20 ng/mL for 10 minutes) with the PAK-CRIB pull-down assay, as described in the Materials and Methods section. D, PDGF stimulation increased the activation of Rac1 in control cells, but had no effect after MARCKS KD. E, Similarly, PDGF stimulation increased the activation of Cdc42 in control cells, but also had no effect after MARCKS KD. F and G, MARCKS KD also prevented the activation of the Rac1 and Cdc42 effector, PAK1. Data are presented as the mean and SE of 3 independent experiments. *P<0.05. CASMCs indicates coronary artery smooth muscle cells; CRIB, Cdc42/Rac interactive binding domain; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21-activated kinase; PDGF, platelet-derived growth factor; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Activation Assay, Transduction, Control, Expressing, Pull Down Assay, Binding Assay, Knockdown, Derivative Assay, Small Interfering RNA

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 3. Markers of differentiation and MARCKS expression in human CASMCs, rat A10 cells, and rat A7r5 cells. In the present investigation, we used CASMCs and A10 cells for loss-of-function experi- ments. A, We chose these cells because they have lower expression of commonly cited markers of smooth muscle cell differentiation and thus more closely resemble the medial VSMCs in intimal hyperplasia. B, These 2 cell lines had higher consti- tutive expression of MARCKS than A7r5 cells, increasing the power of the KD experiments. A7r5 cells had increased expression of the markers of differentiation and lower constitutive MARCKS expression. Thus, these cells were used in the gain- of-function experiments. CASMCs indicates coro- nary artery smooth muscle cells; MARCKS, myris- toylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; PDGFR, platelet- derived growth factor receptor; SM, smooth muscle; VSMC, vascular smooth muscle cell.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, Cell Differentiation, Derivative Assay

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 4. Ectopic expression of wild-type (WT) MARCKS, but not the pseudophosphorylation mutant rescues the migration defects observed in MARCKS KD in dedifferentiated VSMCs. A, Dedifferentiated rat A10 VSMCs were treated with control siRNA (Ctl) or MARCKS siRNA (KD) and cotransfected with plasmids for either pEGFP-N1 vector alone (Vec), WT bovine MARCKS (MARCKS WT), the myristoylation-deficient MARCKS mutant (G2A), the phosphorylation-deficient ED domain MARCKS mutant (S4G), or the pseudophosphorylated ED domain MARCKS mutant (S4D), respectively. B, Cell migration was determined by the wound-healing assay in normal growth media as described in the Materials and Methods section. Cotransfection with WT MARCKS, myristolation-deficient MARCKS, and phosphorylation-deficient MARCKS all rescued migration. The pseudophosphorylation mutant did not rescue migration. Data are presented as the mean and SE of 3 independent experiments and normalized to the control treatment (Ctl+Vec). *P<0.05. C, Activation of the GTPases Rac1 and Cdc42 was determined in the presence of MARCKS KD and cotransfection with WT plasmids and each of the 3 mutant plasmids. D, The WT MARCKS, G2A, and S4G, but not the pseudophosphorylated S4D mutant, rescued both Rac1 and (E) cdc42 activation. Data are presented as the mean and SE of 3 independent experiments normalized to the quiescent condition (PDGF) in control cells (Ctl+Vec). *P<0.05. F, PDGF stimulates MARCKS phosphorylation (Pi-MARCKS) in CASMCs in a time-dependent manner. Maximal MARCKS protein expression was observed after 10 minutes of stimulation. G, MARCKS phosphorylation isalso dependenton the dose ofPDGF.Exposure of starved cells to50 ng/mL yieldedthe greatestphosphorylation of MARCKS at 10 minutes (right panel). CaM indicates calmodulin; ED, effector domain; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; PDGF, platelet-derived growth factor; pEGFP, enhanced green fluorescent protein plasmid; siRNA, small interfering RNA; VSMC, vascular smooth muscle cell; WT, wild type.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, Mutagenesis, Migration, Control, Plasmid Preparation, Phospho-proteomics, Wound Healing Assay, Cotransfection, Activation Assay, Knockdown, Derivative Assay, Small Interfering RNA

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 5. MARCKS KD decreases membrane bound PIP2 in dedifferentiated VSMCs. A, GFP-tagged pleckstrin homology domain of PLC-d (GFP-PH) was used as a live cell biosensor to detect PIP2 levels in human CASMCs. Representative images of cells in each treatment are presented. Scale bars=10 lm. B, Mean intensity values were determined by measuring the pixels along the plasma membrane (Fpm) and in the cytoplasm (Fc). Fluorescence of GFP-PH in plasma membrane is normalized by the mean fluorescence value in the cytoplasm yielding the proportion of PIP2 at the membrane (Fpm/Fc). Cells were treated with MARCKS siRNA (MARCKS KD) or control siRNA (Control). Starved quiescent cells were treated with (+PDGF) or without (PDGF) PDGF (20 ng/mL for 10 minutes). Before exposure to PDGF, significantly more PIP2 localized to the plasma membrane incontrolcells,comparedtoMARCKSKDcells(Fpm/Fc=1.880.42andFpm/Fc=1.170.11,respectively).*P<0.001;n=8.However,afterstimulation with PDGF, there was no significant difference of proportion of PIP2 localized to the plasma membrane (Fpm/Fc=1.080.05 and Fpm/Fc=1.080.11, respectively; n=8). CASMCs indicates coronary artery smooth muscle cells; Fc, fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; KD, knockdown; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; PLC, phospholipase C; siRNA, small interfering RNA.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Membrane, Clinical Proteomics, Fluorescence, Control, Knockdown, Derivative Assay, Small Interfering RNA

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 6. Overexpressing MARCKS increases differentiated vascular smooth muscle cell (VSMC) motility. Differentiated rat VSMC A7r5 cells that express low constituative levels of MARCKS protein were transfected with GFP-vector (Vec) or WT bovine MARCKS-GFP (+WT). A, Cells were cotransfected with RFP-PH and with either GFP vector or MARCKS-GFP plasmids. Membrane-associated PIP2 level (Fpm/Fc) was measured and quantified as described in the Materials and Methods section. B, Membrane-associated PIP2 level increased in A7r5 cells overexpressing MARCKS (MARCKS-GFP) compared to control (Vector). *P<0.001, n=8. There was no difference between the 2 treatments in membrane- associated PIP2 after stimulation with PDGF; n=8. Representative images of subcellular location of the RFP-PH biosensor in each treatment are shown on the right panel. Scale bars=10 lm. C, Cell migration increased in A7r5 cells overexpressing MARCKS as determined by the wound- healing assay. *P<0.05; n=3. D, A7r5 Cells were starved for 48 hours before stimulation with PDGF (+PDGF, 10 ng/mL for 10 minutes). Cells were fixed and stained with phalloidin (red) to label the actin cytoskeleton. White arrows denote dorsal ruffles and lamellipodia. Shown are representative images of 3 experiments with independent cell preparations. E, Overexpression of MARCKS promotes dorsal ruffle and lamellipodia formation in differentiated VSMCs. Seventy cells were counted in each group per experiment. *P<0.01. Scale bars=10 lm. F, Activation of the small GTPases Rac1 and Cdc42 was assessed using the PAK-CRIB pull-down assay. G, Overexpression of MARCKS increased Rac1 activity, but not significantly. H, MARCKS overexpression significantly increased Cdc42 activity. Activity was normalized to unstimulated control cells (Vector, PDGF). CRIB indicates Cdc42/Rac interactive binding domain; Fc indicates fluorescence in cytosol; Fpm, fluorescence in plasma membrane; GFP, green fluorescent protein; MARCKS, myristoylated alanine-rich protein kinase substrate; NS, not significant; PAK, p21- activated kinase; PDGF, platelet-derived growth factor; PH, pleckstrin homology; PIP2, phosphatidylinositol 4,5-bisphosphate; RFP, red fluorescent protein; VSMC, vascular smooth muscle cell; WT, wild type. *P<0.05.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Transfection, Plasmid Preparation, Membrane, Control, Migration, Wound Healing Assay, Staining, Over Expression, Activation Assay, Pull Down Assay, Activity Assay, Binding Assay, Clinical Proteomics, Derivative Assay

Journal: Journal of the American Heart Association

Article Title: Myristoylated Alanine‐Rich Protein Kinase Substrate (MARCKS) Regulates Small GTPase Rac1 and Cdc42 Activity and Is a Critical Mediator of Vascular Smooth Muscle Cell Migration in Intimal Hyperplasia Formation

doi: 10.1161/jaha.115.002255

Figure Lengend Snippet: Figure 7. Decreased MARCKS expression inhibits intimal hyperplasia formation in vivo. A, MARCKS protein is up-regulated during the formation of intimal hyperplasia in the murine carotid ligation model. Common carotid arteries in WT mice were ligated at the carotid bifurcation and harvested at days 0 (untreated), 7, 14, and 28. Total lysates from 3 arteries (6 lg of total protein) were used to assess phospho-MARCKS (Pi-MARCKS), MARCKS, a- SMA, and b-actin expression at each time point with Western blot analysis. B, Protein expression was normalized to GAPDH. MARCKS expression increases early in the formation of intimal hyperplasia. Maximal MARCKS expression occurs at 14 days. C, The caroitd artery was examined with immunostaining of frozen sections at an early phase of intimal hyperplasia formation (day 7 postligation). Confocal microscopy was used to identify MARCKS (red), a-SMA (green), and the murine endothelial cell marker, CD31 (white). At day 7,theinjuredcarotidarteriesinWTmice had developeda significanthyperplasticlesion.The merged confocal images demonstrate that MARCKS was highly expressed in the injured arteries. MARCKS is localized in the polarized leading edge (white arrows) of invading VSMCs. Scale bars=20 lm. D, MARCKS expression increased in WT mice as a result of carotid ligation (day 14). MARCKS expression increased only slightly after carotid ligation in MARCKS+/ mice (M+/). Rac1 and PAK1 activation were lower in the ligated carotid artery (day 14) of MARCKS+/ mice (M+/) as compared to WT mice. E, Representative VVG-stained sections of ligated carotid artery are shown at day 14 in WT (left) and MARCKS +/ (right) mice. There was a significantly greater hyperplastic response observed in WT mice compared to MARCKS+/ mice (M +/). Scale bars=200 lm. F, Intimal hyperplasia was quantified with intima area/media area ratio, n=3. CD indicates cluster of differentiation; MARCKS, myristoylated alanine-rich protein kinase substrate; PAK, p21- activated kinase; SM, smoothelin; SMA, smooth muscle actin; VSMC, vascular smooth muscle cell; VVG, Verhoeff-Van Gieson; WT, wild type. *P<0.0001.

Article Snippet: Antibodies used in this study were from the following sources: anti-human MARCKS (Catalog No.: 5607S; Cell Signaling Technology, Danvers, MA); polyclonal antimurine MARCKS (Catalog No.: AB9298; EMD Millipore, Billerica, MA); phospho-MARCKS (Catalog No.: 07-1238; EMD Millipore); GAPDH (Catalog No.: 2118L; Cell Signaling Technology); monoclonal anti-Rac1 (Catalog No.: 05389, EMD Millipore); polyclonal anti-Cdc42 (Catalog No.: 2462; Cell Signaling Technology); polyclonal anti-PAK (Catalog No.: DOI: 10.1161/JAHA.115.002255 Journal of the American Heart Association 2 MARCKS Regulates VSMC Migration Yu et al O R IG IN A L R E S E A R C H by guest on January 7, 2016http://jaha.ahajournals.org/Downloaded from 4570; Cell Signaling Technology); polyclonal anti-phosphoPAK (Catalog No.: 2601; Cell Signaling Technology); monoclonal anti-cortactin (Catalog No.: 05-180; EMD Millipore); monoclonal anti-beta-actin (Catalog No.:A5316; SigmaAldrich, St. Louis, MO); monoclonal anti–alpha-smooth muscle actin (aSMA; Catalog No.: A5228; Sigma-Aldrich); anti– platelet endothelial cell adhesion molecule-1 (PECAM-1/ CD31; Catalog No.: 550 274; BD, Franklin Lakes, NJ); antiplatelet-derived growth factor (PDGF) receptor alpha (Catalog No.: mab322; R&D Systems, Minneapolis, MN); anti-PDGF receptor beta (Catalog No.: 05-1135; EMD Millipore); polyclonal anti-calponin (Catalog No.: TA300720; OriGene Technologies, Inc., Rockville, MD); monoclonal anti-smoothelin (Catalog No.: MAB3242, EMD Millipore); and anti-SM 22-a (Catalog No.: ab14106; Abcam, Cambridge, MA).

Techniques: Expressing, In Vivo, Ligation, Western Blot, Immunostaining, Confocal Microscopy, Marker, Activation Assay, Staining

Journal: Biology Direct

Article Title: Cepharanthine alleviated chronic prostatitis/chronic pelvic pain syndrome by activating Nrf2 and inhibiting the NF-κB pathway

doi: 10.1186/s13062-026-00744-0

Figure Lengend Snippet: CEP alleviates prostate inflammation and pain in EAP mice. ( A ) Flowchart of the experimental design. ( B ) HE staining of prostate tissues from mice in the EAP group and the CEP treatment group (scale bar: 100 μm). ( C ) Prostate inflammation scores of mice in the EAP group and the CEP treatment group. ( D ) Body weights of mice in the EAP group and the CEP treatment group. ( E ) Tactile response frequencies of mice in the EAP group and the CEP treatment group. ( F ) Serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ, GM-CSF, CCL2, IL-17 A) in mice from the EAP group and the CEP treatment group. ( G ) Immunohistochemical staining images and quantification of CD4 and CD68 in prostate tissues of mice in the EAP group and the CEP treatment group (scale bar: 100 μm). ( H ) Immunofluorescence staining images of CCL2 in prostate tissues of mice from the EAP group and the CEP treatment group (scale bar: 100 μm). ( I ) Quantification of CCL2 fluorescence intensity in prostate tissues of mice from the EAP group and the CEP treatment group. Data are presented as mean ± SD ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, “ns” P > 0.05

Article Snippet: After blocking with 5% skim milk for 2 h, membranes were incubated with primary antibodies, COX-2 (1:2000, 12375-1-AP, Proteintech), iNOS (1:1000, 22226-1-AP, Proteintech), IL-1β (1:1000, 16806-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), CCL2 (1:1000, ab214819, Abcam), Nrf2 (1:10000, 16396-1-AP, Proteintech), HO-1 (1:3000, 10701-1-AP, Proteintech), NQO1 (1:4000, 11451-1-AP, Proteintech), P65 (1:1000, 10745-1-AP, Proteintech), P-P65 (1:5000, 82335-1-RR, Proteintech), IκBα (1:10000, 10268-1-AP, Proteintech), P-IκBα (1:2000, 82349-1-RR, Proteintech), Bax (1:10000, 50599-1-Ig, Proteintech), Bcl-2 (1:1000, 12789-1-AP, Proteintech), caspase-3 (1:500, 19677-1-AP, Proteintech), Lamin B (1:2000, 129871-AP, Proteintech), β-Tubulin (1:5000, 10094-1-AP, Proteintech), and GAPDH (1:50000, 60004-1-Ig, Proteintech), protein bands were visualized using chemiluminescent HRP substrate (Millipore, USA) and detected with the Fusion FX imaging system (Vilber Lourmat, France).

Techniques: Staining, Immunohistochemical staining, Immunofluorescence, Fluorescence

Journal: Biology Direct

Article Title: Cepharanthine alleviated chronic prostatitis/chronic pelvic pain syndrome by activating Nrf2 and inhibiting the NF-κB pathway

doi: 10.1186/s13062-026-00744-0

Figure Lengend Snippet: CEP ameliorates inflammatory response, oxidative stress, and mitochondrial dysfunction in LPS-induced RWPE-1 cells. ( A ) Chemical structure of CEP. ( B ) CCK8 assay was performed to analyze the effect of different concentrations of CEP on cells. ( C ) qRT-PCR was used to detect the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ, GM-CSF, CCL2, IL-17 A) in the control group, CEP group, LPS group, and LPS + CEP group. ( D ) Western blot was used to detect the expression levels of iNOS, COX-2, IL-1β, IL-6, and CCL2 in the control group, CEP group, LPS group, and LPS + CEP group. ( E ) Quantitative analysis of Western blot bands. ( F ) Levels of SOD, MDA, and GSH in the control group, CEP group, LPS group, and LPS + CEP group. ( G ) Intracellular ROS levels in the control, CEP, LPS, and LPS + CEP groups were detected using DCFH probe (scale bar: 100 μm). ( H ) Mitochondrial membrane potential levels in the control, CEP, LPS, and LPS + CEP groups were measured by JC-1 probe (scale bar: 100 μm). ( I ) Mitochondrial ROS levels in the control, CEP, LPS, and LPS + CEP groups were determined using MitoTracker Green and MitoSOX Red (scale bar: 100 μm). ( J ) TUNEL staining images of cells in the control, CEP, LPS, and LPS + CEP groups. ( K ) The expression levels of Caspase-3, BAX, and Bcl-2 in the control, CEP, LPS, and LPS + CEP groups were detected by Western blot. ( L ) Quantitative analysis of Western blot bands. Data are presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01

Article Snippet: After blocking with 5% skim milk for 2 h, membranes were incubated with primary antibodies, COX-2 (1:2000, 12375-1-AP, Proteintech), iNOS (1:1000, 22226-1-AP, Proteintech), IL-1β (1:1000, 16806-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), CCL2 (1:1000, ab214819, Abcam), Nrf2 (1:10000, 16396-1-AP, Proteintech), HO-1 (1:3000, 10701-1-AP, Proteintech), NQO1 (1:4000, 11451-1-AP, Proteintech), P65 (1:1000, 10745-1-AP, Proteintech), P-P65 (1:5000, 82335-1-RR, Proteintech), IκBα (1:10000, 10268-1-AP, Proteintech), P-IκBα (1:2000, 82349-1-RR, Proteintech), Bax (1:10000, 50599-1-Ig, Proteintech), Bcl-2 (1:1000, 12789-1-AP, Proteintech), caspase-3 (1:500, 19677-1-AP, Proteintech), Lamin B (1:2000, 129871-AP, Proteintech), β-Tubulin (1:5000, 10094-1-AP, Proteintech), and GAPDH (1:50000, 60004-1-Ig, Proteintech), protein bands were visualized using chemiluminescent HRP substrate (Millipore, USA) and detected with the Fusion FX imaging system (Vilber Lourmat, France).

Techniques: CCK-8 Assay, Quantitative RT-PCR, Expressing, Control, Western Blot, Membrane, TUNEL Assay, Staining

Journal: Biology Direct

Article Title: Cepharanthine alleviated chronic prostatitis/chronic pelvic pain syndrome by activating Nrf2 and inhibiting the NF-κB pathway

doi: 10.1186/s13062-026-00744-0

Figure Lengend Snippet: CEP alleviates inflammatory response in LPS-induced RWPE-1 cells by activating the Nrf2 signaling pathway. ( A , B ) Western blot analysis was performed to detect the expression levels of Nucl-Nrf2, Total-Nrf2, HO-1, and NQO1 in the Control, LPS, LPS + CEP, and LPS + CEP+ML385 groups, with subsequent quantitative analysis of the bands. ( C , D ) Western blot was used to examine the expression levels of p-IκBα, IκBα, Nucl-P65, P-P65, and Total-P65 in the Control, LPS, LPS + CEP, and LPS + CEP+ML385 groups, followed by quantitative analysis of the bands. ( E ) Fluorescent expression and localization of Nrf2 in Control, LPS, LPS + CEP, and LPS + CEP+ML385 groups (Scale bar: 100 μm). ( F ) Fluorescent expression and localization of P65 in Control, LPS, LPS + CEP, and LPS + CEP+ML385 groups (Scale bar: 100 μm). ( G ) Quantitative analysis of cellular Nrf2 and P65 fluorescence intensity. ( H ) qRT-PCR was performed to detect the mRNA expression levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ, GM-CSF, CCL2, IL-17 A) in cells from the control group, LPS group, LPS + CEP group, and LPS + CEP+ML385 group. ( I , G ) Western blot analysis was conducted to determine the expression levels of iNOS, COX-2, IL-1β, IL-6, and CCL2 in cells from the control group, LPS group, LPS + CEP group, and LPS + CEP+ML385 group, followed by quantitative analysis of the bands. Data are presented as mean ± SD ( n = 3). * P < 0.05, ** P < 0.01,“ns” P > 0.05

Article Snippet: After blocking with 5% skim milk for 2 h, membranes were incubated with primary antibodies, COX-2 (1:2000, 12375-1-AP, Proteintech), iNOS (1:1000, 22226-1-AP, Proteintech), IL-1β (1:1000, 16806-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), CCL2 (1:1000, ab214819, Abcam), Nrf2 (1:10000, 16396-1-AP, Proteintech), HO-1 (1:3000, 10701-1-AP, Proteintech), NQO1 (1:4000, 11451-1-AP, Proteintech), P65 (1:1000, 10745-1-AP, Proteintech), P-P65 (1:5000, 82335-1-RR, Proteintech), IκBα (1:10000, 10268-1-AP, Proteintech), P-IκBα (1:2000, 82349-1-RR, Proteintech), Bax (1:10000, 50599-1-Ig, Proteintech), Bcl-2 (1:1000, 12789-1-AP, Proteintech), caspase-3 (1:500, 19677-1-AP, Proteintech), Lamin B (1:2000, 129871-AP, Proteintech), β-Tubulin (1:5000, 10094-1-AP, Proteintech), and GAPDH (1:50000, 60004-1-Ig, Proteintech), protein bands were visualized using chemiluminescent HRP substrate (Millipore, USA) and detected with the Fusion FX imaging system (Vilber Lourmat, France).

Techniques: Western Blot, Expressing, Control, Fluorescence, Quantitative RT-PCR

Journal: Biology Direct

Article Title: Cepharanthine alleviated chronic prostatitis/chronic pelvic pain syndrome by activating Nrf2 and inhibiting the NF-κB pathway

doi: 10.1186/s13062-026-00744-0

Figure Lengend Snippet: The alleviating effects of CEP on EAP mice depend on the Nrf2 signaling pathway. ( A ) HE staining of prostate tissues from mice in the EAP group and the CEP treatment group (scale bar: 100 μm). ( B ) Prostate inflammation scores of mice in the EAP group and the CEP treatment group. ( C ) Tactile response frequencies of mice in the EAP group and the CEP treatment group. ( D ) Serum levels of pro-inflammatory cytokines (TNF-α, IL-1β, IL-6, IFN-γ, GM-CSF, CCL2, IL-17 A) in mice from the EAP group and the CEP treatment group. ( E ) Immunohistochemical staining images of CD4 and CD68 in prostate tissues of mice from the EAP group and the CEP treatment group (scale bar: 100 μm). ( F , G ) Immunofluorescence staining images and quantitative analysis of fluorescence intensity for CCL2 in prostate tissues of mice from the EAP group and the CEP treatment group (scale bar: 100 μm). ( H ) Serum levels of SOD, MDA, and GSH in mice from the EAP group and the CEP treatment group. ( I ) Mitochondrial Fluoroscopy electron microscopy images of prostate tissues from mice in the EAP group and the CEP treatment group (scale bar: 2 μm). ( J ) Serum ATP levels in mice from the EAP group and the CEP treatment group. ( K , M ) TUNEL staining images and quantitative analysis of fluorescence intensity of prostate tissues from mice in the EAP group and the CEP treatment group. Data are presented as mean ± SD ( n = 6). * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, “ns” P > 0.05

Article Snippet: After blocking with 5% skim milk for 2 h, membranes were incubated with primary antibodies, COX-2 (1:2000, 12375-1-AP, Proteintech), iNOS (1:1000, 22226-1-AP, Proteintech), IL-1β (1:1000, 16806-1-AP, Proteintech), IL-6 (1:1000, 21865-1-AP, Proteintech), CCL2 (1:1000, ab214819, Abcam), Nrf2 (1:10000, 16396-1-AP, Proteintech), HO-1 (1:3000, 10701-1-AP, Proteintech), NQO1 (1:4000, 11451-1-AP, Proteintech), P65 (1:1000, 10745-1-AP, Proteintech), P-P65 (1:5000, 82335-1-RR, Proteintech), IκBα (1:10000, 10268-1-AP, Proteintech), P-IκBα (1:2000, 82349-1-RR, Proteintech), Bax (1:10000, 50599-1-Ig, Proteintech), Bcl-2 (1:1000, 12789-1-AP, Proteintech), caspase-3 (1:500, 19677-1-AP, Proteintech), Lamin B (1:2000, 129871-AP, Proteintech), β-Tubulin (1:5000, 10094-1-AP, Proteintech), and GAPDH (1:50000, 60004-1-Ig, Proteintech), protein bands were visualized using chemiluminescent HRP substrate (Millipore, USA) and detected with the Fusion FX imaging system (Vilber Lourmat, France).

Techniques: Staining, Immunohistochemical staining, Immunofluorescence, Fluorescence, Electron Microscopy, TUNEL Assay

Journal: ACS Nano

Article Title: An Engineered Bionic Nanoparticle Sponge as a Cytokine Trap and Reactive Oxygen Species Scavenger to Relieve Disc Degeneration and Discogenic Pain

doi: 10.1021/acsnano.3c08097

Figure Lengend Snippet: Validation of adsorption capacity of nanomaterials with different concentration gradients on inflammatory factors/LPS/NGF. The remaining concentrations of TNF-α (A), IL-1β (B), IL-6 (C), IFN-γ (D), LPS (E), and NGF (F) were detected by an enzyme-linked immunosorbent assay (ELISA), after coculturing with MnO 2 nanoparticles, MnO 2 @MNP nanoparticles, and MnO 2 @TMNP nanoparticles at different concentrations. The initial concentrations of cytokines, LPS and NGF were 500 pg/mL. Data are presented as the mean ± SD ( n = 3): ns, not significant; ****, p < 0.0001 between groups.

Article Snippet: The cytokines used in this study (TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)), lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress (MCE, China).

Techniques: Biomarker Discovery, Adsorption, Concentration Assay, Enzyme-linked Immunosorbent Assay

Journal: ACS Nano

Article Title: An Engineered Bionic Nanoparticle Sponge as a Cytokine Trap and Reactive Oxygen Species Scavenger to Relieve Disc Degeneration and Discogenic Pain

doi: 10.1021/acsnano.3c08097

Figure Lengend Snippet: Validation of the adsorption capacity of fixed concentrations of nanomaterials to inflammatory cytokines/LPS/NGF with different concentration gradients. The remaining concentrations of TNF-α (A), IL-1β (B), IL-6 (C), IFN-γ (D), LPS (E), and NGF (F) at different initial concentrations were detected, after coculturing with 50 μg/mL MnO 2 @TMNP. Data are presented as the mean ± SD ( n = 3): ns, not significant; ****, p < 0.0001 between groups.

Article Snippet: The cytokines used in this study (TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)), lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress (MCE, China).

Techniques: Biomarker Discovery, Adsorption, Concentration Assay

Journal: ACS Nano

Article Title: An Engineered Bionic Nanoparticle Sponge as a Cytokine Trap and Reactive Oxygen Species Scavenger to Relieve Disc Degeneration and Discogenic Pain

doi: 10.1021/acsnano.3c08097

Figure Lengend Snippet: MnO 2 @TMNP inhibits the LPS-induced M1 polarization of macrophages. (A) Schematic diagram of the experimental design. (B) Flow cytometry detecting F4/80+CD86+ cells and F4/80+CD206+ cells to evaluate the polarization of macrophages. (C) Quantification of the CD86 geomean fluorescence intensity of macrophages according to flow cytometry. (D) Quantification of the proportion of M1 macrophages in each group. The levels of mRNA encoding iNOS (E), TNF-α (F), and IL-6 (G) in macrophages treated with LPS, LPS+MnO 2 , LPS+TMNP, or LPS+MnO 2 @TMNP, respectively. The fold change was normalized to the control group. The concentrations of TNF-α (H), IL-1β (I), IL-6 (J), and IFN-γ (K) in the supernatant after treating macrophages with LPS, LPS+MnO 2 , LPS+TMNP, or LPS+MnO 2 @TMNP. Data are presented as the mean ± SD ( n = 3): ns, not significant; ****, p < 0.0001 between groups.

Article Snippet: The cytokines used in this study (TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)), lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress (MCE, China).

Techniques: Flow Cytometry, Fluorescence, Control

Journal: ACS Nano

Article Title: An Engineered Bionic Nanoparticle Sponge as a Cytokine Trap and Reactive Oxygen Species Scavenger to Relieve Disc Degeneration and Discogenic Pain

doi: 10.1021/acsnano.3c08097

Figure Lengend Snippet: MnO 2 @TMNP inhibits H 2 O 2 -induced M1 macrophage polarization. (A) Schematic illustration of the establishment of H 2 O 2 -induced macrophage M1 polarization to assess the effects of MnO 2 @TMNP on alleviating the inflammatory microenvironment. (B) Flow cytometry of macrophage polarization after treatment with H 2 O 2 , H 2 O 2 +MnO 2 , H 2 O 2 +TMNP, or H 2 O 2 +MnO 2 @TMNP. (C) CD86 geomean fluorescence intensity of macrophages according to flow cytometry. (D) Quantification of the proportion of M1 macrophages in each group. The mRNA content of TNF-α (E), iNOS (F), and IL-6 (G) in macrophages treated with H 2 O 2 , H 2 O 2 +MnO 2 , H 2 O 2 +TMNP, or H 2 O 2 +MnO 2 @TMNP. The fold change was normalized to the control group. The concentrations of TNF-α (H), IL-1β (I), IL-6 (J), and IFN-γ (K) in the supernatant after treating macrophages with H 2 O 2 , H 2 O 2 +MnO 2 , H 2 O 2 +TMNP, or H 2 O 2 +MnO 2 @TMNP. Data are presented as the mean ± SD ( n = 3): ns, not significant; *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001 between groups.

Article Snippet: The cytokines used in this study (TNF-α, IL-1β, IL-6, interferon-γ (IFN-γ)), lipopolysaccharide (LPS), and NGF were purchased from MedChemExpress (MCE, China).

Techniques: Flow Cytometry, Fluorescence, Control

Journal: Immunology and Cell Biology

Article Title: Phorbol myristate acetate, but not CD40L, induces the differentiation of CLL B cells into Ab-secreting cells

doi: 10.1038/icb.2014.37

Figure Lengend Snippet: Day 7 analysis of the mRNAs for transcription factors involved in B cell-to-plasma cell differentiation. ( a ) The expression of the PAX5 , BCL6 , BACH2 , IRF8 , IRF4 , PRDM1 , IRE1 and XBP1s genes was evaluated by quantitative real-time RT-PCR on D0 and D7. Results are expressed relative to gene expression in CLL B cells on D0, according to the 2 −ΔΔCT method. Bars represent mean values±s.e.m. from five independent experiments. Statistical significance was calculated using the Wilcoxon test: * P <0.05. ns, not significant.

Article Snippet: The TaqMan Gene Expression assays for PRDM1 (BLIMP1) (Assay ID Hs00153357_m1), PAX5 (Hs00172003_m1), BCL6 (Hs00277037_m1), XBP1 (Hs00231936_m1), XBP1s (Hs03929085_g1), IRF4 (Hs01056533_m1), IRF8 (Hs01128710_m1), BACH2 (Hs00222364_m1), ERN1 (Hs00176385_m1), GAS6 (Hs01090305_m1), CD38 (Hs01120071_m1) and CD138 (Hs00896423_m1) were purchased from Applied Biosystems.

Techniques: Clinical Proteomics, Cell Differentiation, Expressing, Quantitative RT-PCR, Gene Expression

Journal: Immunology and Cell Biology

Article Title: Phorbol myristate acetate, but not CD40L, induces the differentiation of CLL B cells into Ab-secreting cells

doi: 10.1038/icb.2014.37

Figure Lengend Snippet: Day 7 proteomic analysis of transcription factors involved in B cell-to-plasma cell differentiation. ( a ) Immunoblot analysis and densitometry quantification of PAX5, IRF8, IRF4, XBP1, XBP1s and BLIMP1 in D0 CLL B cells, D7 non-stimulated cells (Medium), D7 PMA/c-stimulated cells and D7 CD40L/c-stimulated cells. The data shown are representative of three independent experiments. Statistical significance was calculated with using Student's t -test. ( b ) Cells were labeled with anti-PAX5 and anti-IRF4 Abs after permeabilization. Upper panel: relative fluorescence intensities (RFIs) were calculated as the ratio of the MFI of cells labeled with a specific Ab to that of cells labeled with a matched isotype control. Bars represent RFI mean values±s.e.m. from three independent experiments. Lower panel: cytometry plots for a representative patient. Significance was calculated using a paired Student's t -test. * P <0.05, ** P <0.01. ns, not significant. D, day.

Article Snippet: The TaqMan Gene Expression assays for PRDM1 (BLIMP1) (Assay ID Hs00153357_m1), PAX5 (Hs00172003_m1), BCL6 (Hs00277037_m1), XBP1 (Hs00231936_m1), XBP1s (Hs03929085_g1), IRF4 (Hs01056533_m1), IRF8 (Hs01128710_m1), BACH2 (Hs00222364_m1), ERN1 (Hs00176385_m1), GAS6 (Hs01090305_m1), CD38 (Hs01120071_m1) and CD138 (Hs00896423_m1) were purchased from Applied Biosystems.

Techniques: Clinical Proteomics, Cell Differentiation, Western Blot, Labeling, Fluorescence, Control, Cytometry