Structured Review

Abcam gapdh
Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, <t>MMP9,</t> E-cadherin, N-cadherin, snail, slug, and <t>GAPDH</t> were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P
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1) Product Images from "Lentivirus-mediated PLCγ1 gene short-hairpin RNA suppresses tumor growth and metastasis of human gastric adenocarcinoma"

Article Title: Lentivirus-mediated PLCγ1 gene short-hairpin RNA suppresses tumor growth and metastasis of human gastric adenocarcinoma

Journal: Oncotarget

doi: 10.18632/oncotarget.6976

Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, MMP9, E-cadherin, N-cadherin, snail, slug, and GAPDH were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P
Figure Legend Snippet: Lentivirus-mediated PLCγ1 shRNA could suppress migration in human gastric adenocarcinoma BGC-823 cells Cells were transduced with lentivirus-mediated PLCγ1 shRNA2/3 vectors. ( A ) The formation of membrane ruffles was detected using Ruffling assay as described in Materials and Methods. The cell nuclei were stained DAPI (blue) and the membrane ruffles were stained rhodamine-conjugated phalloidin (red). Scale bar = 10 μm. ( B and C ) The migration ability was measured using Transwell assay (B, magnification × 100) and Scratch assay (C, magnification × 400) as described in Materials and Methods. ( D ) The protein levels of MMP2, MMP9, E-cadherin, N-cadherin, snail, slug, and GAPDH were detected with Western blotting analysis, and the pro and active forms of MMP2/9 were observed using gelatin zymography assay as described in Materials and Methods. ( E ) The mRNA levels of PLCG1, MMP2, MMP9, CDHI, CDH2, SNAIL, SLUG, and GAPDH were detected using Real-time PCR analysis as described in Materials and Methods. ( F ) The level of VEGF in extracellular matrix was detected using ELISA as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (* P

Techniques Used: shRNA, Migration, Transduction, Staining, Transwell Assay, Wound Healing Assay, Western Blot, Zymography Assay, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Lentivirus-mediated PLCγ1 shRNA could block proliferation in human gastric adenocarcinoma BGC-823 cells BGC-823 cell line of stable expressing PLCγ1shRNA was established with the transduction of four types PLCγ1shRNAs using a lentiviral transduction strategy. ( A ) The effect of PLCγ1shRNAs on the level of PLCγ1 protein was detected with Western blotting analysis as described in Materials and Methods. ( B ) The effect of PLCγ1shRNAs on cell growth rate was measured with MTT assay as described in Materials and Methods. ( C ) The effect of PLCγ1 shRNA2/3 on cloning formation was detected with Colony formation assay as described in Materials and Methods. ( D ) The levels of PCNA, cleaved-PARP, PARP, Bcl-2, PLCγ1, and GAPDH protein were detected with Western blotting analysis as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (** P
Figure Legend Snippet: Lentivirus-mediated PLCγ1 shRNA could block proliferation in human gastric adenocarcinoma BGC-823 cells BGC-823 cell line of stable expressing PLCγ1shRNA was established with the transduction of four types PLCγ1shRNAs using a lentiviral transduction strategy. ( A ) The effect of PLCγ1shRNAs on the level of PLCγ1 protein was detected with Western blotting analysis as described in Materials and Methods. ( B ) The effect of PLCγ1shRNAs on cell growth rate was measured with MTT assay as described in Materials and Methods. ( C ) The effect of PLCγ1 shRNA2/3 on cloning formation was detected with Colony formation assay as described in Materials and Methods. ( D ) The levels of PCNA, cleaved-PARP, PARP, Bcl-2, PLCγ1, and GAPDH protein were detected with Western blotting analysis as described in Materials and Methods. Data are reported as means ± S.D. of three independent experiments (** P

Techniques Used: shRNA, Blocking Assay, Expressing, Transduction, Western Blot, MTT Assay, Clone Assay, Colony Assay

2) Product Images from "ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity"

Article Title: ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity

Journal: bioRxiv

doi: 10.1101/443010

Partial ADAR2 knockdown mimics and occludes TTX-evoked KAR upscaling. A. Representative western blot of total and surface levels of GluK2, EGFR and GAPDH in scrambled or ADAR2 KD infected cells in the presence or absence of TTX. EGFR was used as a negative control while GAPDH was used as a control to show only surface proteins were labelled with biotin. B. Quantification of (A) surface levels of GluK2 from 10 independent experiments. Surface levels were normalised to their total levels. Statistical Analysis: Two-Way ANOVA with Tukey’s Multiple comparisons test; *
Figure Legend Snippet: Partial ADAR2 knockdown mimics and occludes TTX-evoked KAR upscaling. A. Representative western blot of total and surface levels of GluK2, EGFR and GAPDH in scrambled or ADAR2 KD infected cells in the presence or absence of TTX. EGFR was used as a negative control while GAPDH was used as a control to show only surface proteins were labelled with biotin. B. Quantification of (A) surface levels of GluK2 from 10 independent experiments. Surface levels were normalised to their total levels. Statistical Analysis: Two-Way ANOVA with Tukey’s Multiple comparisons test; *

Techniques Used: Western Blot, Infection, Negative Control

TTX promotes proteasomal degradation of ADAR2. A. RT-qPCR analysis of mRNA levels of ADAR2 post TTX treatment showing no changes in the ADAR2 mature mRNA transcripts from 7 independent experiments. Statistical analysis: Unpaired t-test; ns > 0.05. B. Representative western blots of total ADAR2, total ubiquitin and GAPDH levels in neurons treated with either DMSO or 1μM Bortezomib (BTZ) for 20 h either in the presence or absence of 24 h TTX. C. Quantification of total ADAR2 immunoblots normalised to GAPDH from 6 independent experiments. Statistical Analysis: Two-way ANOVA with Tukey’s multiple comparisons test: **
Figure Legend Snippet: TTX promotes proteasomal degradation of ADAR2. A. RT-qPCR analysis of mRNA levels of ADAR2 post TTX treatment showing no changes in the ADAR2 mature mRNA transcripts from 7 independent experiments. Statistical analysis: Unpaired t-test; ns > 0.05. B. Representative western blots of total ADAR2, total ubiquitin and GAPDH levels in neurons treated with either DMSO or 1μM Bortezomib (BTZ) for 20 h either in the presence or absence of 24 h TTX. C. Quantification of total ADAR2 immunoblots normalised to GAPDH from 6 independent experiments. Statistical Analysis: Two-way ANOVA with Tukey’s multiple comparisons test: **

Techniques Used: Quantitative RT-PCR, Western Blot

Chronic suppression of network activity decreases ADAR2 levels. A. Representative western blots of total ADAR2 and GAPDH levels in hippocampal neurons with or without 24 h TTX treatment to suppress synaptic activity. B. Quantification of (A) total ADAR2 normalised to GAPDH from 5 independent experiments. ADAR2 levels normalised to loading control GAPDH. Statistical Analysis: Unpaired t-test; ****
Figure Legend Snippet: Chronic suppression of network activity decreases ADAR2 levels. A. Representative western blots of total ADAR2 and GAPDH levels in hippocampal neurons with or without 24 h TTX treatment to suppress synaptic activity. B. Quantification of (A) total ADAR2 normalised to GAPDH from 5 independent experiments. ADAR2 levels normalised to loading control GAPDH. Statistical Analysis: Unpaired t-test; ****

Techniques Used: Activity Assay, Western Blot

3) Product Images from "The expression and function of RASAL2 in renal cell carcinoma angiogenesis"

Article Title: The expression and function of RASAL2 in renal cell carcinoma angiogenesis

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0898-x

RASAL2 could modulate RCC angiogenesis. a Real-time quantitative PCR and western blot analysis of RASAL2 levels in ACHN cells transfected with RASAL2 shRNAs (KD) and negative control (NC), or 786O cells infected with RASAL2 lentivirus and negative control (NC). GAPDH was used as loading control. b , c Representative pictures and quantification analysis of the recruitment of HUVECs using the conditioned mediums (CMs) collected from ACHN sublines and cocultured system with ACHN sublines. The scale bar is 40 µm. b, d Representative pictures and quantification analysis of the recruitment of HUVECs using CMs collected from 786O sublines and cocultured system with 786O sublines. The scale bar is 40 µm. e MTT assay of HUVECs treated with SFM or diluted CMs from 786O sublines for 48 h. f , g Representative pictures and quantification analysis of HUVECs tube formation treated with serum free medium (SFM) or CMs from ACHN or 786O sublines. * P
Figure Legend Snippet: RASAL2 could modulate RCC angiogenesis. a Real-time quantitative PCR and western blot analysis of RASAL2 levels in ACHN cells transfected with RASAL2 shRNAs (KD) and negative control (NC), or 786O cells infected with RASAL2 lentivirus and negative control (NC). GAPDH was used as loading control. b , c Representative pictures and quantification analysis of the recruitment of HUVECs using the conditioned mediums (CMs) collected from ACHN sublines and cocultured system with ACHN sublines. The scale bar is 40 µm. b, d Representative pictures and quantification analysis of the recruitment of HUVECs using CMs collected from 786O sublines and cocultured system with 786O sublines. The scale bar is 40 µm. e MTT assay of HUVECs treated with SFM or diluted CMs from 786O sublines for 48 h. f , g Representative pictures and quantification analysis of HUVECs tube formation treated with serum free medium (SFM) or CMs from ACHN or 786O sublines. * P

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Transfection, Negative Control, Infection, MTT Assay

RASAL2 modulated VEGFA expression via p-GSK3β/c-FOS pathway in RCC. a Western blot analysis of c-FOS expression in ACHN or 786O sublines. b Western blot analysis of c-FOS and VEGFA expression in ACHN sublines transfected with c-FOS siRNA. c Western blot analysis of p-GSK3β S9 and GSK3β in ACHN or 786O sublines. d , e Western blot analysis of c-FOS and VEGFA in ACHN sublines treated with XAV-939 or 786O sublines treated with CT99021. The densitometric analysis of VEGFA expression was shown. f Immunoprecipitation assay of RASAL2 and GSK3β in 786O sublines. GAPDH was used as loading control
Figure Legend Snippet: RASAL2 modulated VEGFA expression via p-GSK3β/c-FOS pathway in RCC. a Western blot analysis of c-FOS expression in ACHN or 786O sublines. b Western blot analysis of c-FOS and VEGFA expression in ACHN sublines transfected with c-FOS siRNA. c Western blot analysis of p-GSK3β S9 and GSK3β in ACHN or 786O sublines. d , e Western blot analysis of c-FOS and VEGFA in ACHN sublines treated with XAV-939 or 786O sublines treated with CT99021. The densitometric analysis of VEGFA expression was shown. f Immunoprecipitation assay of RASAL2 and GSK3β in 786O sublines. GAPDH was used as loading control

Techniques Used: Expressing, Western Blot, Transfection, Immunoprecipitation

RASAL2 regulated the expression of VEGFA in RCC. a Western blot analysis of VEGFA expression in ACHN or 786O sublines. GAPDH was used as loading control, and the densitometric analysis of VEGFA expression was shown. b ELISA assay of VEGFA expression in CMs from ACHN or 786O sublines. * P
Figure Legend Snippet: RASAL2 regulated the expression of VEGFA in RCC. a Western blot analysis of VEGFA expression in ACHN or 786O sublines. GAPDH was used as loading control, and the densitometric analysis of VEGFA expression was shown. b ELISA assay of VEGFA expression in CMs from ACHN or 786O sublines. * P

Techniques Used: Expressing, Western Blot, Enzyme-linked Immunosorbent Assay

4) Product Images from "Jinhong Tablet Reduces Damage of Intestinal Mucosal Barrier in Rats with Acute Biliary Infection via Bcl-2/Bax mRNA and Protein Regulation"

Article Title: Jinhong Tablet Reduces Damage of Intestinal Mucosal Barrier in Rats with Acute Biliary Infection via Bcl-2/Bax mRNA and Protein Regulation

Journal: Evidence-based Complementary and Alternative Medicine : eCAM

doi: 10.1155/2017/4985926

Relative protein expression analysis: protein expression of bcl-2 and bax in intestinal tissue at days 3 and 5, respectively (a), and the densitometric quantification of the protein expression levels, which are expressed as a ratio to the expression of GAPDH of bcl-2, bax, and bcl-2/bax protein expression ratio at day 3 (b) and day 5 (c). Values were expressed as mean ± SD; n = 6 in each group; ∗∗ P
Figure Legend Snippet: Relative protein expression analysis: protein expression of bcl-2 and bax in intestinal tissue at days 3 and 5, respectively (a), and the densitometric quantification of the protein expression levels, which are expressed as a ratio to the expression of GAPDH of bcl-2, bax, and bcl-2/bax protein expression ratio at day 3 (b) and day 5 (c). Values were expressed as mean ± SD; n = 6 in each group; ∗∗ P

Techniques Used: Expressing

5) Product Images from "Budesonide and Calcitriol Synergistically Inhibit Airway Remodeling in Asthmatic Mice"

Article Title: Budesonide and Calcitriol Synergistically Inhibit Airway Remodeling in Asthmatic Mice

Journal: Canadian Respiratory Journal

doi: 10.1155/2018/5259240

Effect of budesonide (Bud) and calcitriol (Cal) on the expression of the TGF β /Smad signaling pathway-related proteins and miR-21 in ovalbumin- (Ova-) treated mice. (a) The lung tissue of the mice was fixed, embedded, and sectioned for immunohistochemical staining (DAB stain, original magnification ×400). (b) The total protein was extracted from the lung tissue of mice for Western blot analysis. (c, d) The expression of pSmad2 and pSmad3 in the lung tissue of mice was determined by Western blot analysis, with GAPDH as an internal reference ( ∗ P
Figure Legend Snippet: Effect of budesonide (Bud) and calcitriol (Cal) on the expression of the TGF β /Smad signaling pathway-related proteins and miR-21 in ovalbumin- (Ova-) treated mice. (a) The lung tissue of the mice was fixed, embedded, and sectioned for immunohistochemical staining (DAB stain, original magnification ×400). (b) The total protein was extracted from the lung tissue of mice for Western blot analysis. (c, d) The expression of pSmad2 and pSmad3 in the lung tissue of mice was determined by Western blot analysis, with GAPDH as an internal reference ( ∗ P

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Western Blot

Effect of budesonide (Bud) and calcitriol (Cal) on the expression of GR, VDR, and RXR in ovalbumin- (Ova-) treated mice. (a) The lung tissue of the mice was fixed, embedded, and sectioned for immunohistochemical staining (DAB stain, original magnification ×400). (b) The total protein was extracted from the lung tissue of mice for Western blot analysis. (c) VDR protein expression in the lung tissue of mice was determined by Western blot analysis, with GAPDH as an internal reference ( ∗ P
Figure Legend Snippet: Effect of budesonide (Bud) and calcitriol (Cal) on the expression of GR, VDR, and RXR in ovalbumin- (Ova-) treated mice. (a) The lung tissue of the mice was fixed, embedded, and sectioned for immunohistochemical staining (DAB stain, original magnification ×400). (b) The total protein was extracted from the lung tissue of mice for Western blot analysis. (c) VDR protein expression in the lung tissue of mice was determined by Western blot analysis, with GAPDH as an internal reference ( ∗ P

Techniques Used: Expressing, Mouse Assay, Immunohistochemistry, Staining, Western Blot

6) Product Images from "Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle"

Article Title: Inhibition of the Activin Receptor Type-2B Pathway Restores Regenerative Capacity in Satellite Cell-Depleted Skeletal Muscle

Journal: Frontiers in Physiology

doi: 10.3389/fphys.2018.00515

RAP-031 treatment rescues the reduction of muscle fiber size induced by satellite cell depletion and increases PICs in satellite cell-depleted muscle. (A) Strategy: the right TA of 10 week-old Pax7 DTR /+ males was injected with diphteria toxin (DT) to deplete satellite cells while the contralateral muscle was injected with PBS 2 weeks before RAP-031 treatment. Mice were intraperitoneally injected with RAP-031 or vehicle (CTL) twice a week for 2 weeks and sacrificed 14 days after the first injection. (B) Western Blot of TA protein extracts with antibodies against phospho-Smad2/3, total Smad2/3 and GAPDH. (i) Representative image of the blots. Molecular size in kDa is shown on the right side. (ii) Ratio between levels of phospho-Smad2 and levels of total Smad2. The graph shows a marked reduction in the ratio pSmad2/Smad2 in RAP-031 muscles, indicating an inhibition of the AcvR2B pathway following systemic RAP-031 treatment. N = 2 muscles per group. (C) Cross-sections images of TA muscles from CTL (Upper) or RAP-031 (Lower) mice injected with PBS (Left) or DT (Right) stained with hematoxylin and eosin. Scale bar, 60 μm. (D) Fiber size distribution in CTL PBS (gray), CTL DT (purple), RAP-031 PBS (blue), and RAP-031 DT (orange) TAs. RAP-031 treatment rescued the reduction of muscle fiber size induced by satellite cell depletion. Values represent the mean number ± s.e.m. per 100 fibers for each size. N = 3 animals for each group. Median of fiber area distributions are shown in the graphs. (E) Quantification of the number of sublaminal nuclei per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT. RAP-031 treatment did not result in myonuclei addition when satellite cells are depleted. Values represent the mean number ± s.e.m. per 100 fibers. At least 300 fibers from randomly chosen fields were counted for each animal, n = 3 animals were considered for each group. (F,G) Quantification of satellite cells (E) and PICs (F) per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT. (H) Ratio between PICs (green) and satellite cells (red) per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT was profoundly altered in DT-injected mice after RAP-031 treatment. In (E–G) , satellite cells were determined as M-Cadherin pos cells underneath the basal lamina, PICs were determined as interstitial PW1 pos M-Cadherin neg cells. In (E–G) , values represent the mean number of positive cells ± s.e.m. per 100 fibers. At least 300 fibers from randomly chosen fields were counted for each animal, n = 3 animals were considered for each group. For all values, * p
Figure Legend Snippet: RAP-031 treatment rescues the reduction of muscle fiber size induced by satellite cell depletion and increases PICs in satellite cell-depleted muscle. (A) Strategy: the right TA of 10 week-old Pax7 DTR /+ males was injected with diphteria toxin (DT) to deplete satellite cells while the contralateral muscle was injected with PBS 2 weeks before RAP-031 treatment. Mice were intraperitoneally injected with RAP-031 or vehicle (CTL) twice a week for 2 weeks and sacrificed 14 days after the first injection. (B) Western Blot of TA protein extracts with antibodies against phospho-Smad2/3, total Smad2/3 and GAPDH. (i) Representative image of the blots. Molecular size in kDa is shown on the right side. (ii) Ratio between levels of phospho-Smad2 and levels of total Smad2. The graph shows a marked reduction in the ratio pSmad2/Smad2 in RAP-031 muscles, indicating an inhibition of the AcvR2B pathway following systemic RAP-031 treatment. N = 2 muscles per group. (C) Cross-sections images of TA muscles from CTL (Upper) or RAP-031 (Lower) mice injected with PBS (Left) or DT (Right) stained with hematoxylin and eosin. Scale bar, 60 μm. (D) Fiber size distribution in CTL PBS (gray), CTL DT (purple), RAP-031 PBS (blue), and RAP-031 DT (orange) TAs. RAP-031 treatment rescued the reduction of muscle fiber size induced by satellite cell depletion. Values represent the mean number ± s.e.m. per 100 fibers for each size. N = 3 animals for each group. Median of fiber area distributions are shown in the graphs. (E) Quantification of the number of sublaminal nuclei per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT. RAP-031 treatment did not result in myonuclei addition when satellite cells are depleted. Values represent the mean number ± s.e.m. per 100 fibers. At least 300 fibers from randomly chosen fields were counted for each animal, n = 3 animals were considered for each group. (F,G) Quantification of satellite cells (E) and PICs (F) per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT. (H) Ratio between PICs (green) and satellite cells (red) per 100 fibers in TA from CTL and RAP-031 mice injected with PBS or DT was profoundly altered in DT-injected mice after RAP-031 treatment. In (E–G) , satellite cells were determined as M-Cadherin pos cells underneath the basal lamina, PICs were determined as interstitial PW1 pos M-Cadherin neg cells. In (E–G) , values represent the mean number of positive cells ± s.e.m. per 100 fibers. At least 300 fibers from randomly chosen fields were counted for each animal, n = 3 animals were considered for each group. For all values, * p

Techniques Used: Injection, Mouse Assay, CTL Assay, Western Blot, Inhibition, Staining

7) Product Images from "Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12"

Article Title: Breast Cancer: An Examination of the Potential of ACKR3 to Modify the Response of CXCR4 to CXCL12

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113592

Western blot and cell-based ELISA show that CXCL12 treatment of transfected CHO cells differentially activates the ERK and AKT pathways. Serum starved ( A ) CHO-CXCR4, ( B ) CHO-ACKR3 and ( C ) CHO-CXCR4-ACKR3 cells were stimulated with 10 nM CXCL12 for 5, 15 and 120 min. ( Top ) Cells were lysed and immunoblots probed with p-ERK or p-Akt, stripped and re-probed for pan-ERK or pan-AKT and GAPDH as a loading control. Images are representative of three independent experiments. ( Bottom ) Cells were fixed with methanol and cell-based ELISA was performed as per protocol using p-ERK and total-ERK antibodies with fluorescence intensity readings at 600 and 450 nm. Data represents the mean ± SEM of three independent experiments and statistical significance was calculated using a one way ANOVA (* p
Figure Legend Snippet: Western blot and cell-based ELISA show that CXCL12 treatment of transfected CHO cells differentially activates the ERK and AKT pathways. Serum starved ( A ) CHO-CXCR4, ( B ) CHO-ACKR3 and ( C ) CHO-CXCR4-ACKR3 cells were stimulated with 10 nM CXCL12 for 5, 15 and 120 min. ( Top ) Cells were lysed and immunoblots probed with p-ERK or p-Akt, stripped and re-probed for pan-ERK or pan-AKT and GAPDH as a loading control. Images are representative of three independent experiments. ( Bottom ) Cells were fixed with methanol and cell-based ELISA was performed as per protocol using p-ERK and total-ERK antibodies with fluorescence intensity readings at 600 and 450 nm. Data represents the mean ± SEM of three independent experiments and statistical significance was calculated using a one way ANOVA (* p

Techniques Used: Western Blot, In-Cell ELISA, Transfection, Fluorescence

8) Product Images from "TGF-β Regulates Endothelial Function During High Salt Intake in Rats"

Article Title: TGF-β Regulates Endothelial Function During High Salt Intake in Rats

Journal: Hypertension

doi: 10.1161/HYPERTENSIONAHA.113.01835

, and GAPDH. These western blot analyses were compared to lysates from HUVEC (lane 1) and ASMC in culture (lane 2). NOS3 was found in HUVEC and in all the endothelial lysates, but not the lysate of cultured aortic smooth muscle cells (ASMC). In contrast, SM22α was not observed in HUVEC but was abundantly expressed in ASMC. This sensitive assay revealed that eight of the twelve endothelial cell lysates contained no SM22α, while four of the twelve lysates demonstrated slight expression of SM22α. When expressed relative to GAPDH, the amount of SM22α in the endothelial cell lysates averaged 2.7±1.6% of that observed in the lysate of the ASMC. These data demonstrated minimal contamination of the endothelial cell lysates with smooth muscle tissue.
Figure Legend Snippet: , and GAPDH. These western blot analyses were compared to lysates from HUVEC (lane 1) and ASMC in culture (lane 2). NOS3 was found in HUVEC and in all the endothelial lysates, but not the lysate of cultured aortic smooth muscle cells (ASMC). In contrast, SM22α was not observed in HUVEC but was abundantly expressed in ASMC. This sensitive assay revealed that eight of the twelve endothelial cell lysates contained no SM22α, while four of the twelve lysates demonstrated slight expression of SM22α. When expressed relative to GAPDH, the amount of SM22α in the endothelial cell lysates averaged 2.7±1.6% of that observed in the lysate of the ASMC. These data demonstrated minimal contamination of the endothelial cell lysates with smooth muscle tissue.

Techniques Used: Western Blot, Cell Culture, Sensitive Assay, Expressing

9) Product Images from "The Bardet–Biedl syndrome-related protein CCDC28B modulates mTORC2 function and interacts with SIN1 to control cilia length independently of the mTOR complex"

Article Title: The Bardet–Biedl syndrome-related protein CCDC28B modulates mTORC2 function and interacts with SIN1 to control cilia length independently of the mTOR complex

Journal: Human Molecular Genetics

doi: 10.1093/hmg/ddt253

CCDC28B modulates mTORC2 activity. ( A and B ) Depletion of CCDC28B in both NIH3T3 cells (A) and zebrafish embryos (B) results in a reduction of phospho-Akt S473 indicating decreased mTORC2 activity. Total Akt or GAPDH were used as loading controls. ( C
Figure Legend Snippet: CCDC28B modulates mTORC2 activity. ( A and B ) Depletion of CCDC28B in both NIH3T3 cells (A) and zebrafish embryos (B) results in a reduction of phospho-Akt S473 indicating decreased mTORC2 activity. Total Akt or GAPDH were used as loading controls. ( C

Techniques Used: Activity Assay

10) Product Images from "Identification of Gangliosides GD1b and GT1b as Receptors for BK Virus"

Article Title: Identification of Gangliosides GD1b and GT1b as Receptors for BK Virus

Journal: Journal of Virology

doi: 10.1128/JVI.80.3.1361-1366.2006

GD1b and GT1b allow BKV infection of LNCaP cells. LNCaP cells were preincubated with medium containing the gangliosides GM1, GM2, GM3, GD1a, GD1b, or GT1b or ganglioside-free medium (mock), and infected with either BKV or SV40 at an MOI of 5 ffu/cell. A total of 25 μg of protein, extracted at 5 days postinfection, was analyzed for the expression of TAg or GAPDH as a loading control. The arrow denotes a background band detected even in mock-infected cells.
Figure Legend Snippet: GD1b and GT1b allow BKV infection of LNCaP cells. LNCaP cells were preincubated with medium containing the gangliosides GM1, GM2, GM3, GD1a, GD1b, or GT1b or ganglioside-free medium (mock), and infected with either BKV or SV40 at an MOI of 5 ffu/cell. A total of 25 μg of protein, extracted at 5 days postinfection, was analyzed for the expression of TAg or GAPDH as a loading control. The arrow denotes a background band detected even in mock-infected cells.

Techniques Used: Infection, Expressing

11) Product Images from "MiR-375-3p regulates rat pulmonary microvascular endothelial cell activity by targeting Notch1 during hypoxia"

Article Title: MiR-375-3p regulates rat pulmonary microvascular endothelial cell activity by targeting Notch1 during hypoxia

Journal: The Journal of International Medical Research

doi: 10.1177/0300060520926851

MiR-375-3p negatively regulated the expression of Notch1 upon hypoxia in PMECs. (a) Quantitative PCR of miR-375-3p expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus. (b) Immunoblotting of Notch1 protein expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus. (c) The relative expression of Notch1 in (b) was quantified and normalized to that of GAPDH. (d) Immunoblotting of Notch1 expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus and subjected to normoxic or hypoxic conditions. (e) The relative expression of Notch1 in (d) was quantified and normalized to that of GAPDH. Data represent the mean ± SD, n = 6. *** P
Figure Legend Snippet: MiR-375-3p negatively regulated the expression of Notch1 upon hypoxia in PMECs. (a) Quantitative PCR of miR-375-3p expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus. (b) Immunoblotting of Notch1 protein expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus. (c) The relative expression of Notch1 in (b) was quantified and normalized to that of GAPDH. (d) Immunoblotting of Notch1 expression in PMECs transfected with GFP-NC or GFP-miR-375-3p lentivirus and subjected to normoxic or hypoxic conditions. (e) The relative expression of Notch1 in (d) was quantified and normalized to that of GAPDH. Data represent the mean ± SD, n = 6. *** P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction, Transfection

MiR-375-3p levels were negatively correlated with the expression of Notch1 following hypoxic exposure for 0, 24, 48, or 72 hours in primary PMECs. (a) Immunohistochemical staining of CD31 expression, a marker of PMECs. (b, c) Quantitative PCR of the miR-375-3p and Notch1 expression levels in PMECs following hypoxic treatment. (d) Immunoblotting of the Notch 1 protein expression in PMECs following hypoxic treatment. (e) The relative expression of Notch1 in (d) was quantified and normalized to that of GAPDH. (e) Dual-luciferase reporter assay illustrating the effect of miR-375-3p on the expression of Notch1. Data represent the mean ± SD, n = 6. ** P
Figure Legend Snippet: MiR-375-3p levels were negatively correlated with the expression of Notch1 following hypoxic exposure for 0, 24, 48, or 72 hours in primary PMECs. (a) Immunohistochemical staining of CD31 expression, a marker of PMECs. (b, c) Quantitative PCR of the miR-375-3p and Notch1 expression levels in PMECs following hypoxic treatment. (d) Immunoblotting of the Notch 1 protein expression in PMECs following hypoxic treatment. (e) The relative expression of Notch1 in (d) was quantified and normalized to that of GAPDH. (e) Dual-luciferase reporter assay illustrating the effect of miR-375-3p on the expression of Notch1. Data represent the mean ± SD, n = 6. ** P

Techniques Used: Expressing, Immunohistochemistry, Staining, Marker, Real-time Polymerase Chain Reaction, Luciferase, Reporter Assay

12) Product Images from "Pathological presentation of cardiac mitochondria in a rat model for chronic kidney disease"

Article Title: Pathological presentation of cardiac mitochondria in a rat model for chronic kidney disease

Journal: PLoS ONE

doi: 10.1371/journal.pone.0198196

CKD-induced mitochondrial damage. (A) Relative expression of CytB as a marker for total mitochondrial content, relative to 18S. (B) Citrate synthase activity as an indicator for intact mitochondrial content. (C) Protein expression of PGC1α as a marker for mitochondrial biogenesis, normalized to GAPDH. (D) ATP synthase (complex V) activity. All data are given for the 3 experimental groups: sham, CKD and ARB (4–6 animals/group). ARB–CKD animals treated with ARB. Data are represented as mean ± SEM.
Figure Legend Snippet: CKD-induced mitochondrial damage. (A) Relative expression of CytB as a marker for total mitochondrial content, relative to 18S. (B) Citrate synthase activity as an indicator for intact mitochondrial content. (C) Protein expression of PGC1α as a marker for mitochondrial biogenesis, normalized to GAPDH. (D) ATP synthase (complex V) activity. All data are given for the 3 experimental groups: sham, CKD and ARB (4–6 animals/group). ARB–CKD animals treated with ARB. Data are represented as mean ± SEM.

Techniques Used: Expressing, Marker, Activity Assay

Potential mechanisms for CKD-induced mitochondrial damage. (A) Relative CytC expression (CytC leakage) in cytosolic fractions of cardiac cells. (B) Protein expression of PARP-1 as a marker for cellular apoptosis, normalized to GAPDH. N = 4–6 animals/group. ARB–CKD animals treated with ARB. Data are represented as mean ± SEM.
Figure Legend Snippet: Potential mechanisms for CKD-induced mitochondrial damage. (A) Relative CytC expression (CytC leakage) in cytosolic fractions of cardiac cells. (B) Protein expression of PARP-1 as a marker for cellular apoptosis, normalized to GAPDH. N = 4–6 animals/group. ARB–CKD animals treated with ARB. Data are represented as mean ± SEM.

Techniques Used: Expressing, Marker

13) Product Images from "Ginsenoside Rg1 Prevents Doxorubicin-Induced Cardiotoxicity through the Inhibition of Autophagy and Endoplasmic Reticulum Stress in Mice"

Article Title: Ginsenoside Rg1 Prevents Doxorubicin-Induced Cardiotoxicity through the Inhibition of Autophagy and Endoplasmic Reticulum Stress in Mice

Journal: International Journal of Molecular Sciences

doi: 10.3390/ijms19113658

Effect of Rg1 on the autophagic pathway activated by endoplasmic reticulum stress in mice heart. ( A ) Expression of p-P70S6K, P70S6K, JNK1 and Beclin1 protein in mouse heart. ( B ) Expression of FAM134B protein in mouse heart, GAPDH was used as the loading control. # p
Figure Legend Snippet: Effect of Rg1 on the autophagic pathway activated by endoplasmic reticulum stress in mice heart. ( A ) Expression of p-P70S6K, P70S6K, JNK1 and Beclin1 protein in mouse heart. ( B ) Expression of FAM134B protein in mouse heart, GAPDH was used as the loading control. # p

Techniques Used: Mouse Assay, Expressing

The mechanisms of Rg1 to improve endoplasmic reticulum stress. ( A ) Expression of TIF1, GRP78 and HRD1 protein in mouse heart. ( B ) Expression of Pre-rRNA level in mouse heart, GAPDH was used as the loading control. # p
Figure Legend Snippet: The mechanisms of Rg1 to improve endoplasmic reticulum stress. ( A ) Expression of TIF1, GRP78 and HRD1 protein in mouse heart. ( B ) Expression of Pre-rRNA level in mouse heart, GAPDH was used as the loading control. # p

Techniques Used: Expressing

14) Product Images from "p53-independent role of MYC mutant T58A in the proliferation and apoptosis of breast cancer cells"

Article Title: p53-independent role of MYC mutant T58A in the proliferation and apoptosis of breast cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2018.9688

Mutant MYC does not induce p14 or Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p14 siRNA construct at the same time. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p14. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p14. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Mutant MYC does not induce p14 or Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p14 siRNA construct at the same time. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p14. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p14. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Mutagenesis, Stable Transfection, Infection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Expression levels of Bim in HCC1937 cells transfected with T58A and WT MYC. HCC1937 cells were stably infected with control, WT MYC or T58A MYC vectors. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of MYC and Bim. (C) Western blot analysis. (D) Protein expression levels of MYC and Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Expression levels of Bim in HCC1937 cells transfected with T58A and WT MYC. HCC1937 cells were stably infected with control, WT MYC or T58A MYC vectors. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of MYC and Bim. (C) Western blot analysis. (D) Protein expression levels of MYC and Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Mutant MYC does not suppress p21 or induce Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p21 siRNA construct simultaneously. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p21. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p21. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Mutant MYC does not suppress p21 or induce Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p21 siRNA construct simultaneously. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p21. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p21. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Mutagenesis, Stable Transfection, Infection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

15) Product Images from "Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway"

Article Title: Emodin, A Chinese Herbal Medicine, Inhibits Reoxygenation-Induced Injury in Cultured Human Aortic Endothelial Cells by Regulating the Peroxisome Proliferator-Activated Receptor-γ (PPAR-γ) and Endothelial Nitric Oxide Synthase (eNOS) Signaling Pathway

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.908237

Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation and time-dependent inflammatory cytokine expression The upper part of left panel shows the immunoblots of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 in cultured HAECs exposed to hypoxia-reoxygenation for 0, 12, and 24 hours. Columns on the lower part of left panel indicate the relative expression levels (normalized to GAPDH) of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in cultured HAECs exposed to hypoxia-reoxygenation for 0, 12 and 24 hours respectively. Columns on the right of this figure indicate the detected concentrations of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in culture medium supernatant of cultured HAECs exposed to hypoxia-reoxygenation for 0, 12 and 24 hours respectively. Differences are significant (p
Figure Legend Snippet: Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation and time-dependent inflammatory cytokine expression The upper part of left panel shows the immunoblots of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 in cultured HAECs exposed to hypoxia-reoxygenation for 0, 12, and 24 hours. Columns on the lower part of left panel indicate the relative expression levels (normalized to GAPDH) of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in cultured HAECs exposed to hypoxia-reoxygenation for 0, 12 and 24 hours respectively. Columns on the right of this figure indicate the detected concentrations of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in culture medium supernatant of cultured HAECs exposed to hypoxia-reoxygenation for 0, 12 and 24 hours respectively. Differences are significant (p

Techniques Used: Cell Culture, Expressing, Western Blot

Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation and expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). ( A ) Columns indicate the detected optical density (O.D) at 450nm indicating peroxisome proliferator-activated receptor-γ (PPAR-γ) activities in cultured HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( B ) The left side shows the captured images of DAF-FM DA fluorescent stain of HAECs. Columns on the right part indicated the fluorescent intensities of DAG-FM DA stain of HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( C ) The immunoblots of pPPAR-γ, PPAR-γ, p-eNOS, eNOS and GAPDH in HAECs are shown on the left. Columns on the right show the relative phosphorylation levels of PPAR-γ (white columns) and eNOS (black columns) in cultured HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( D ) The immunoblots on the right show the immunoprecipitation analysis of heat shock protein (HSP)90/eNOS interaction in HAECs. Columns on the right indicate the eNOS/HSP90 association (normalized to HSP90) in HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. Differences were significant (p
Figure Legend Snippet: Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation and expression of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). ( A ) Columns indicate the detected optical density (O.D) at 450nm indicating peroxisome proliferator-activated receptor-γ (PPAR-γ) activities in cultured HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( B ) The left side shows the captured images of DAF-FM DA fluorescent stain of HAECs. Columns on the right part indicated the fluorescent intensities of DAG-FM DA stain of HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( C ) The immunoblots of pPPAR-γ, PPAR-γ, p-eNOS, eNOS and GAPDH in HAECs are shown on the left. Columns on the right show the relative phosphorylation levels of PPAR-γ (white columns) and eNOS (black columns) in cultured HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. ( D ) The immunoblots on the right show the immunoprecipitation analysis of heat shock protein (HSP)90/eNOS interaction in HAECs. Columns on the right indicate the eNOS/HSP90 association (normalized to HSP90) in HAECS exposed to hypoxia-reoxygenation for 0, 12, and 24 hours, respectively. Differences were significant (p

Techniques Used: Cell Culture, Expressing, Staining, Western Blot, Immunoprecipitation

Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation, expression of inflammatory cytokines, and treatment with emodin and inhibitors of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). The upper panel of left part of this figure shows the immunoblots of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 in cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation. Columns in the lower panel of the left part of this figure indicate the relative expression levels (normalized to GAPDH) of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (bleu columns) in HAECS exposed to hypoxia-reoxygenation treated with emodin and inhibitors of PPARγ/eNOS respectively. Columns on the right part of this figure indicated the detected concentrations of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in culture medium supernatant of HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS respectively. Differences were significant (p
Figure Legend Snippet: Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation, expression of inflammatory cytokines, and treatment with emodin and inhibitors of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). The upper panel of left part of this figure shows the immunoblots of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-8 in cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation. Columns in the lower panel of the left part of this figure indicate the relative expression levels (normalized to GAPDH) of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (bleu columns) in HAECS exposed to hypoxia-reoxygenation treated with emodin and inhibitors of PPARγ/eNOS respectively. Columns on the right part of this figure indicated the detected concentrations of TNF-α (light blue columns), IL-6 (deep blue columns) and IL-8 (blue columns) in culture medium supernatant of HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS respectively. Differences were significant (p

Techniques Used: Cell Culture, Expressing, Western Blot

Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation, expression of inflammatory cytokines, and treatment with emodin and inhibitors of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). ( A ) Columns show the detected O.D. at 450nm indicating peroxisome proliferator-activated receptor-γ (PPAR-γ) activities in cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/endothelial nitric oxide synthase (eNOS), respectively. ( B ) The left part of this panel demonstrates the captured fluorescent images of diaminofluorescein-FM diacetate (DAF-FM DA) fluorescence staining of HAECs. Columns on the right part of this panel indicated the fluorescent intensities of DAG-FM DA stain of HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS, respectively. ( C ) The immunoblots of p-PPAR, PPAR, p-eNOS, eNOS and GAPDH are demonstrated on the right part of this panel. Columns on the right part indicate the relative phosphorylation levels of PPAR-γ (light blue columns) and eNOS (deep blue columns) in HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS, respectively. Differences were significant (p
Figure Legend Snippet: Cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation, expression of inflammatory cytokines, and treatment with emodin and inhibitors of peroxisome proliferator-activated receptor-γ (PPAR-γ) and endothelial nitric oxide synthase (eNOS). ( A ) Columns show the detected O.D. at 450nm indicating peroxisome proliferator-activated receptor-γ (PPAR-γ) activities in cultured human aortic endothelial cells (HAECs) exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/endothelial nitric oxide synthase (eNOS), respectively. ( B ) The left part of this panel demonstrates the captured fluorescent images of diaminofluorescein-FM diacetate (DAF-FM DA) fluorescence staining of HAECs. Columns on the right part of this panel indicated the fluorescent intensities of DAG-FM DA stain of HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS, respectively. ( C ) The immunoblots of p-PPAR, PPAR, p-eNOS, eNOS and GAPDH are demonstrated on the right part of this panel. Columns on the right part indicate the relative phosphorylation levels of PPAR-γ (light blue columns) and eNOS (deep blue columns) in HAECS exposed to hypoxia-reoxygenation co-treated with emodin and inhibitors of PPARγ/eNOS, respectively. Differences were significant (p

Techniques Used: Cell Culture, Expressing, Fluorescence, Staining, Western Blot

16) Product Images from "Proteasome subunit-α type-6 protein is post-transcriptionally repressed by the microRNA-4490 in diabetic nephropathy"

Article Title: Proteasome subunit-α type-6 protein is post-transcriptionally repressed by the microRNA-4490 in diabetic nephropathy

Journal: Bioscience Reports

doi: 10.1042/BSR20180815

Down-regulation of PSMA1 expression in NRK-52E cells maintained under high glucose occurs independently of changes in PSMA6 mRNA levels ( A ) Relative expression of PSMA6 mRNA in NRK-52E cells maintained in culture with high glucose over 3 days. ( B ) Relative expression of PSMA6 protein under the same conditions as ( A ). Blots were probed with GAPDH antibody to confirm equal loading across the different lanes.
Figure Legend Snippet: Down-regulation of PSMA1 expression in NRK-52E cells maintained under high glucose occurs independently of changes in PSMA6 mRNA levels ( A ) Relative expression of PSMA6 mRNA in NRK-52E cells maintained in culture with high glucose over 3 days. ( B ) Relative expression of PSMA6 protein under the same conditions as ( A ). Blots were probed with GAPDH antibody to confirm equal loading across the different lanes.

Techniques Used: Expressing

17) Product Images from "α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis"

Article Title: α-ketoglutarate dehydrogenase inhibition counteracts breast cancer-associated lung metastasis

Journal: Cell Death & Disease

doi: 10.1038/s41419-018-0802-8

AA6 prevents metastasization targeting the TET–miR200–Zeb1/CtBP1–MMP3 axis. a Relative enrichment of 5mC in selected CCpGG sites of miR-200 family promoter regions for cluster 1 (left panel) and cluster 2 (right panel) in 4T1-injected mice DNA treated with AA6 (50 mg/kg; grey bars) versus control mice DNA (black bars); n = 5. b Pri-miR-200 cluster 1 (left panel) and cluster 2 (right panel) level of AA6 (50 mg/kg; grey bars) treated 4T1-injected mice expressed as fold-induction compared to untreated mice (black bars); n = 3. c Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1-injected mice treated with AA6 (50 mg/kg; grey bars), the graph represents average fold changes versus controls (black bars); n = 4. d Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1 cells treated with AA6 (50 µM; grey bars) for 6, 16, and 24 h, bar graphs represent average fold changes versus vehicle-treated cells (black bars); n = 4. e Representative WB (left panel) and relative densitometry (right panel; n = 5) of ZEB1 protein level in AA6 (50 mg/kg; grey bar) treated mice compared to controls (black bar). GAPDH and Red Ponceau were used as loading controls. f , g Zeb1 mRNA expression levels ( f ) and representative western blotting analysis of ZEB1 protein expression ( g ) in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); the right panel shows the relative densitometry as fold-change versus vehicle. α-tubulin was used as loading control; n = 4. h Representative WB (left panel) and relative densitometry (right panel) of ZEB1 protein expression level in AA6 treated 4T1 cells compared to vehicle-treated cells after transfection either with scramble-LNA (vehicle: black bar; AA6 50 µM: light grey bar) or anti-miR-200c-LNA (vehicle: dark grey bar; AA6 50 µM: medium grey bar). α-tubulin was used as loading control; n = 4. Data are presented as mean ± SE; * p
Figure Legend Snippet: AA6 prevents metastasization targeting the TET–miR200–Zeb1/CtBP1–MMP3 axis. a Relative enrichment of 5mC in selected CCpGG sites of miR-200 family promoter regions for cluster 1 (left panel) and cluster 2 (right panel) in 4T1-injected mice DNA treated with AA6 (50 mg/kg; grey bars) versus control mice DNA (black bars); n = 5. b Pri-miR-200 cluster 1 (left panel) and cluster 2 (right panel) level of AA6 (50 mg/kg; grey bars) treated 4T1-injected mice expressed as fold-induction compared to untreated mice (black bars); n = 3. c Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1-injected mice treated with AA6 (50 mg/kg; grey bars), the graph represents average fold changes versus controls (black bars); n = 4. d Cluster 1 (miR-200b, miR-200a and miR-429; left panel) and cluster 2 (miR-200c and miR-141; right panel) expression in 4T1 cells treated with AA6 (50 µM; grey bars) for 6, 16, and 24 h, bar graphs represent average fold changes versus vehicle-treated cells (black bars); n = 4. e Representative WB (left panel) and relative densitometry (right panel; n = 5) of ZEB1 protein level in AA6 (50 mg/kg; grey bar) treated mice compared to controls (black bar). GAPDH and Red Ponceau were used as loading controls. f , g Zeb1 mRNA expression levels ( f ) and representative western blotting analysis of ZEB1 protein expression ( g ) in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); the right panel shows the relative densitometry as fold-change versus vehicle. α-tubulin was used as loading control; n = 4. h Representative WB (left panel) and relative densitometry (right panel) of ZEB1 protein expression level in AA6 treated 4T1 cells compared to vehicle-treated cells after transfection either with scramble-LNA (vehicle: black bar; AA6 50 µM: light grey bar) or anti-miR-200c-LNA (vehicle: dark grey bar; AA6 50 µM: medium grey bar). α-tubulin was used as loading control; n = 4. Data are presented as mean ± SE; * p

Techniques Used: Injection, Mouse Assay, Expressing, Western Blot, Transfection

AA6 administration decreases metastasis-associated transcripts and interferes with 4T1 cells migration. a Heatmap showing the 53 most differentially regulated genes in tumour mass derived from AA6 injected mice (50 mg/kg), or untreated mice; n = 3 each group. Yellow and blue represent over- and under-expressed genes, respectively. b mRNA expression analysis of Matrix metallopeptidase 3 (Mmp3), Glycoprotein transmembrane non-metastatic B (Gpnmb), C-terminal binding protein 1 (Ctbp1), Plasminogen activator, urokinase receptor (Plaur) and Rous sarcoma oncogene (Src) genes in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. c Representative western blot (upper panels) and relative densitometry (lower panel) of MMP3, GPNMB, CtBP1 and SRC protein levels in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). GRB2 and GAPDH were used as loading controls; n = 5 each group. d Representative phase contrast microscopy images (upper panel) depicting 4T1 cells motility after 24 h treatment with AA6 (50 µM) or vehicle alone; the graph (lower panel) shows the percentage of closure in 4T1 cells after 24 h treatment with AA6 (50 µM; grey bar) or vehicle (black bar). Scale bar 100 μm; n = 5 each group. e Representative pictures (upper panel) showing 4T1 cell invasiveness after AA6 (50 µM) treatment versus vehicle; the graphs (lower panel) represent migrated cells counted after 24 h treatment with AA6 (50 µM; grey bar) or vehicle alone (black bar). Scale bar 50 μm; n = 3. Data are presented as mean ± SE; * p
Figure Legend Snippet: AA6 administration decreases metastasis-associated transcripts and interferes with 4T1 cells migration. a Heatmap showing the 53 most differentially regulated genes in tumour mass derived from AA6 injected mice (50 mg/kg), or untreated mice; n = 3 each group. Yellow and blue represent over- and under-expressed genes, respectively. b mRNA expression analysis of Matrix metallopeptidase 3 (Mmp3), Glycoprotein transmembrane non-metastatic B (Gpnmb), C-terminal binding protein 1 (Ctbp1), Plasminogen activator, urokinase receptor (Plaur) and Rous sarcoma oncogene (Src) genes in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. c Representative western blot (upper panels) and relative densitometry (lower panel) of MMP3, GPNMB, CtBP1 and SRC protein levels in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). GRB2 and GAPDH were used as loading controls; n = 5 each group. d Representative phase contrast microscopy images (upper panel) depicting 4T1 cells motility after 24 h treatment with AA6 (50 µM) or vehicle alone; the graph (lower panel) shows the percentage of closure in 4T1 cells after 24 h treatment with AA6 (50 µM; grey bar) or vehicle (black bar). Scale bar 100 μm; n = 5 each group. e Representative pictures (upper panel) showing 4T1 cell invasiveness after AA6 (50 µM) treatment versus vehicle; the graphs (lower panel) represent migrated cells counted after 24 h treatment with AA6 (50 µM; grey bar) or vehicle alone (black bar). Scale bar 50 μm; n = 3. Data are presented as mean ± SE; * p

Techniques Used: Migration, Derivative Assay, Injection, Mouse Assay, Expressing, Binding Assay, Western Blot, Microscopy

KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p
Figure Legend Snippet: KGDH inhibition increases TET expression and modulates 5mC/5hmC global levels both in vivo and in vitro. a Ten-eleven translocation hydroxylases (Tet) -1, 2, 3 mRNA expression levels in AA6 injected mice (50 mg/kg; grey bars) and control mice (black bars); n = 5. b Representative western blot (left panel) and relative densitometry (right panel; n = 4) of TET1, 2, 3 in AA6 (50 mg/kg; grey bars) treated mice compared to controls (black bars). α-tubulin and Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) were used as a loading controls. c Representative confocal images depicting the intracellular content of TET1, 2, 3 enzymes in 4T1 cells treated with AA6 (50 µM) or vehicle alone. Cells were probed by an anti-TET1 antibody (red; monoclonal), TET2 (green; polyclonal), TET3 (green; polyclonal) and counterstained by DAPI (blue). Scale bar 25 μm; n = 3. d TET activity quantification performed in 4T1 cells treated with AA6 (50 µM; grey bar) for 48 h indicated as percentage versus vehicle-treated cells (black bar); n = 3. e Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1-injected mice after AA6 administration (50 mg/kg; grey bars) compared to untreated mice (black bars); n = 5 each group. f Quantification of 5mC (left panel) and 5hmC (right panel) global levels in 4T1 cells exposed to AA6 (50 µM; grey bars) for 48 h indicated as fold-change versus vehicle-treated cells (black bars); n = 3 each group. Data are presented as mean ± SE; * p

Techniques Used: Inhibition, Expressing, In Vivo, In Vitro, Translocation Assay, Injection, Mouse Assay, Western Blot, Activity Assay

18) Product Images from "SORCS1 and SORCS3 control energy balance and orexigenic peptide production"

Article Title: SORCS1 and SORCS3 control energy balance and orexigenic peptide production

Journal: EMBO Reports

doi: 10.15252/embr.201744810

Deficiency for SORCS 1 and SORCS 3 enhances BDNF signaling in primary cortical neurons and acute hypothalamic slices Representative Western blots showing phosphorylated TrkB (pTrk) and total TrkB levels in DIV7 primary cortical neurons treated with BDNF (100 ng/ml) for the indicated periods of time. Detection of tubulin served as loading control. Quantification by densitometric scanning of replicate blots ( n = 4 biological replicates/genotype), as exemplified in panel (A), documents an increase in pTrkB levels in S1/3 KO neurons compared to WT neurons 10 min after BNDF application. Representative Western blots showing phosphorylated TrkB (pTrk) and total TrkB levels in acute hypothalamic slices of the indicated genotypes treated with BDNF (200 ng/ml) for 1 h. Detection of GAPDH served as a loading control. Quantification by densitometric scanning of replicate blots ( n = 14–15 mice/genotype), as exemplified in panel (C), documents an increase in pTrkB levels in S1/3 KO compared to WT hypothalamic slices. Expression of the indicated genes as analyzed by qRT–PCR in FACS‐sorted NPY/AgRP neurons (GFP + ) from fasted Npy ‐GFP mice. Expression in the sorted neurons was related to the expression in the whole (dissociated, but not FACS‐sorted) arcuate nucleus (Arc). Tissue from 10 mice was pooled for the experiment. Expression of neuropeptides as analyzed by qRT–PCR in DIV8 primary hypothalamic neurons treated with BDNF (100 ng/ml) for 48 h. The log2‐fold change in expression relative to the expression levels in vehicle‐treated neurons is shown. n = 5–6 technical replicates/treatment; data are from two neuronal culture preparations; 8‐9 hypothalami pooled in a single preparation. Expression of immediate early genes and neuropeptides as analyzed by qRT–PCR in DIV8 primary hypothalamic neurons treated with BDNF (100 ng/ml) for 1.5 h. The log2‐fold change in expression relative to the expression level in vehicle‐treated neurons is shown. n = 4 technical replicates/treatment; data are from two neuronal culture preparations; 8‐9 hypothalami pooled in a single preparation. Data information: Data in (B, D, F, and G) are shown as mean ± SEM and analyzed using two‐way ANOVA with Bonferroni post‐test (B) or a two‐tailed unpaired t ‐test (D, F, G). * P
Figure Legend Snippet: Deficiency for SORCS 1 and SORCS 3 enhances BDNF signaling in primary cortical neurons and acute hypothalamic slices Representative Western blots showing phosphorylated TrkB (pTrk) and total TrkB levels in DIV7 primary cortical neurons treated with BDNF (100 ng/ml) for the indicated periods of time. Detection of tubulin served as loading control. Quantification by densitometric scanning of replicate blots ( n = 4 biological replicates/genotype), as exemplified in panel (A), documents an increase in pTrkB levels in S1/3 KO neurons compared to WT neurons 10 min after BNDF application. Representative Western blots showing phosphorylated TrkB (pTrk) and total TrkB levels in acute hypothalamic slices of the indicated genotypes treated with BDNF (200 ng/ml) for 1 h. Detection of GAPDH served as a loading control. Quantification by densitometric scanning of replicate blots ( n = 14–15 mice/genotype), as exemplified in panel (C), documents an increase in pTrkB levels in S1/3 KO compared to WT hypothalamic slices. Expression of the indicated genes as analyzed by qRT–PCR in FACS‐sorted NPY/AgRP neurons (GFP + ) from fasted Npy ‐GFP mice. Expression in the sorted neurons was related to the expression in the whole (dissociated, but not FACS‐sorted) arcuate nucleus (Arc). Tissue from 10 mice was pooled for the experiment. Expression of neuropeptides as analyzed by qRT–PCR in DIV8 primary hypothalamic neurons treated with BDNF (100 ng/ml) for 48 h. The log2‐fold change in expression relative to the expression levels in vehicle‐treated neurons is shown. n = 5–6 technical replicates/treatment; data are from two neuronal culture preparations; 8‐9 hypothalami pooled in a single preparation. Expression of immediate early genes and neuropeptides as analyzed by qRT–PCR in DIV8 primary hypothalamic neurons treated with BDNF (100 ng/ml) for 1.5 h. The log2‐fold change in expression relative to the expression level in vehicle‐treated neurons is shown. n = 4 technical replicates/treatment; data are from two neuronal culture preparations; 8‐9 hypothalami pooled in a single preparation. Data information: Data in (B, D, F, and G) are shown as mean ± SEM and analyzed using two‐way ANOVA with Bonferroni post‐test (B) or a two‐tailed unpaired t ‐test (D, F, G). * P

Techniques Used: Western Blot, Mouse Assay, Expressing, Quantitative RT-PCR, FACS, Two Tailed Test

19) Product Images from "Long noncoding RNA ROR promotes breast cancer by regulating the TGF-β pathway"

Article Title: Long noncoding RNA ROR promotes breast cancer by regulating the TGF-β pathway

Journal: Cancer Cell International

doi: 10.1186/s12935-018-0638-4

Linc-ROR modulates the TGF-β signaling pathway. a Western blot probing TGF-β, Smad2 and α-SMA in the MCF-7 cells. The cells were transfected with si-ROR or si-nc. GAPDH was blotted as an internal reference. b Quantitative comparison of the expression levels of TGF-β, Smad2 and α-SMA. The protein expression levels in the MCF-7 cells transfected with si-nc were normalized to 1. The cells transfected with si-ROR showed reduced expression of all three proteins. c , d Comparison of the protein expression levels of TGF-β, Smad2 and α-SMA in the MDA-MB-231 cells. The panels were arranged as a , b . Data were Mean ± SD derived from three independent experiments. * p
Figure Legend Snippet: Linc-ROR modulates the TGF-β signaling pathway. a Western blot probing TGF-β, Smad2 and α-SMA in the MCF-7 cells. The cells were transfected with si-ROR or si-nc. GAPDH was blotted as an internal reference. b Quantitative comparison of the expression levels of TGF-β, Smad2 and α-SMA. The protein expression levels in the MCF-7 cells transfected with si-nc were normalized to 1. The cells transfected with si-ROR showed reduced expression of all three proteins. c , d Comparison of the protein expression levels of TGF-β, Smad2 and α-SMA in the MDA-MB-231 cells. The panels were arranged as a , b . Data were Mean ± SD derived from three independent experiments. * p

Techniques Used: Western Blot, Transfection, Expressing, Multiple Displacement Amplification, Derivative Assay

20) Product Images from "Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling"

Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

Journal: Nature Communications

doi: 10.1038/s41467-018-07660-6

Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm
Figure Legend Snippet: Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm

Techniques Used: Migration, Imaging, Immunohistochemistry, Expressing, Staining, Western Blot, Immunofluorescence

21) Product Images from "Human cytomegalovirus UL7, miR-US5-1 and miR-UL112-3p inactivation of FOXO3a protects CD34+ hematopoietic progenitor cells from apoptosis"

Article Title: Human cytomegalovirus UL7, miR-US5-1 and miR-UL112-3p inactivation of FOXO3a protects CD34+ hematopoietic progenitor cells from apoptosis

Journal: bioRxiv

doi: 10.1101/2020.09.28.317859

pUL7 induces FOXO3a translocation from the nucleus to the cytoplasm in RS4;11 and CD34 + HPC cells. A) RS4;11 cells were serum-starved, pre-treated with PD98059 (50 μM), LY294002 (20 μM), LY303511 (25 μM) for 1 hour and then stimulated for 10 min with Fc+DMSO or pUL7. Protein lysates were generated and immunoblotted for phosphorylation of Flt-3R (Tyr591), Akt (Thr308), ERK1/2 (Thr202/Tyr204), FOXO3a (Ser 294), and S6 (Ser235/236). Equal loading was confirmed by Flt-3R, Akt, ERK1, FOXO3a, S6 and GAPDH antibody staining. Numbers under the immunoblots indicate relative phosphorylation of Flt-3R, Akt, FOXO3a, ERK1/2 or S6 normalized to the amount of total Flt-3R, Akt, FOXO3a, ERK1, and S6 and compared to the value of the Fc control. B) Subcellular localization of FOXO3a in RS4;11 cells stimulated with Fc and pUL7 (50 ng/ml) at the indicated time points (representative images). FOXO3a (red, secondary antibody conjugated to AF594) and nucleus (DAPI, blue). Images were quantified using ImageJ and the ratio of nuclear to cytoplasmic FOXO3a over time is shown. The dotted line indicate transition from higher signal in the nucleus (above) to cytoplasm (below). Average of 30 cells were analyzed per condition. Values are means±standard error of the means (SEM) (error bars) from three independent experiments. Statistical significance was determined by unpaired Student’s t-test (*, P
Figure Legend Snippet: pUL7 induces FOXO3a translocation from the nucleus to the cytoplasm in RS4;11 and CD34 + HPC cells. A) RS4;11 cells were serum-starved, pre-treated with PD98059 (50 μM), LY294002 (20 μM), LY303511 (25 μM) for 1 hour and then stimulated for 10 min with Fc+DMSO or pUL7. Protein lysates were generated and immunoblotted for phosphorylation of Flt-3R (Tyr591), Akt (Thr308), ERK1/2 (Thr202/Tyr204), FOXO3a (Ser 294), and S6 (Ser235/236). Equal loading was confirmed by Flt-3R, Akt, ERK1, FOXO3a, S6 and GAPDH antibody staining. Numbers under the immunoblots indicate relative phosphorylation of Flt-3R, Akt, FOXO3a, ERK1/2 or S6 normalized to the amount of total Flt-3R, Akt, FOXO3a, ERK1, and S6 and compared to the value of the Fc control. B) Subcellular localization of FOXO3a in RS4;11 cells stimulated with Fc and pUL7 (50 ng/ml) at the indicated time points (representative images). FOXO3a (red, secondary antibody conjugated to AF594) and nucleus (DAPI, blue). Images were quantified using ImageJ and the ratio of nuclear to cytoplasmic FOXO3a over time is shown. The dotted line indicate transition from higher signal in the nucleus (above) to cytoplasm (below). Average of 30 cells were analyzed per condition. Values are means±standard error of the means (SEM) (error bars) from three independent experiments. Statistical significance was determined by unpaired Student’s t-test (*, P

Techniques Used: Translocation Assay, Generated, Staining, Western Blot

22) Product Images from "The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility"

Article Title: The MK2/3 cascade regulates AMPAR trafficking and cognitive flexibility

Journal: Nature Communications

doi: 10.1038/ncomms5701

The p38-MK2/3-cofilin1 cascade is required in DHPG-LTD. ( a – f ) Representative blots of non-stimulated (C), DHPG-incubated (100 μM, 10 min; D), pre-incubated with p38 inhibitor SB 203580 (5 μM) alone (SB) or pre-incubated with DHPG application (SB+D) cultures from WT and MK2/3 DKO hippocampus. ( a ) DHPG causes a significant increase in p-p38 at (Thr180/Tyr182) and ( b ) a significant reduction in p-cofilin1 in WT cells, an effect that is blocked in DKO cells. Note that no change in total cofilin1 ( c ) or mGluR5 ( d ) expression is detected between genotypes. ( e ) Blot showing a significant increase in phosphorylation of MK2 (Thr334) after DHPG-exposure in WT cells, an effect blocked by pre-incubation with SB. ( f ) Blot showing that the DHPG-dependent reduction in levels of p-cofilin1 (Ser3) is blocked by pre-incubation of p38 inhibitor SB 203580 (5 μM) in WT hippocampal cultures. Incubation of SB alone does not promote any significant changes in cofilin1 phosphorylation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin were used as loading controls. Western blot band densitometry analyses were obtained from a minimum of three different primary hippocampal preparations from the WT and DKO mice. T -tests, one-way analysis of variance and the appropriate post-hoc test were conducted accordingly for each data set. Error bars indicate ±s.e.m. and * P
Figure Legend Snippet: The p38-MK2/3-cofilin1 cascade is required in DHPG-LTD. ( a – f ) Representative blots of non-stimulated (C), DHPG-incubated (100 μM, 10 min; D), pre-incubated with p38 inhibitor SB 203580 (5 μM) alone (SB) or pre-incubated with DHPG application (SB+D) cultures from WT and MK2/3 DKO hippocampus. ( a ) DHPG causes a significant increase in p-p38 at (Thr180/Tyr182) and ( b ) a significant reduction in p-cofilin1 in WT cells, an effect that is blocked in DKO cells. Note that no change in total cofilin1 ( c ) or mGluR5 ( d ) expression is detected between genotypes. ( e ) Blot showing a significant increase in phosphorylation of MK2 (Thr334) after DHPG-exposure in WT cells, an effect blocked by pre-incubation with SB. ( f ) Blot showing that the DHPG-dependent reduction in levels of p-cofilin1 (Ser3) is blocked by pre-incubation of p38 inhibitor SB 203580 (5 μM) in WT hippocampal cultures. Incubation of SB alone does not promote any significant changes in cofilin1 phosphorylation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) and β-tubulin were used as loading controls. Western blot band densitometry analyses were obtained from a minimum of three different primary hippocampal preparations from the WT and DKO mice. T -tests, one-way analysis of variance and the appropriate post-hoc test were conducted accordingly for each data set. Error bars indicate ±s.e.m. and * P

Techniques Used: Incubation, Expressing, Western Blot, Mouse Assay

23) Product Images from "Bepridil decreases Aβ and calcium levels in the thalamus after middle cerebral artery occlusion in rats"

Article Title: Bepridil decreases Aβ and calcium levels in the thalamus after middle cerebral artery occlusion in rats

Journal: Journal of Cellular and Molecular Medicine

doi: 10.1111/j.1582-4934.2012.01599.x

Bepridil treatment does not affect IDE, NEP or LRP expression, or the inflammatory response in the ipsilateral thalamus in MCAO rats. (A) Western blot showing the protein levels of IDE, NEP and LRP in the contralateral (C) and ipsilateral (I) thalamus of the vehicle- (MCAO) and bepridil-treated (MCAO+BEP) MCAO rats as well as sham-operated (SHAM) rats. Total protein lysate from human SH-SY5Y cells stably overexpressing the APP751 isoform (SH-SY5Y-APP751) was used as a control. GAPDH-normalized protein levels in the ipsilateral thalamus were compared relative to the contralateral thalamus (=100%). Statistical comparison was performed between ipsilateral and contralateral sides as well as between different treatment groups. (B) Western blot analysis of GFAP levels in the contralateral (C) and ipsilateral (I) thalamus of the vehicle- (MCAO) and bepridil-treated (MCAO+BEP) MCAO rats as well as sham-operated (SHAM) rats. TNF-α and GAPDH mRNA levels were quantified using qPCR. GAPDH-normalized GFAP and TNF-α levels in the ipsilateral thalamus were compared relative to the contralateral thalamus (=100%). Data are shown as mean ± S.D. * P
Figure Legend Snippet: Bepridil treatment does not affect IDE, NEP or LRP expression, or the inflammatory response in the ipsilateral thalamus in MCAO rats. (A) Western blot showing the protein levels of IDE, NEP and LRP in the contralateral (C) and ipsilateral (I) thalamus of the vehicle- (MCAO) and bepridil-treated (MCAO+BEP) MCAO rats as well as sham-operated (SHAM) rats. Total protein lysate from human SH-SY5Y cells stably overexpressing the APP751 isoform (SH-SY5Y-APP751) was used as a control. GAPDH-normalized protein levels in the ipsilateral thalamus were compared relative to the contralateral thalamus (=100%). Statistical comparison was performed between ipsilateral and contralateral sides as well as between different treatment groups. (B) Western blot analysis of GFAP levels in the contralateral (C) and ipsilateral (I) thalamus of the vehicle- (MCAO) and bepridil-treated (MCAO+BEP) MCAO rats as well as sham-operated (SHAM) rats. TNF-α and GAPDH mRNA levels were quantified using qPCR. GAPDH-normalized GFAP and TNF-α levels in the ipsilateral thalamus were compared relative to the contralateral thalamus (=100%). Data are shown as mean ± S.D. * P

Techniques Used: Expressing, Western Blot, Stable Transfection, Real-time Polymerase Chain Reaction

24) Product Images from "Sirt1-deficiency causes defective protein quality control"

Article Title: Sirt1-deficiency causes defective protein quality control

Journal: Scientific Reports

doi: 10.1038/srep12613

Proteasome activities are not impaired in Sirt1 –/– MEFs. ( A ) Lysates from wild-type and Sirt1 –/– MEFs were fractionated by 8–32% glycerol gradient centrifugation. An aliquot of each fraction was used for an assay of chymotryptic activity of proteasomes using succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (Suc-LLVY-AMC) as a substrate and immunoblotting for Rpt6 and α6. ( B ) Ubiquitin-independent and -dependent protease activities of proteasomes. Lysates from wild-type and Sirt1 –/– MEFs were subjected to an in vitro protein degradation assay. Antizyme-dependent degradation of 35 S-labeled ODC and ubiquitin-dependent degradation of 35 S-labeled cIAP1 were measured. The data represent means ± standard error of the mean (SEM) from three independent experiments. Statistical comparisons were made by Student’s t -test for two tailed unpaired samples. N.S. indicates not significant. ( C ) Lysates of wild-type and Sirt1 –/– MEFs treated with or without 10 μM MG132 for 30 min were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). ( D ) Lysates of wild-type and Sirt1 –/– MEFs treated with or without 100 nM bafilomycin A1 for 20 h were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure .
Figure Legend Snippet: Proteasome activities are not impaired in Sirt1 –/– MEFs. ( A ) Lysates from wild-type and Sirt1 –/– MEFs were fractionated by 8–32% glycerol gradient centrifugation. An aliquot of each fraction was used for an assay of chymotryptic activity of proteasomes using succinyl-Leu-Leu-Val-Tyr-7-amino-4-methyl-coumarin (Suc-LLVY-AMC) as a substrate and immunoblotting for Rpt6 and α6. ( B ) Ubiquitin-independent and -dependent protease activities of proteasomes. Lysates from wild-type and Sirt1 –/– MEFs were subjected to an in vitro protein degradation assay. Antizyme-dependent degradation of 35 S-labeled ODC and ubiquitin-dependent degradation of 35 S-labeled cIAP1 were measured. The data represent means ± standard error of the mean (SEM) from three independent experiments. Statistical comparisons were made by Student’s t -test for two tailed unpaired samples. N.S. indicates not significant. ( C ) Lysates of wild-type and Sirt1 –/– MEFs treated with or without 10 μM MG132 for 30 min were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). ( D ) Lysates of wild-type and Sirt1 –/– MEFs treated with or without 100 nM bafilomycin A1 for 20 h were subjected to immunoblotting with the indicated antibodies. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure .

Techniques Used: Gradient Centrifugation, Activity Assay, In Vitro, Degradation Assay, Labeling, Two Tailed Test

Ubiquitinated proteins accumulate in Sirt1 –/– MEFs. ( A ) Immunoblotting of immortalized MEF lysates of the indicated genotype. Actin serves as a loading control. Values represent the relative band intensities of ubiquitin (Ub) (normalized to GAPDH). ( B ) Lysates from wild-type and Sirt1 –/– MEFs were subjected to immunoprecipitation with anti-Rpt6 antibodies, followed by immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). ( C ) Immunoblotting of lysates from Flag-ubiquitin-introduced wild-type MEFs and Sirt1 –/– MEFs transfected with wild-type Sirt1 or H355Y Sirt1. Values represent the relative band intensities of Ub (normalized to Actin). Uncropped gel images are shown in Supplementary Figure .
Figure Legend Snippet: Ubiquitinated proteins accumulate in Sirt1 –/– MEFs. ( A ) Immunoblotting of immortalized MEF lysates of the indicated genotype. Actin serves as a loading control. Values represent the relative band intensities of ubiquitin (Ub) (normalized to GAPDH). ( B ) Lysates from wild-type and Sirt1 –/– MEFs were subjected to immunoprecipitation with anti-Rpt6 antibodies, followed by immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). ( C ) Immunoblotting of lysates from Flag-ubiquitin-introduced wild-type MEFs and Sirt1 –/– MEFs transfected with wild-type Sirt1 or H355Y Sirt1. Values represent the relative band intensities of Ub (normalized to Actin). Uncropped gel images are shown in Supplementary Figure .

Techniques Used: Immunoprecipitation, Transfection

Downregulation of Hsp70 is not the sole cause of aberrant protein quality control in Sirt1 –/– MEFs. ( A ) Immunoblotting of lysates from wild-type and Sirt1 –/– MEFs transfected with empty vector or HA-tagged Hsp70. Values represent the relative band intensities of Ub (normalized to Actin). ( B ) Immunoblotting of lysates from wild-type, Sirt1 –/– , and Sirt1 –/– transfected with wild-type Sirt1 MEFs. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure .
Figure Legend Snippet: Downregulation of Hsp70 is not the sole cause of aberrant protein quality control in Sirt1 –/– MEFs. ( A ) Immunoblotting of lysates from wild-type and Sirt1 –/– MEFs transfected with empty vector or HA-tagged Hsp70. Values represent the relative band intensities of Ub (normalized to Actin). ( B ) Immunoblotting of lysates from wild-type, Sirt1 –/– , and Sirt1 –/– transfected with wild-type Sirt1 MEFs. Values represent the relative band intensities of Ub (normalized to GAPDH). Uncropped gel images are shown in Supplementary Figure .

Techniques Used: Transfection, Plasmid Preparation

Sirt1-deficiency does not lead to impairment in the induction of Hsp70 after heat shock. ( A ) Immunofluorescence with anti-ubiquitin antibodies of wild-type and Sirt1 –/– MEFs treated with or without heat shock (42 °C for 30 min). Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. Values represent the relative intensity of Ub staining per cell. ( B ) Wild-type and Sirt1 –/– MEFs were treated with or without heat shock (42 °C for 30 min). Heated cells were then immediately harvested or allowed to recover at 37 °C for 5 h, and cell lysates were subjected to immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). ( C , D ) Wild-type and Sirt1 –/– mice were treated with or without heat shock (42 °C for 45 min). Each group consisted of four or six littermates. Heated mice were then immediately sacrificed or allowed to recover at room temperature for 7 h. The lysates from their lung ( C ) and liver ( D ) were subjected to immunoblotting (left panels). Values represent the relative band intensities of Ub (normalized to GAPDH). Statistical comparisons were made by Student’s t -test for two tailed unpaired samples. The data represent means ± SEM. N.S. indicates not significant. Uncropped gel images are shown in Supplementary Figure .
Figure Legend Snippet: Sirt1-deficiency does not lead to impairment in the induction of Hsp70 after heat shock. ( A ) Immunofluorescence with anti-ubiquitin antibodies of wild-type and Sirt1 –/– MEFs treated with or without heat shock (42 °C for 30 min). Nuclei were stained with Hoechst 33342. Scale bar, 50 μm. Values represent the relative intensity of Ub staining per cell. ( B ) Wild-type and Sirt1 –/– MEFs were treated with or without heat shock (42 °C for 30 min). Heated cells were then immediately harvested or allowed to recover at 37 °C for 5 h, and cell lysates were subjected to immunoblotting. Values represent the relative band intensities of Ub (normalized to GAPDH). ( C , D ) Wild-type and Sirt1 –/– mice were treated with or without heat shock (42 °C for 45 min). Each group consisted of four or six littermates. Heated mice were then immediately sacrificed or allowed to recover at room temperature for 7 h. The lysates from their lung ( C ) and liver ( D ) were subjected to immunoblotting (left panels). Values represent the relative band intensities of Ub (normalized to GAPDH). Statistical comparisons were made by Student’s t -test for two tailed unpaired samples. The data represent means ± SEM. N.S. indicates not significant. Uncropped gel images are shown in Supplementary Figure .

Techniques Used: Immunofluorescence, Staining, Mouse Assay, Two Tailed Test

25) Product Images from "Effects of miR-106b-3p on cell proliferation and epithelial-mesenchymal transition, and targeting of ZNRF3 in esophageal squamous cell carcinoma"

Article Title: Effects of miR-106b-3p on cell proliferation and epithelial-mesenchymal transition, and targeting of ZNRF3 in esophageal squamous cell carcinoma

Journal: International Journal of Molecular Medicine

doi: 10.3892/ijmm.2019.4107

miR-106b-3p promoted adhesion and epithelial-mesenchymal transition of ESCC cells. (A) Cell morphology was observed using a microscope. (B) miR-106b-3p mimics promoted cell adhesion. (C) Western blot analysis was performed to analyze the protein expressions of Slug, Snail, E-cadherin, N-cadherin and Vimentin in ESCC cells. GAPDH was used the internal control. The data are presented as the mean ± standard deviation of three independent experiments. * P
Figure Legend Snippet: miR-106b-3p promoted adhesion and epithelial-mesenchymal transition of ESCC cells. (A) Cell morphology was observed using a microscope. (B) miR-106b-3p mimics promoted cell adhesion. (C) Western blot analysis was performed to analyze the protein expressions of Slug, Snail, E-cadherin, N-cadherin and Vimentin in ESCC cells. GAPDH was used the internal control. The data are presented as the mean ± standard deviation of three independent experiments. * P

Techniques Used: Microscopy, Western Blot, Standard Deviation

26) Product Images from "In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation"

Article Title: In vivo imaging of endothelial cell adhesion molecule expression after radiosurgery in an animal model of arteriovenous malformation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0185393

Quantitative real-time PCR and western analysis of ICAM-1 and VCAM-1 expression in irradiated bEnd.3 cells. qRT-PCR analysis of ICAM-1 (A) and VCAM-1 (E) gene expression, n = 4 independent experiments. Representative western blots (time course) of ICAM-1 (B) and VCAM-1 (F) protein at 25 Gy. Representative western blots (dose response) of ICAM-1 expression (120 h) (C) and VCAM-1 expression (72 h) (G) post-irradiation. ICAM-1 (D) and VCAM-1 (H) protein expression quantitated using Image J, n = 4. Values are mean ± SEM. Data were normalized to GAPDH (westerns) or HPRT (qRT-PCR).
Figure Legend Snippet: Quantitative real-time PCR and western analysis of ICAM-1 and VCAM-1 expression in irradiated bEnd.3 cells. qRT-PCR analysis of ICAM-1 (A) and VCAM-1 (E) gene expression, n = 4 independent experiments. Representative western blots (time course) of ICAM-1 (B) and VCAM-1 (F) protein at 25 Gy. Representative western blots (dose response) of ICAM-1 expression (120 h) (C) and VCAM-1 expression (72 h) (G) post-irradiation. ICAM-1 (D) and VCAM-1 (H) protein expression quantitated using Image J, n = 4. Values are mean ± SEM. Data were normalized to GAPDH (westerns) or HPRT (qRT-PCR).

Techniques Used: Real-time Polymerase Chain Reaction, Western Blot, Expressing, Irradiation, Quantitative RT-PCR

27) Product Images from "Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells"

Article Title: Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells

Journal: Nucleic Acids Research

doi: 10.1093/nar/gky149

USP9X activity regulates ZBTB38 protein abundance. ( A ) Depletion of USP9X by siRNA causes a decrease in ZBTB38 protein abundance. Upper panel: western blot in HeLa cells; two independent siRNAs were used, GAPDH is a loading control. WCE: whole-cell extract. Lower panel: ZBTB38 mRNA abundance is not affected by USP9X depletion. qRT-PCR on HeLa cells after siRNA transfection, values normalized to three housekeeping genes (MAPK14, TBP and TFRC), n = 3. ( B ) The amount of ZBTB38 is cell-cycle regulated, and USP9X stabilizes ZBTB38 in both mitosis and S-phase. Western blotting on HeLa cells transfected with the indicated siRNAs, then synchronized in mitosis (with nocodazole), or in S-phase (with hydroxyurea). ( C ) ZBTB38 is stabilized by MG132 (20 mM, 8 h). Western blot as in panels A and B ( D ) Treatment with a USP9X inhibitor, WP1130, causes a decrease in ZBTB38 protein abundance. The cells were harvested immediately after a 2-h treatment with WP1130 (lower concentration: 1μM; higher concentration: 2μM), then western blotting was carried out with WCE. ( E ) Treatment with WP1130 does not affect ZBTB38 mRNA abundance. RT-qPCR as in panel A. ( F ) Treatment with WP1130 does not affect cell cycle distribution or viability, as determined by FACS analysis. ( G ) Treatment with WP1130 leads to an increase of ZBTB38 ubiquitination. Cells were tranfected with an HA-ubiquitin expression vector, treated with MG132 (20mM, 8hrs), and ubiquitinated ZBTB38 was precipitated followed by immunoblotting with anti-HA. ( H ) Depletion of USP9X, but not USP7, leads to an increase of ZBTB38 ubiquitination. As in panel G.
Figure Legend Snippet: USP9X activity regulates ZBTB38 protein abundance. ( A ) Depletion of USP9X by siRNA causes a decrease in ZBTB38 protein abundance. Upper panel: western blot in HeLa cells; two independent siRNAs were used, GAPDH is a loading control. WCE: whole-cell extract. Lower panel: ZBTB38 mRNA abundance is not affected by USP9X depletion. qRT-PCR on HeLa cells after siRNA transfection, values normalized to three housekeeping genes (MAPK14, TBP and TFRC), n = 3. ( B ) The amount of ZBTB38 is cell-cycle regulated, and USP9X stabilizes ZBTB38 in both mitosis and S-phase. Western blotting on HeLa cells transfected with the indicated siRNAs, then synchronized in mitosis (with nocodazole), or in S-phase (with hydroxyurea). ( C ) ZBTB38 is stabilized by MG132 (20 mM, 8 h). Western blot as in panels A and B ( D ) Treatment with a USP9X inhibitor, WP1130, causes a decrease in ZBTB38 protein abundance. The cells were harvested immediately after a 2-h treatment with WP1130 (lower concentration: 1μM; higher concentration: 2μM), then western blotting was carried out with WCE. ( E ) Treatment with WP1130 does not affect ZBTB38 mRNA abundance. RT-qPCR as in panel A. ( F ) Treatment with WP1130 does not affect cell cycle distribution or viability, as determined by FACS analysis. ( G ) Treatment with WP1130 leads to an increase of ZBTB38 ubiquitination. Cells were tranfected with an HA-ubiquitin expression vector, treated with MG132 (20mM, 8hrs), and ubiquitinated ZBTB38 was precipitated followed by immunoblotting with anti-HA. ( H ) Depletion of USP9X, but not USP7, leads to an increase of ZBTB38 ubiquitination. As in panel G.

Techniques Used: Activity Assay, Western Blot, Quantitative RT-PCR, Transfection, Concentration Assay, FACS, Expressing, Plasmid Preparation

28) Product Images from "Rab27-dependent exosome production inhibits chronic inflammation and enables acute responses to inflammatory stimuli"

Article Title: Rab27-dependent exosome production inhibits chronic inflammation and enables acute responses to inflammatory stimuli

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

doi: 10.4049/jimmunol.1700904

miR-155 containing exosome rescue LPS responsiveness in Rab27DKO mice (A) Rab27DKO mice were either i.p injected with a Rab27DKO, WT, or miR-155−/− exosome pellets 24 hours before an LPS challenge. Serum was taken 2 hours post LPS injection for ELISAs. Values are set relative to Rab27DKO +Rab27DKO exosomal pellets +LPS set to 1. (B) Representative TNFα concentrations. (C–D) Levels of SHIP1 mRNA in resting WT and Rab27DKO mice in the spleen and bone marrow relative to L32 loading control. P values are from Welsh’s corrected t- tests. (E) Westerns of SHIP1 in resting spleen and bone marrow with GAPDH as a loading control. (F) IRAK–M mRNA levels in resting bone marrow relative to L32. P values are from Welsh’s corrected t- tests. (G–H) Levels of SHIP1 and IRAK-M mRNA in Rab27DKO mice BM that received Rab27DKO, WT, or miR-155−/− exosomal pellets then were treated with LPS for 72 hours. (I) Representative flow plots of the myeloid (GR1+ CD11b+) population for each condition in the bone marrow compartment from the same experiment conditions of H. (J) Ratio of the CD11b+ GR1+ population is shown of the LPS treatment group compared to the no LPS treatment group. Dotted line marks no change between LPS and no LPS treatments. Adjusted p values are either stated or *, p
Figure Legend Snippet: miR-155 containing exosome rescue LPS responsiveness in Rab27DKO mice (A) Rab27DKO mice were either i.p injected with a Rab27DKO, WT, or miR-155−/− exosome pellets 24 hours before an LPS challenge. Serum was taken 2 hours post LPS injection for ELISAs. Values are set relative to Rab27DKO +Rab27DKO exosomal pellets +LPS set to 1. (B) Representative TNFα concentrations. (C–D) Levels of SHIP1 mRNA in resting WT and Rab27DKO mice in the spleen and bone marrow relative to L32 loading control. P values are from Welsh’s corrected t- tests. (E) Westerns of SHIP1 in resting spleen and bone marrow with GAPDH as a loading control. (F) IRAK–M mRNA levels in resting bone marrow relative to L32. P values are from Welsh’s corrected t- tests. (G–H) Levels of SHIP1 and IRAK-M mRNA in Rab27DKO mice BM that received Rab27DKO, WT, or miR-155−/− exosomal pellets then were treated with LPS for 72 hours. (I) Representative flow plots of the myeloid (GR1+ CD11b+) population for each condition in the bone marrow compartment from the same experiment conditions of H. (J) Ratio of the CD11b+ GR1+ population is shown of the LPS treatment group compared to the no LPS treatment group. Dotted line marks no change between LPS and no LPS treatments. Adjusted p values are either stated or *, p

Techniques Used: Mouse Assay, Injection, Flow Cytometry

29) Product Images from "MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways"

Article Title: MutT Homolog 1 (MTH1) maintains multiple KRAS-driven pro-malignant pathways

Journal: Oncogene

doi: 10.1038/onc.2014.195

Molecular effects of MTH1 suppression in KRAS-mutant NSCLC cells (A)-(C) Immunoblotting using 20 μg protein lysates was carried out against the indicated proteins. Cell pellets were harvested approximately 14 days post-transduction (the A549 cells were harvested 8 days post-transduction). (A) MTH1 suppression elevates p53/p21 cip1/waf1 in p53-competent NSCLC cells. GAPDH is used as a loading normalization control. (B) MTH1 suppression elevates p27 kip1 and reduces pAkt in p53-noncompetent H358 and H23 NSCLC cells. Tubulin is used as a normalization control. (C) In vivo shMTH1 effects in H358 xenograft tumors recapitulate in vitro effects. Immunoblotting using 20 μg protein lysate from H358 xenograft tumor samples was carried out using the indicated antibodies. Coomassie blue total protein staining was used as a loading control. (D) H358 cells were transduced with either pBneo (pBn) or pBn. Myr-Akt and then selected in G418-containing media. Following successful selection, each counterpart was transduced with either shGFP or shMTH1. Cells were harvested and total protein lysates produced approximately 16 days following shRNA transduction. Immunoblotting was carried out on 20 μg of total protein against the indicated antibodies. (E) Relative cell proliferation rates. The four cell lines from (D) were each seeded at 5×10 4 on Day 0 and the number of cells counted (in triplicate) for the next four days.
Figure Legend Snippet: Molecular effects of MTH1 suppression in KRAS-mutant NSCLC cells (A)-(C) Immunoblotting using 20 μg protein lysates was carried out against the indicated proteins. Cell pellets were harvested approximately 14 days post-transduction (the A549 cells were harvested 8 days post-transduction). (A) MTH1 suppression elevates p53/p21 cip1/waf1 in p53-competent NSCLC cells. GAPDH is used as a loading normalization control. (B) MTH1 suppression elevates p27 kip1 and reduces pAkt in p53-noncompetent H358 and H23 NSCLC cells. Tubulin is used as a normalization control. (C) In vivo shMTH1 effects in H358 xenograft tumors recapitulate in vitro effects. Immunoblotting using 20 μg protein lysate from H358 xenograft tumor samples was carried out using the indicated antibodies. Coomassie blue total protein staining was used as a loading control. (D) H358 cells were transduced with either pBneo (pBn) or pBn. Myr-Akt and then selected in G418-containing media. Following successful selection, each counterpart was transduced with either shGFP or shMTH1. Cells were harvested and total protein lysates produced approximately 16 days following shRNA transduction. Immunoblotting was carried out on 20 μg of total protein against the indicated antibodies. (E) Relative cell proliferation rates. The four cell lines from (D) were each seeded at 5×10 4 on Day 0 and the number of cells counted (in triplicate) for the next four days.

Techniques Used: Mutagenesis, Transduction, In Vivo, In Vitro, Staining, Selection, Produced, shRNA

30) Product Images from "CR6-interacting factor 1 is a key regulator in Aβ-induced mitochondrial disruption and pathogenesis of Alzheimer's disease"

Article Title: CR6-interacting factor 1 is a key regulator in Aβ-induced mitochondrial disruption and pathogenesis of Alzheimer's disease

Journal: Cell Death and Differentiation

doi: 10.1038/cdd.2014.184

A β -induced ROS decreased the transcription of Crif1 gene by facilitating sumoylation of Sp1. ( a ) Treatment with H 2 O 2 -reduced Crif1 levels. 10–750 μ M of H 2 O 2 were administered for 6 h into SH-SY5Y cells, and then WB was performed to measure Crif1 levels. GAPDH is used as a loading control. Representative images are shown. ( b ) Treatment with a ROS scavenger (NAC; 1 mM) rescued A β -induced Crif1 downregulation. β -Actin is used as a loading control. Representative images are shown. Data are represented as mean±S.E.M. from three independent experiments. ** P
Figure Legend Snippet: A β -induced ROS decreased the transcription of Crif1 gene by facilitating sumoylation of Sp1. ( a ) Treatment with H 2 O 2 -reduced Crif1 levels. 10–750 μ M of H 2 O 2 were administered for 6 h into SH-SY5Y cells, and then WB was performed to measure Crif1 levels. GAPDH is used as a loading control. Representative images are shown. ( b ) Treatment with a ROS scavenger (NAC; 1 mM) rescued A β -induced Crif1 downregulation. β -Actin is used as a loading control. Representative images are shown. Data are represented as mean±S.E.M. from three independent experiments. ** P

Techniques Used: Western Blot

31) Product Images from "Osteogenic potential of cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells using concave microwells"

Article Title: Osteogenic potential of cell spheroids composed of varying ratios of gingiva-derived and bone marrow stem cells using concave microwells

Journal: Experimental and Therapeutic Medicine

doi: 10.3892/etm.2018.6462

Runx2 and osteocalcin protein expression in gingiva-derived and bone marrow stem cells co-cultures on day 7. (A) Western blot for Runx2, osteocalcin and GAPDH expression on day 7. Quantitative analysis of (B) Runx2 and (C) osteocalcin protein expression normalized to GAPDH. Runx2, runt-related transcription factor 2.
Figure Legend Snippet: Runx2 and osteocalcin protein expression in gingiva-derived and bone marrow stem cells co-cultures on day 7. (A) Western blot for Runx2, osteocalcin and GAPDH expression on day 7. Quantitative analysis of (B) Runx2 and (C) osteocalcin protein expression normalized to GAPDH. Runx2, runt-related transcription factor 2.

Techniques Used: Expressing, Derivative Assay, Western Blot

32) Product Images from "Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop"

Article Title: Nuclear expression of FLT1 and its ligand PGF in FUS-DDIT3 carrying myxoid liposarcomas suggests the existence of an intracrine signaling loop

Journal: BMC Cancer

doi: 10.1186/1471-2407-10-249

Nuclear FLT1 localization in MLS tumors and cultured MLS cells . (a) Immunohistochemical analysis of FLT1 expression in tissue sections of a representative MLS tumor and in an MLS 402-91 SCID mouse xenograft. Brown staining indicates FLT1 expression while blue staining shows negatively staining nuclei. Cytoplasmic FLT1 expression in angiosarcoma was used as a positive control. (b) FLT1 expression in cultured MLS 402-91 cells analyzed by immunofluorescence. The merge image shows nuclear DAPI staining in blue. Scale bar indicates 10 μm. (c) Western blot analysis of nuclear (N) and cytoplasmic (CP) fractions of MLS 402-91 cells. Lamin A was used as a control for the nuclear fraction and GAPDH was used as control for the cytoplasmic fraction.
Figure Legend Snippet: Nuclear FLT1 localization in MLS tumors and cultured MLS cells . (a) Immunohistochemical analysis of FLT1 expression in tissue sections of a representative MLS tumor and in an MLS 402-91 SCID mouse xenograft. Brown staining indicates FLT1 expression while blue staining shows negatively staining nuclei. Cytoplasmic FLT1 expression in angiosarcoma was used as a positive control. (b) FLT1 expression in cultured MLS 402-91 cells analyzed by immunofluorescence. The merge image shows nuclear DAPI staining in blue. Scale bar indicates 10 μm. (c) Western blot analysis of nuclear (N) and cytoplasmic (CP) fractions of MLS 402-91 cells. Lamin A was used as a control for the nuclear fraction and GAPDH was used as control for the cytoplasmic fraction.

Techniques Used: Cell Culture, Immunohistochemistry, Expressing, Staining, Positive Control, Immunofluorescence, Western Blot

Increased FLT1 transcription in FUS-DDIT3 expressing cell lines . Bars show mean relative FLT1 expression by quantitative real-time PCR analysis of three independent biological replicates compared to wild type HT1080 with FLT1 expression set to 1. The geometric mean of ACTB and GAPDH expression was used to normalize FLT1 expression between samples. Error bars show standard error of the mean. Asterisks indicate statistical significance with p
Figure Legend Snippet: Increased FLT1 transcription in FUS-DDIT3 expressing cell lines . Bars show mean relative FLT1 expression by quantitative real-time PCR analysis of three independent biological replicates compared to wild type HT1080 with FLT1 expression set to 1. The geometric mean of ACTB and GAPDH expression was used to normalize FLT1 expression between samples. Error bars show standard error of the mean. Asterisks indicate statistical significance with p

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

33) Product Images from "p53-independent role of MYC mutant T58A in the proliferation and apoptosis of breast cancer cells"

Article Title: p53-independent role of MYC mutant T58A in the proliferation and apoptosis of breast cancer cells

Journal: Oncology Letters

doi: 10.3892/ol.2018.9688

Mutant MYC does not induce p14 or Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p14 siRNA construct at the same time. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p14. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p14. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Mutant MYC does not induce p14 or Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p14 siRNA construct at the same time. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p14. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p14. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Mutagenesis, Stable Transfection, Infection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

Expression levels of Bim in HCC1937 cells transfected with T58A and WT MYC. HCC1937 cells were stably infected with control, WT MYC or T58A MYC vectors. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of MYC and Bim. (C) Western blot analysis. (D) Protein expression levels of MYC and Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Expression levels of Bim in HCC1937 cells transfected with T58A and WT MYC. HCC1937 cells were stably infected with control, WT MYC or T58A MYC vectors. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of MYC and Bim. (C) Western blot analysis. (D) Protein expression levels of MYC and Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Expressing, Transfection, Stable Transfection, Infection, Real-time Polymerase Chain Reaction, Western Blot, Standard Deviation

Mutant MYC does not suppress p21 or induce Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p21 siRNA construct simultaneously. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p21. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p21. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P
Figure Legend Snippet: Mutant MYC does not suppress p21 or induce Bim. HCC1937 cells were stably infected with control, p21siRNA, WT MYC, WT and p21siRNA construct, T58A MYC vectors, and with T58A and p21 siRNA construct simultaneously. (A) Reverse transcription-semi-quantitative polymerase chain reaction analysis. (B) mRNA expression levels of p21. (C) mRNA expression levels of Bim. (D) Western blot analysis. (E) Protein expression levels of p21. (F) Protein expression levels of Bim. GAPDH and β-actin were used as controls. Each value represents the mean ± standard deviation of three observations. **P

Techniques Used: Mutagenesis, Stable Transfection, Infection, Construct, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Standard Deviation

34) Product Images from "Frontotemporal dementia mutant tau (P301L) locks Fyn in an open, active conformation conducive to nanoclustering"

Article Title: Frontotemporal dementia mutant tau (P301L) locks Fyn in an open, active conformation conducive to nanoclustering

Journal: bioRxiv

doi: 10.1101/2020.09.04.282855

Overexpression of tau (WT, P301L) decreases the mobility and increases activity of Fyn. A . Intensity map of Fyn-mEos2 co-expressed with GFP, tau-WT-GFP and tau-P301L-GFP in the spines of mouse hippocampal neurons (DIV19-22). Scale bar = 1 µm. B . Frequency distribution (%) of Log 10 [D] of Fyn-mEos2 with immobile fraction (%). C . Mean square displacement (MSD) of Fyn-mEos2 over time (0.14 sec) and area under curve (AUC) [(µm 2 s) × 100]. D . Increased phosphorylation at the Y16 epitope in the presence of tau P301L. Western blot of Fyn in protein extracts derived from HEK293 cells co-transfected with Fyn-WT-mEos2 and GFP, tau-WT-GFP or tau-P301L-GFP. Fyn-Y420F-mEos2 was also co-transfected with GFP as a control. Activity of Fyn determined by quantifying proportion of active Fyn (anti-Y416) relative to total Fyn, normalised to GAPDH. Co-expression of tau-P301L increased the proportion of active Fyn. Error bars are standard errors of the mean (SEM). Mean ± SEM values were obtained from neurons transfected with Fyn-mEos2+GFP (n = 12), Fyn-mEos2+tau-WT-GFP (n=4) or Fyn-mEos2+tau-P301L-GFP (n=15), and HEK293 protein lysates (n=3 for each group). Statistical comparisons between tau-transfections (A-C) were performed using a one-way ANOVA and Tukey’s test for multiple comparisons. A Student’s t test was performed for statistical comparisons of Y16 phosphorylation (D).
Figure Legend Snippet: Overexpression of tau (WT, P301L) decreases the mobility and increases activity of Fyn. A . Intensity map of Fyn-mEos2 co-expressed with GFP, tau-WT-GFP and tau-P301L-GFP in the spines of mouse hippocampal neurons (DIV19-22). Scale bar = 1 µm. B . Frequency distribution (%) of Log 10 [D] of Fyn-mEos2 with immobile fraction (%). C . Mean square displacement (MSD) of Fyn-mEos2 over time (0.14 sec) and area under curve (AUC) [(µm 2 s) × 100]. D . Increased phosphorylation at the Y16 epitope in the presence of tau P301L. Western blot of Fyn in protein extracts derived from HEK293 cells co-transfected with Fyn-WT-mEos2 and GFP, tau-WT-GFP or tau-P301L-GFP. Fyn-Y420F-mEos2 was also co-transfected with GFP as a control. Activity of Fyn determined by quantifying proportion of active Fyn (anti-Y416) relative to total Fyn, normalised to GAPDH. Co-expression of tau-P301L increased the proportion of active Fyn. Error bars are standard errors of the mean (SEM). Mean ± SEM values were obtained from neurons transfected with Fyn-mEos2+GFP (n = 12), Fyn-mEos2+tau-WT-GFP (n=4) or Fyn-mEos2+tau-P301L-GFP (n=15), and HEK293 protein lysates (n=3 for each group). Statistical comparisons between tau-transfections (A-C) were performed using a one-way ANOVA and Tukey’s test for multiple comparisons. A Student’s t test was performed for statistical comparisons of Y16 phosphorylation (D).

Techniques Used: Over Expression, Activity Assay, Western Blot, Derivative Assay, Transfection, Expressing

35) Product Images from "Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways"

Article Title: Antitumor effect of Quercetin on Y79 retinoblastoma cells via activation of JNK and p38 MAPK pathways

Journal: BMC Complementary and Alternative Medicine

doi: 10.1186/s12906-017-2023-6

Effect of Quercetin induced apoptosis in RB Y79 cells mediated by JNK and p38 MAPK. a and b The RB Y79 cells were exposed in the presence or absence of JNK inhibitor or MAPK inhibitor for 1 h followed by treatment with 100 μM Quercetin for time period 24 h. Western blot analysis was done for levels of caspase-9 and -3, GAPDH was loading control. ** P
Figure Legend Snippet: Effect of Quercetin induced apoptosis in RB Y79 cells mediated by JNK and p38 MAPK. a and b The RB Y79 cells were exposed in the presence or absence of JNK inhibitor or MAPK inhibitor for 1 h followed by treatment with 100 μM Quercetin for time period 24 h. Western blot analysis was done for levels of caspase-9 and -3, GAPDH was loading control. ** P

Techniques Used: Western Blot

Quercetin causes apoptosis of cancerous RB Y79 cells via intrinsic pathways. a and b The Y79 cells were exposed to Quercetin (0-100 μM). The obtained cell lysates after 24 h were analyzed by western blot using specific antibodies against caspase-9, caspase-3 and cytochrome c , GAPDH was used as a loading control. * P
Figure Legend Snippet: Quercetin causes apoptosis of cancerous RB Y79 cells via intrinsic pathways. a and b The Y79 cells were exposed to Quercetin (0-100 μM). The obtained cell lysates after 24 h were analyzed by western blot using specific antibodies against caspase-9, caspase-3 and cytochrome c , GAPDH was used as a loading control. * P

Techniques Used: Western Blot

36) Product Images from "Baicalin Suppresses Bilirubin-Induced Apoptosis and Inflammation by Regulating p38 Mitogen-Activated Protein Kinases (MAPK) Signaling in Neonatal Neurons"

Article Title: Baicalin Suppresses Bilirubin-Induced Apoptosis and Inflammation by Regulating p38 Mitogen-Activated Protein Kinases (MAPK) Signaling in Neonatal Neurons

Journal: Medical Science Monitor : International Medical Journal of Experimental and Clinical Research

doi: 10.12659/MSM.926441

( A ) Immunofluorescent staining was used to evaluate the nuclear translocation of p65. p65 was tagged with green fluorescence (Alexa488) and nuclei were tagged with blue fluorescence (DAPI). Columns indicate the relative nuclear translocation rate of p65 in neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. ( B ) Western blotting was used to determine the relative expression levels of proteins. Immunoblots of cleaved caspase3 (c-caspase3), TNF-α, and GAPDH (internal reference) of neurons. Columns indicate the relative expression levels of c-caspase3 and TNF-α in neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. ( C ) ELISA was used to detect concentration of cytokines. Columns indicate the concentrations of TNF-α in cell medium of neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. [n=10; a difference was significant when compared with neurons incubated with control; b difference was significant when compared with neurons incubated with Bf at 70 nmol/L; c difference was significant when compared with neurons incubated with Bf at 140 nmol/L].
Figure Legend Snippet: ( A ) Immunofluorescent staining was used to evaluate the nuclear translocation of p65. p65 was tagged with green fluorescence (Alexa488) and nuclei were tagged with blue fluorescence (DAPI). Columns indicate the relative nuclear translocation rate of p65 in neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. ( B ) Western blotting was used to determine the relative expression levels of proteins. Immunoblots of cleaved caspase3 (c-caspase3), TNF-α, and GAPDH (internal reference) of neurons. Columns indicate the relative expression levels of c-caspase3 and TNF-α in neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. ( C ) ELISA was used to detect concentration of cytokines. Columns indicate the concentrations of TNF-α in cell medium of neurons incubated with Bf at concentrations of 0, 70, 140, and 280 nmol/L. [n=10; a difference was significant when compared with neurons incubated with control; b difference was significant when compared with neurons incubated with Bf at 70 nmol/L; c difference was significant when compared with neurons incubated with Bf at 140 nmol/L].

Techniques Used: Staining, Translocation Assay, Fluorescence, Incubation, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Concentration Assay

( A ) p65 was tagged with green fluorescence (Alexa488) and nuclei were tagged with blue fluorescence (DAPI). Columns indicated the nuclear translocation rate of p65 in Bf-incubated neurons treated with BAI at concentrations of 0, 0.5, 1.0, and 2.0 μmol/L. ( B ) The immunoblots of p-MKK3, MKK3, p-MKK6, MKK6, p-p38, and p38 of neurons were demonstrated. Columns indicate the relative phosphorylation levels of MKK3, MKK6, and p38. ( C ) Immunoblots of c-caspase3, TNF-α, and GAPDH of neurons. Columns indicate the relative expression levels of c-caspase3 and TNF-α in neurons. ( D ) Columns indicate the concentrations of TNF-α in cell medium of neurons. [n=10; a difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 0 μmol/L; b difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 0.5 μmol/L; c difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 1.0 μmol/L].
Figure Legend Snippet: ( A ) p65 was tagged with green fluorescence (Alexa488) and nuclei were tagged with blue fluorescence (DAPI). Columns indicated the nuclear translocation rate of p65 in Bf-incubated neurons treated with BAI at concentrations of 0, 0.5, 1.0, and 2.0 μmol/L. ( B ) The immunoblots of p-MKK3, MKK3, p-MKK6, MKK6, p-p38, and p38 of neurons were demonstrated. Columns indicate the relative phosphorylation levels of MKK3, MKK6, and p38. ( C ) Immunoblots of c-caspase3, TNF-α, and GAPDH of neurons. Columns indicate the relative expression levels of c-caspase3 and TNF-α in neurons. ( D ) Columns indicate the concentrations of TNF-α in cell medium of neurons. [n=10; a difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 0 μmol/L; b difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 0.5 μmol/L; c difference was significant when compared with neurons incubated with Bf at 280 nmol/L and BAI at 1.0 μmol/L].

Techniques Used: Fluorescence, Translocation Assay, Incubation, Western Blot, Expressing

37) Product Images from "Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells"

Article Title: Influence of the cystic fibrosis transmembrane conductance regulator on expression of lipid metabolism-related genes in dendritic cells

Journal: Respiratory Research

doi: 10.1186/1465-9921-10-26

Confirmation of microarray results by real-time RT-PCR . DC from WT and CF mice were infected in vitro with P. aeruginosa for 4 h. RNA levels for three genes were measured by quantitative real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. A , C and E . The y -axis represents the relative gene expression level for Cav1, Dhcr7 and Scd2 in the uninfected control DC (gray) and P. aeruginosa infected DC (black). B, D, and F . The y -axis represents fold change of Cav1, Dhcr7 and Scd2 expression upon P. aeruginosa infection compared to the control in both groups. Shown are the means ± SEM of three pairs of DC samples from WT and CF mice with or without P. aeruginosa infection. *denotes p
Figure Legend Snippet: Confirmation of microarray results by real-time RT-PCR . DC from WT and CF mice were infected in vitro with P. aeruginosa for 4 h. RNA levels for three genes were measured by quantitative real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. A , C and E . The y -axis represents the relative gene expression level for Cav1, Dhcr7 and Scd2 in the uninfected control DC (gray) and P. aeruginosa infected DC (black). B, D, and F . The y -axis represents fold change of Cav1, Dhcr7 and Scd2 expression upon P. aeruginosa infection compared to the control in both groups. Shown are the means ± SEM of three pairs of DC samples from WT and CF mice with or without P. aeruginosa infection. *denotes p

Techniques Used: Microarray, Quantitative RT-PCR, Mouse Assay, Infection, In Vitro, Expressing

Cav1 and SREBP expression in DC from WT and CF mice . A . RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y -axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B . Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C . Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D . Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p
Figure Legend Snippet: Cav1 and SREBP expression in DC from WT and CF mice . A . RNA was extracted from DC from WT and CF mice and Cav1 gene expression was measured by Real-time RT-PCR. Relative expression levels in the samples were calculated using the ΔΔCt method, with GAPDH as internal normalization control. The y -axis represents Cav1 cDNA transcription level in terms of relative quantity value (RQ). B . Western analysis of Cav1 and SREBP in DC from WT and CF mice and corresponding GAPDH expression. C . Quantification of Cav1 and SREBP expression by image intensity analysis normalized to GAPDH. D . Luciferase assay of SRE transcriptional activity in CF and WT DC. DC were infected with AdZ-SRE-luc for 48 h and harvested for luci-ferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. *denotes p

Techniques Used: Expressing, Mouse Assay, Quantitative RT-PCR, Western Blot, Luciferase, Activity Assay, Infection

Cav1 and SREBP expression in DC from WT and CF mice infected with P. aeruginosa . DC from WT and CF mice were infected in vitro with P. aeruginosa for 4 h, and uninfected cells served as the control (Co). A. Western analysis of Cav1 and corresponding GAPDH. B. Western analysis of SREBP and corresponding GAPDH. C. Luciferase assay of SRE transcriptional activity of CF and WT DC infected with P. aeruginosa . DC were infected with AdZ-SRE-luc for 48 h, and then infected with P. aeruginosa for 4 h. DC were harvested for luciferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. **denotes p
Figure Legend Snippet: Cav1 and SREBP expression in DC from WT and CF mice infected with P. aeruginosa . DC from WT and CF mice were infected in vitro with P. aeruginosa for 4 h, and uninfected cells served as the control (Co). A. Western analysis of Cav1 and corresponding GAPDH. B. Western analysis of SREBP and corresponding GAPDH. C. Luciferase assay of SRE transcriptional activity of CF and WT DC infected with P. aeruginosa . DC were infected with AdZ-SRE-luc for 48 h, and then infected with P. aeruginosa for 4 h. DC were harvested for luciferase assay and β-galactosidase assay. Data is shown luciferase activity (RLU) normalized to β-galactosidase. Shown is the mean ± SEM of three of independent samples. **denotes p

Techniques Used: Expressing, Mouse Assay, Infection, In Vitro, Western Blot, Luciferase, Activity Assay

38) Product Images from "RBM5 inhibits tumorigenesis of gliomas through inhibition of Wnt/β-catenin signaling and induction of apoptosis"

Article Title: RBM5 inhibits tumorigenesis of gliomas through inhibition of Wnt/β-catenin signaling and induction of apoptosis

Journal: World Journal of Surgical Oncology

doi: 10.1186/s12957-016-1084-1

RBM5 inactivated Wnt/β-catenin signaling and induced apoptosis in SHG44 cells. Proteins were extracted from SHG44 cells with or without RBM5 overexpression. a Expression of β-catenin, P-GSK-3β, Cyclin D1, DKK1, Bax, Bcl-2, cleaved caspase3, and TNF-α were determined by western blot. GAPDH was used internal control. b FACS analysis of the apoptotic rate of SHG44 cells without RBM5 overexpression. c FACS analysis of the apoptotic rate of SHG44 cells with RBM5 overexpression. All experiments were repeated in triplicates. * P
Figure Legend Snippet: RBM5 inactivated Wnt/β-catenin signaling and induced apoptosis in SHG44 cells. Proteins were extracted from SHG44 cells with or without RBM5 overexpression. a Expression of β-catenin, P-GSK-3β, Cyclin D1, DKK1, Bax, Bcl-2, cleaved caspase3, and TNF-α were determined by western blot. GAPDH was used internal control. b FACS analysis of the apoptotic rate of SHG44 cells without RBM5 overexpression. c FACS analysis of the apoptotic rate of SHG44 cells with RBM5 overexpression. All experiments were repeated in triplicates. * P

Techniques Used: Over Expression, Expressing, Western Blot, FACS

39) Product Images from "Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury"

Article Title: Acute administration of catalase targeted to ICAM-1 attenuates neuropathology in experimental traumatic brain injury

Journal: Scientific Reports

doi: 10.1038/s41598-017-03309-4

CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).
Figure Legend Snippet: CCI-TBI increases BBB permeability to fibrinogen and decreases tight junction protein detection with a rescue or preservation of barrier function with anti-ICAM-1/catalase. Immunohistochemical detection of plasma protein fibrinogen in brain parenchyma at 48 hr following moderate CCI-TBI. ( A,B ) Naive and sham controls show absent fibrinogen detection in the brain parenchyma, as the BBB is healthy and intact. Absence of staining maintained at 20X. ( C ) Following CCI-TBI, BBB hyperpermeability permits the extravasation of fibrinogen into the brain tissue. Black arrowheads indicate areas of dense fibrinogen staining in the perivascular space. ( D ) Anti-ICAM-1/catalase reduces parenchymal and perivascular fibrinogen detection in the brain by 48 hrs post-CCI-TBI. ( E , F ) Anti-ICAM-1 antibody and catalase alone do not appear to reduce fibrinogen extravasation as indicated by intense fibrinogen detection in the impact site with dense staining in perivascular space. Scale bar equals 50 microns. (Top panels 2X, bottom panels 20X). ( G ) Western blot analysis of tight junction protein expression for occludin and claudin-5 in the cortex ipsilateral to the site of CCI-TBI for sham, CCI-TBI and CCI-TBI+anti-ICAM-1/catalase groups. The whole membrane was cut at 75 kDa and between 37 and 25 kDa to minimize antibody use. Membranes were separately probed for occludin and claudin-5. The membrane probed for occludin was striped, reblocked, and then probed for GAPDH. Full length blots are presented in Supplementary Figure 1 . ( H , I ) Densitometry quantification for occludin and claudin-5 normalized to GAPDH presented as mean ± SD (Ordinary one-way ANOVA with multiple comparisons occludin F = 5.779, P = 0.0399. Claudin-5 F = 17.42, P = 0.0032).

Techniques Used: Permeability, Preserving, Immunohistochemistry, Staining, Western Blot, Expressing

40) Product Images from "β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells"

Article Title: β-Catenin/CBP–Dependent Signaling Regulates TGF-β–Induced Epithelial to Mesenchymal Transition of Lens Epithelial Cells

Journal: Investigative Ophthalmology & Visual Science

doi: 10.1167/iovs.16-20162

Effect on E-cadherin by inhibition of β-catenin/TCF interaction. ( A ) E-cadherin stained untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs imaged using Zeiss LSM 510 confocal microscope. Scale bar : 10 μm. ( B ) Total protein from untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs was subjected to Western blot analysis for E-cadherin and GAPDH ( n = 3). ( C ) Graph showing average relative density ± SD of E-cadherin normalized to untreated LECs ( n = 3, * P
Figure Legend Snippet: Effect on E-cadherin by inhibition of β-catenin/TCF interaction. ( A ) E-cadherin stained untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs imaged using Zeiss LSM 510 confocal microscope. Scale bar : 10 μm. ( B ) Total protein from untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs was subjected to Western blot analysis for E-cadherin and GAPDH ( n = 3). ( C ) Graph showing average relative density ± SD of E-cadherin normalized to untreated LECs ( n = 3, * P

Techniques Used: Inhibition, Staining, Microscopy, Western Blot

Effect of inhibition of β-catenin/CBP–dependent signaling by ICG-001 on TGF-β–induced EMT of lens explants. ( A ) Untreated, ICG-only, TGF-β2–only, and TGF-β2– and ICG-001-treated lens explants were immunostained for fascin and α-SMA. Slides were imaged using ×40 lens of Zeiss Apotome microscope and analyzed using Zeiss Zen software. Scale bar : 100 μm. ( B ) Western blot analysis for fascin, α-SMA, and GAPDH was carried out using protein lysates extracted from untreated, ICG-treated, TGF-β2–treated, and TGF-β2– and ICG-001-treated lens explants. ( C ) Densitometric quantification of α-SMA indicating fold reduction in α-SMA expression normalized to untreated lens explants ( n = 3, ** P
Figure Legend Snippet: Effect of inhibition of β-catenin/CBP–dependent signaling by ICG-001 on TGF-β–induced EMT of lens explants. ( A ) Untreated, ICG-only, TGF-β2–only, and TGF-β2– and ICG-001-treated lens explants were immunostained for fascin and α-SMA. Slides were imaged using ×40 lens of Zeiss Apotome microscope and analyzed using Zeiss Zen software. Scale bar : 100 μm. ( B ) Western blot analysis for fascin, α-SMA, and GAPDH was carried out using protein lysates extracted from untreated, ICG-treated, TGF-β2–treated, and TGF-β2– and ICG-001-treated lens explants. ( C ) Densitometric quantification of α-SMA indicating fold reduction in α-SMA expression normalized to untreated lens explants ( n = 3, ** P

Techniques Used: Inhibition, Microscopy, Software, Western Blot, Expressing

Inhibition of β-catenin/CBP interaction by ICG-001 prevents MMP9 expression. ( A ) Untreated, ICG-001–treated, TGF-β2–treated, and TGF-β2– and ICG-001–cotreated lens explants were subjected to Western blot analysis for MMP9 and GAPDH ( n = 3). ( B ) Graph representing the relative density ± SD of MMP9 normalized to untreated LECs ( n = 3, * P
Figure Legend Snippet: Inhibition of β-catenin/CBP interaction by ICG-001 prevents MMP9 expression. ( A ) Untreated, ICG-001–treated, TGF-β2–treated, and TGF-β2– and ICG-001–cotreated lens explants were subjected to Western blot analysis for MMP9 and GAPDH ( n = 3). ( B ) Graph representing the relative density ± SD of MMP9 normalized to untreated LECs ( n = 3, * P

Techniques Used: Inhibition, Expressing, Western Blot

Effect of inhibition of β-catenin/TCF–dependent signaling on fascin. ( A ) Untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs were stained for fascin and imaged using ×40 lens of Leica DMRA2 fluorescent microscope ( n = 3). Scale bar : 100 μm. ( B ) Western blot analysis for fascin and GAPDH was carried out using protein lysates from untreated, TGF-β2–treated, PNU-treated, and TGF-β2– and PNU-cotreated LECs ( n = 3). ( C ) Graph representing the relative density ± SD of fascin normalized to untreated LECs ( n = 3, * P
Figure Legend Snippet: Effect of inhibition of β-catenin/TCF–dependent signaling on fascin. ( A ) Untreated, TGF-β2–treated, and TGF-β2– and PNU-cotreated LECs were stained for fascin and imaged using ×40 lens of Leica DMRA2 fluorescent microscope ( n = 3). Scale bar : 100 μm. ( B ) Western blot analysis for fascin and GAPDH was carried out using protein lysates from untreated, TGF-β2–treated, PNU-treated, and TGF-β2– and PNU-cotreated LECs ( n = 3). ( C ) Graph representing the relative density ± SD of fascin normalized to untreated LECs ( n = 3, * P

Techniques Used: Inhibition, Staining, Microscopy, Western Blot

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In Vivo:

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other:

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Incubation:

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Immunofluorescence:

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    Abcam genes rabbit gapdh
    Diabetes alters key signaling molecules involved in skeletal muscle mass regulation. (a) Representative immunoblots and quantifications of phosphorylated Akt, S6, and 4E-BP1 protein levels in muscles of control and GK diabetic rats. (b) Total phosphorylated levels of FoxO1 and its nuclear localization relative to <t>GAPDH</t> and histone, respectively, were analyzed by Western blot. (c) Expression of MuRF1 and Atrogin 1 mRNAs relative to GAPDH was examined by real-time PCR. (d) Expression of miRNA 486 relative to U6 was determined using real-time PCR (e) PTEN protein level (e) and activity (f) were measured as described in Materials and Methods. (f) Key regulators of myogenesis including IGF-1 and <t>PAX7</t> (g) were assessed in muscle using real-time RT-PCR-based technique. Abbreviation: C: control; D: diabetic. Values are means ± SEM for at least 6 animals/group. ∗ Significantly different from corresponding control values at P ≤ 0.05.
    Genes Rabbit Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 93/100, based on 3 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Abcam gapdh
    Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. <t>GAPDH</t> was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). <t>CTTN</t> ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm
    Gapdh, supplied by Abcam, used in various techniques. Bioz Stars score: 99/100, based on 4311 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Diabetes alters key signaling molecules involved in skeletal muscle mass regulation. (a) Representative immunoblots and quantifications of phosphorylated Akt, S6, and 4E-BP1 protein levels in muscles of control and GK diabetic rats. (b) Total phosphorylated levels of FoxO1 and its nuclear localization relative to GAPDH and histone, respectively, were analyzed by Western blot. (c) Expression of MuRF1 and Atrogin 1 mRNAs relative to GAPDH was examined by real-time PCR. (d) Expression of miRNA 486 relative to U6 was determined using real-time PCR (e) PTEN protein level (e) and activity (f) were measured as described in Materials and Methods. (f) Key regulators of myogenesis including IGF-1 and PAX7 (g) were assessed in muscle using real-time RT-PCR-based technique. Abbreviation: C: control; D: diabetic. Values are means ± SEM for at least 6 animals/group. ∗ Significantly different from corresponding control values at P ≤ 0.05.

    Journal: Oxidative Medicine and Cellular Longevity

    Article Title: Hydrogen Sulfide Donor NaHS Improves Metabolism and Reduces Muscle Atrophy in Type 2 Diabetes: Implication for Understanding Sarcopenic Pathophysiology

    doi: 10.1155/2018/6825452

    Figure Lengend Snippet: Diabetes alters key signaling molecules involved in skeletal muscle mass regulation. (a) Representative immunoblots and quantifications of phosphorylated Akt, S6, and 4E-BP1 protein levels in muscles of control and GK diabetic rats. (b) Total phosphorylated levels of FoxO1 and its nuclear localization relative to GAPDH and histone, respectively, were analyzed by Western blot. (c) Expression of MuRF1 and Atrogin 1 mRNAs relative to GAPDH was examined by real-time PCR. (d) Expression of miRNA 486 relative to U6 was determined using real-time PCR (e) PTEN protein level (e) and activity (f) were measured as described in Materials and Methods. (f) Key regulators of myogenesis including IGF-1 and PAX7 (g) were assessed in muscle using real-time RT-PCR-based technique. Abbreviation: C: control; D: diabetic. Values are means ± SEM for at least 6 animals/group. ∗ Significantly different from corresponding control values at P ≤ 0.05.

    Article Snippet: Antibodies Primary antibodies used in the current study included the following: rabbit anti-PTEN, 9552; rabbit ant-phospho S6 (Ser235/236), 4858; S6, 2217, rabbit anti-phospho-4E-BP1 (Thr37/46), 2855; rabbit anti-phospho-Akt (Ser473), 4060; rabbit anti-phospho-Akt (Thr308), 4056; rabbit anti-Akt, 9272; rabbit anti-FoxO1, 2880; rabbit anti-phospho-FoxO1 (Ser256), 8419; rabbit anti-Smad2, 5339; rabbit anti-phospho-Smad2 (Ser465/467), 18338 (Cell Signaling Technology, Bever, USA); rabbit anti-NOX2, NB2-41291 and -NOX4, NB110-58849 (Novus Biologicals, USA); rabbit ant-CSE (MyBioSource, USA MBS 769567); rabbit anti-PAX7, ab92317; and the housekeeping genes rabbit GAPDH, ab181602, and histone H3, ab201456 (Abcam, USA).

    Techniques: Western Blot, Expressing, Real-time Polymerase Chain Reaction, Activity Assay, Quantitative RT-PCR

    Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm

    Journal: Nature Communications

    Article Title: Cytoplasmic LIF reprograms invasive mode to enhance NPC dissemination through modulating YAP1-FAK/PXN signaling

    doi: 10.1038/s41467-018-07660-6

    Figure Lengend Snippet: Cytoplasmic LIF regulates EMT and invadopodia formation. a Migration pattern analysis using time-lapse live imaging in wound-healing experiments. Scale bars, 100 μm. See also Supplementary movies 1–3. b 3D-gelatin invasion assays. The invasion depth of cancer cells through a stiff gelatin matrix (6%) were measured by time-lapse phase contrast vertical scanning (Olympus IX83). Values are presented as means ± SD of triplicate experiments. c Representative IHC images of LIF expression in paraffin-embedded mouse tumor xenografts. Scale bars, 50 μm. d Quantification of mouse tumor xenografts with events of local invasion based on hematoxylin eosin (HE) staining analysis. e Western blot of EMT and invadopodia markers in wild-type parental, cLIF and LIF +/− cancer cells. GAPDH was used as a loading control. f – h Immunofluorescence staining of invadopodia markers: TKS5 ( f ). CTTN ( g ). MMP2 ( h ). Alexa Fluor 488 phalloidin (green) was used to stain F-actin. Blue, nuclear staining. Scale bars, 10 μm

    Article Snippet: Blots were probed with specific primary antibodies against LIF (Abcam, ab135629, 1:500), LIFR (Santa Cruz Biotechnology, sc-515337, 1:500), phospho-YAP1 (Cell Signaling, 13008, 1:2000), YAP1 (Cell Signaling, 14074, 1:2000), phospho-FAK (Invitrogen, 700255, 1:5000), FAK (Santa Cruz Biotechnology, sc-558, 1:500), PECAM-1 (CD31) (Santa Cruz Biotechnology, sc-376764, 1:500), VE-cad (Santa Cruz Biotechnology, sc-9989, 1:1000), E-cad (BD Transduction Laboratories, 610181, 1:5000), phospho-PXN (Y118) (Abcam, ab109547, 1:4000), PXN (BD Transduction Laboratories, 610052, 1:10,000), phospho-p70S6K1 (T389) (Cell Signaling, 9234, 1:2000), p70S6K1 (Abcam, ab32529, 1:10,000), N-cad (Abcam, ab76011, 1:10,000), VIM (Sigma-Aldrich, V5255, 1:500), IQGAP1 (Santa Cruz Biotechnology, sc-81906, 1:1000), phospho-SRC (Cell Signaling, 6942, 1:2000), SRC (Cell Signaling, 2109, 1:2000), TKS5 (Proteintech, 18976-1-AP, 1:1500), CTTN (Abcam, ab81208, 1:10,000), MMP2 (Proteintech, 10373-2-AP, 1:800), and GAPDH (Abcam, ab8245, 1:5000) by incubation with horseradish peroxidase-conjugated secondary antibody and developed with enhanced chemiluminescence detection reagent (GE Healthcare).

    Techniques: Migration, Imaging, Immunohistochemistry, Expressing, Staining, Western Blot, Immunofluorescence

    Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Journal: International Journal of Molecular Medicine

    Article Title: Gastrodin protects MC3T3-E1 osteoblasts from dexamethasone-induced cellular dysfunction and promotes bone formation via induction of the NRF2 signaling pathway

    doi: 10.3892/ijmm.2018.3414

    Figure Lengend Snippet: Gastrodin (GSTD) increased osteogenic transcription Runx2, decreased adipogenic factor peroxisome proliferator-activated receptor (PPAR)γ isoform 2 and activated nuclear factor-like 2 (NRF2) pathway protein expression levels. Representative western blot analyses for total protein was analyzed by western blotting in dexamethasone (DEX)-treated MC3T3-E1 cells with or without GSTD pretreatment at different doses. The relative protein expression levels were measured using the fold-change in each protein relative to β-actin from the same sample. The data are expressed as the mean ± standard deviation. ## P

    Article Snippet: Rabbit anti-HO-1 (cat. no. ab68477; 1:1,000), rabbit anti-NQO-1 (cat. no. ab76956; 1:1,000), and mouse anti-β-actin (cat. no. ab8245; 1:1,000), Anti-OCN (cat. no. ab93876; 1:1,000) monoclonal antibodies were purchased from Abcam (Cambridge, MA, USA).

    Techniques: Expressing, Western Blot, Standard Deviation

    QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Journal: OncoTargets and therapy

    Article Title: circSMAD2 inhibits the epithelial–mesenchymal transition by targeting miR-629 in hepatocellular carcinoma

    doi: 10.2147/OTT.S158008

    Figure Lengend Snippet: QRT-PCR and Western blot assays showed no significant changes in the mRNA ( A ) or protein levels ( B ) of SMAD2 in overexpressed or knocked down circSMAD2 HepG2 cells. Note: Data are presented as mean ± SEM. Abbreviations: QRT-PCR, quantitative real-time polymerase chain reaction; circSMAD2, circRNA SMAD2; SEM, standard error of the mean; NC, negative control; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.

    Article Snippet: After blocking with 5% non-fat milk, the membranes were incubated with primary antibodies: anti-SMAD2 (Abcam, Cambridge, MA, USA), anti-GAPDH (Abcam), anti-E-cadherin (Cell Signaling Technology, Danvers, MA, USA), anti-N-cadherin (Cell Signaling Technology), anti-Snail (Cell Signaling Technology), and anti-Vimentin (Cell Signaling Technology).

    Techniques: Quantitative RT-PCR, Western Blot, Real-time Polymerase Chain Reaction, Negative Control