Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 4. HSP27 regulates IL-1-induced p38 MAPK and JNK activity but not ERK activation. Cells were transfected as indicated and treated with IL-1 for different times. Cells were harvested and lysed. A, p38 MAPK was immunoprecipitated from lysates and assayed for its ability to phosphorylate His-tagged MK2. Graph shows mean p38 MAPK activity from three independent experiments (S.E.) as a percentage of peak activity in Scr-transfected cells. A portion of the lysates was Western-blotted for HSP27 and p38 MAPK. B, JNK was immu- noprecipitated from lysates, and JNK activity was assayed with GST-ATF-2 as a substrate. Graph shows JNK activity (as for A). An aliquot of lysate was analyzed by Western blotting for HSP27 and -tubulin. C, activation of ERK in lysates was detected by Western blotting with an antibody that recognizes ERK phos- phorylated on Thr-185 and Tyr-187. The blot was stripped and re-probed for total ERK. D, cells were transfected with Scr, HSP27#2, or HSP27#5 twice (with a 24-h interval between transfections) and harvested and lysed 24 h after the second transfection. Phosphorylation of p38 MAPK on Thr-180 and Tyr-182, JNK1 on Thr-183 and Tyr-185, JNK2 on Thr-221 and Tyr-223, and ERK (as in C) in lysates was examined by Western blotting with phosphospecific antibodies. p38 MAPK was blotted as a loading control. Data in each panel (A–D) are repre- sentative of at least two independent experiments.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activity Assay, Activation Assay, Transfection, Immunoprecipitation, Western Blot, Phospho-proteomics, Control

Journal: Journal of Biological Chemistry

Article Title: Heat Shock Protein 27 Functions in Inflammatory Gene Expression and Transforming Growth Factor-β-activated Kinase-1 (TAK1)-mediated Signaling

doi: 10.1074/jbc.m610987200

Figure Lengend Snippet: FIGURE 5. HSP27 is needed for activation by IL-1 of the p38 MAPK activators (MKK3 and MKK6) and the JNK activators (MKK4 and MKK7). Cells were transfected and treated as before. A, activation of MKK3 and MKK6 was detected with an antibody that recognizes phosphorylation of Ser-189 and Thr-193 of MKK3 and Ser-207 and Thr-211 of MKK6. The blot was stripped and re-probed with an antibody that recognizes total MKK3 and MKK6. B, kinase assay for MKK4 using a GST-JNK2 (K55R) (a mutant form that is incapable of auto- phosphorylation) and Western blots for HSP27 and -tubulin. C, kinase assay for MKK7 (as for B). Similar results were obtained in two separate experiments for each panel.

Article Snippet: The membranes were stained with rabbit anti-COX-2 antibody (Alexis), a mouse anti- -tubulin antibody DM-1A (Sigma-Aldrich), a mouse monoclonal antibody against actin (Sigma), a rabbit antiserum raised against MKK3/6 (32), a rabbit antiserum to human HSP27 (7), a rabbit antiserum that recognizes both human HSP27 and murine HSP25 (Stressgen), a rabbit antiserum to the C-terminal peptide of p38 MAPK (33), a rabbit antiserum against TAK1 (sc-7162; Santa Cruz Biotechnology), and a rabbit antiserum against I B (sc-203; Santa Cruz Biotechnology).

Techniques: Activation Assay, Transfection, Phospho-proteomics, Kinase Assay, Mutagenesis, Western Blot

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO, bars 1-4) or 660 nM TSA (TSA, bars 5-8) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of seven independent experiments. Asterisks denote significant differences (***p<0.001). NS, not significant. ## p<0.01 bar 5 vs 1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with increasing TSA doses (0, 6.6, 66, 660, 6600 nM) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of three independent experiments. Asterisks denote significant differences (p<0.05). NS, not significant. # p<0.05 vs 0 nM control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were transfected with non-targeting luciferase siRNA (Luc), or siRNA against SMRT, NCoR, or PPARγ. The levels of mRNA were determined by qPCR. Each point represents the mean ± S.E. of at least three independent experiments. (B) 3T3-L1 adipocytes were transfected with control (Luc), SMRT, NCoR, or PPARγ siRNA. 24 h post transfection, cells were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα) for additional 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01). NS, not significant.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: Transfection, Luciferase, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were treated with DMSO, 660 nM TSA, 20 µM SAHA, 10 µM MS275, or 5 µM MC1568 for 24 h. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. Samples were treated in duplicate. Representative immunoblots from three independent experiments are shown. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA, 10 µM MS275, 5 µM MC1568, or combination of MS275 and MC1568 (MS275+MC1568) for 24 h. (C) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with vehicle (DMSO), 660 nM TSA (TSA), 5 or 20 µM SAHA (SAHA) for 24 h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (*p<0.05; **p<0.01; ***p<0.001). NS: not significant. # p<0.05, ## p<0.01 vs the corresponding DMSO Control.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A and B) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 660 nM TSA, together with or without 1 µM Rosi (Rosi) for 24h. Cells were then treated with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western analysis with the indicated antibodies. Representative immunoblots and quantification data from five independent experiments are shown in 5A and B, respectively. (C) ERK phosphorylation correlates highly with lipolysis in the treatments of Rosi, TNFα, or both in the presence or absence of TSA, as shown by fitting with linear regression. Individual values were obtained from the experiments described in Figures. 1 and 7B.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Phospho-proteomics

Journal: PLoS ONE

Article Title: Trichostatin A Modulates Thiazolidinedione-Mediated Suppression of Tumor Necrosis Factor α-Induced Lipolysis in 3T3-L1 Adipocytes

doi: 10.1371/journal.pone.0071517

Figure Lengend Snippet: (A) 3T3-L1 adipocytes were pretreated with vehicle (DMSO) or 25 µM U0126 for 1 h, followed by treatment with or without 10 ng/ml TNFα for 30 min. Cellular proteins were solubilized and subjected to SDS-PAGE and Western blot analysis with the indicated antibodies. (B) 3T3-L1 adipocytes were treated with vehicle (Control), 1 µM Rosi (Rosi), 10 ng/ml TNFα (TNFα), or both (Rosi+TNFα), together with DMSO (bars 1-4), 660 nM TSA (bars 5-8), 25 µM U0126 (bars 9-12), or both (bars 13-16) for 24h. Glycerol released into the media and protein concentrations of cell lysate were determined as described in Materials and Methods . Each point represents the mean ± S.E. of four independent experiments. Asterisks denote significant differences (**: p<0.01). NS, not significant. ## p<0.01 vs Treatment No.1.

Article Snippet: Recombinant murine TNFα (No. 410-MT) was from R & D Systems (Minneapolis, MN).

Techniques: SDS Page, Western Blot, Control

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 1. RASEF expression in tumor tissues and cell lines. A, expression of RASEF in 12 clinical lung cancers (T; 4 clinical lung ADC, 4 clinical lung SCC, and 4 clinical SCLC) and corresponding normal lung tissues (N) detected by semiquantitative RT-PCR analysis. B, expression of RASEF in 22 lung cancer cell lines and a bronchial epithelial cell line BEAS-2B detected by semiquantitative RT-PCR analysis. ASC indicates lung adenosquamous cell carcinoma; LCC, large cell carcinoma. C, Western blot analysis of RASEF protein using anti-RASEF antibody. IB, immunoblotting. D, expression and subcellular localization of endogenous RASEF protein in RASEF-positive and RASEF-negative lung cancer cell lines, and bronchial epithelial cells. RASEF was stained mainly at the cytoplasm in A549 and NCI-H2170 cells, whereas no staining was observed in DMS114 and bronchial epithelia–derived BEAS-2B cell lines.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Western Blot, Staining, Derivative Assay

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 4. Enhanced phosphorylation of ERK1/2 by RASEF in lung cancer cells. A, expression of MAPK signal molecules and their phosphorylation levels in DMS114 cells transfected with RASEF expression vector or mock plasmid. B, expression of MAPK signal molecules and their phosphorylation levels in NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC). C and D, expression levels of downstream target genes of MAPK cascade were regulated by RASEF expression in lung cancer cells. Total RNA from BEAS-2B and DMS114 cells transfected with RASEF expression vector or mock plasmid (C) and A549 and NCI-H2170 cells transfected with siRNAs for RASEF (si-RASEF#2) or control siRNAs (si-LUC; D) were subjected to reverse-transcription reaction, followed by PCR reaction to evaluate the expression levels of CCND1, CCNB1, and CDKN1A transcription. Western blotting with antiphosphorylated ERK1/2 antibody was conducted to confirm the change of ERK1/2 phosphorylation according to RASEF expression.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Phospho-proteomics, Expressing, Transfection, Plasmid Preparation, Control, Reverse Transcription, Western Blot

Journal: Molecular cancer research : MCR

Article Title: RASEF is a novel diagnostic biomarker and a therapeutic target for lung cancer.

doi: 10.1158/1541-7786.MCR-12-0685-T

Figure Lengend Snippet: Figure 5. Identification of ERK1/2-interacting sites on RASEF. A, interaction of endogenous RASEF with endogenous ERK1/2. The immunoprecipitates obtained using anti-RASEF antibody were subjected to Western blotting with anti-ERK1/2 antibody. B, schematic representation of various partial constructs of RASEF expression vector. C and D, determination of the ERK1/2-interacting regions on RASEF by immunoprecipitation experiments using DMS114 cells transfected with vectors expressing partial RASEF protein. COOH-terminal part of RASEF (codons 520–575) was likely to be ERK1/2-interacting region.

Article Snippet: The cells were then incubated overnight at 4 C with a rabbit polyclonal antibody to RASEF (Catalog No. 11569-1-AP, Proteintech Group, Inc.) diluted in PBS containing 1% bovine serum albumin (BSA).

Techniques: Western Blot, Construct, Expressing, Plasmid Preparation, Immunoprecipitation, Transfection

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Diagram of coronal rat brain section showing the site of injury and photographed region (4 A). Representative immunofluorescence images (10 ×) of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity (green) combined with DAPI (blue) at 14 days post-TBI (10 days post-alcohol exposure) (4 B).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Immunofluorescence

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Quantification of ipsilateral cortex GFAP, ED-1, and HMGB1 immunoreactivity at 14 days post-TBI (10 days post-alcohol exposure). Images are quantified as % area of positive staining in 1.035 mm2 (10×). Values are shown as means ± SEM. For GFAP and ED-1, Sham/Air n = 4, Sham/Alcohol n = 4, TBI/Air n = 4, TBI/Alcohol n = 6. For HMGB1, Sham/Air n = 4, Sham/Alcohol n = 5, TBI/Air n = 5, TBI/Alcohol n = 7 (each n represents the average of 3 images taken from each animal) * p < 0.05 of the TBI groups vs. time-matched sham controls; $ p < 0.05 of the TBI/Alcohol group vs. TBI/Air group, by two-way ANOVA (5 A, 5 C, and 5 E).

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Staining

Journal: Brain, behavior, and immunity

Article Title: Alcohol Exposure after Mild Focal Traumatic Brain Injury Impairs Neurological Recovery and Exacerbates Localized Neuroinflammation

doi: 10.1016/j.bbi.2014.11.006

Figure Lengend Snippet: Primary and Secondary Antibodies.

Article Snippet: Anti-HMGB1 Rabbit polyclonal (Abcam, Cambridge, UK) , 1:100 , Donkey anti-Rabbit Alexa Fluor 488 (Invitrogen, Carlsbad, CA) , 1:200.

Techniques: Concentration Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 2. Changes in mRNA expression of BCL2, E2F1, E2F3, RB1 and P53 in K562 cells after cisplatin treatment. BCL2, E2F1, E2F3, RB1 and P53 were detected by (A) RT-PCR, (B) real-time PCR and (C) ELISA.

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 4. Correlative expression of miRNAs and oncogenes using antisense oligos (ASO). (A) RT-PCR, (B) real-time PCR. Correlative expression of (C) E2F1 and its targeted miR-17-5p, (D) E2F3 and its targeted miRNAs and (E) Bcl-2 and its targeted miRNAs (miR-16, 34a-c) using ELISA is shown. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing, Reverse Transcription Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay

Journal: Oncology reports

Article Title: miRNA-regulated expression of oncogenes and tumor suppressor genes in the cisplatin-inhibited growth of K562 cells.

doi: 10.3892/or_00000813

Figure Lengend Snippet: Figure 5. Expression of E2F1, E2F3, BCL2, RB1 and P53 genes regulated by miRNAs. Detection of the expression of (A) E2F1, (B) E2F3, (C) BCL2, (D) RB1 and (E) P53. *Significant difference (p<0.05).

Article Snippet: K562 cells were lysed [lysis buffer: 0.15 M NaCl, 5 mM EDTA (pH 8.0), 1% Triton X-100, 10 mM Tris-Cl (pH 7.4), 100 mM PMSF and 5 M DTT) and incubated in a 96-well plate, followed by the addition of goat anti-human antibodies against BCL2 (1:400), E2F1, E2F3 (1:400, 1:400), RB1 (1:500) and P53 (1:300, 1:400) (all from Immunoleader, Boster) according to the manufacturer's instructions.

Techniques: Expressing

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 3 Immunohistochemical analysis of the distinct presence of Cx43 and BMP-15 in the oophorons of each group (× 200) A-F: Cx43; G-L: BMP-15. A, G: control group; B, H: model group; C, I: positive group; D, J: low dose of BSJPP group; E, K: moderate dose of BSJPP group; F, L: high dose of BSJPP group. Positive group treated with premarin (0.03 mg/kg) once daily for 90 d. Con- trol and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Immunohistochemi- cal analysis clearly revealed the presence of Cx43 and BMP-15 in the oophorons of control mice and those treated with BSJPP (D, E) and premarin compared with the model group. BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Immunohistochemical staining, Control, In Vivo

Journal: Journal of Traditional Chinese Medicine

Article Title: Efficacy of Bushenjianpi prescription on autoimmune premature ovarian failure in mice

doi: 10.1016/S0254-6272(17)30321-7

Figure Lengend Snippet: Figure 4 Expression Cx43 and BMP-15 mRNA in the ovaries of mice in each group. 1: control group; 2: model group; 3: positive group; 4: low dose of BSJPP group; 5: moderate dose of BSJPP group; 6: high dose of BSJPP group. Positive group treated with pre- marin (0.03 mg/kg) once daily for 90 d. Control and model group received the same volume of distilled water. Low (8.1 mg/kg), moderate (16.2 mg/kg, the dose used in vivo in human studies), and high (32.4 mg/kg) dose of BSJPP groups treated by gastrogavage once daily for 90 d. Compared with the model group, significantly upregulated expression of both Cx43 and BMP-15 was detected in the control group and the groups treated with BSJPP (M and L groups). BSJPP: Bushenjianpi prescription; Cx43: connexin 43.

Article Snippet: Anti-connexin 43 (Cx43) antibody (product lot No. BA1727) and anti-bone morphogenetic protein 15 (BMP-15) antibody (product lot No. BA2018) were purchased from Boster Biological Engineering (Wuhan, China).

Techniques: Expressing, Control, In Vivo

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Knockout of MAP4K3 promotes autophagy induction and flux. a Validation of MAP4K3 knockout (k.o.) cell lines. Wild-type (WT) and HEK293A cells gene-edited with either of two different sgRNAs (M1 and M4) were lysed, and protein lysates were immunoblotted for MAP4K3. Immunoblotting of β-actin served as a loading control. b , c Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells, M1-1 MAP4K3 k.o. cells, and M4-6 MAP4K3 k.o. cells (not shown) were cultured in complete media (CM) or subjected to amino acid starvation (– AA), and remained untreated or were treated with ammonium chloride. Protein lysates were immunoblotted for LC3 and β-actin, which served as a loading control b . The ratio of LC3-II:actin was determined by densitometry using ImageJ and normalized to WT CM, which was arbitrarily set to 1 c . One-way ANOVA with post-hoc Tukey’s test; * P < 0.05. d , e Knockout of MAP4K3 promotes autophagy induction. LC-3 immunostaining of WT HEK293A cells and three different MAP4K3 k.o. cell lines, all cultured in CM d . Quantification of LC3 puncta area per cell area was determined using ImageJ. n > 100 cells per genotype. One-way ANOVA with post-hoc Tukey’s test; ** P < 0.01, *** P < 0.001. f – h Knockout of MAP4K3 promotes autophagy flux. WT HEK293A cells and MAP4K3 k.o. cells were cultured in CM or amino acid starved, and were transfected with a GFP-mCherry-LC3 expression construct ( f ). Note the predominance of red puncta indicative of autolysosomes in MAP4K3 k.o. cells. Quantification of autophagosome number per cell was determined by counting yellow puncta GFP-mCherry-LC3-expressing cell ( g ). Quantification of autolysosome number per cell was determined by counting red puncta/GFP-mCherry-LC3-expressing cell ( h ). n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’ test; ** P < 0.01, *** P < 0.001. All experiments were performed in triplicate. Error bars = SEM. Scale bars = 10 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Knock-Out, Biomarker Discovery, Western Blot, Control, Cell Culture, Immunostaining, Transfection, Expressing, Construct

Journal: Nature Communications

Article Title: MAP4K3 mediates amino acid-dependent regulation of autophagy via phosphorylation of TFEB

doi: 10.1038/s41467-018-03340-7

Figure Lengend Snippet: Phosphorylation of TFEB at serine 3 is a key determinant of TFEB cellular regulation and autophagy function. a TFEB serine 3 phosphorylation is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG, TFEB-S3A-FLAG, or TFEB-S3E-FLAG, and Torin1 treated. FLAG immunoprecipitates were immunoblotted and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. b MAP4K3 phosphorylation of TFEB is required for mTORC1 phosphorylation at serine 211. WT HEK293A and MAP4K3 k.o. cells were transfected with TFEB-FLAG and either no MAP4K3 (--), WT-MAP4K3, or KD-MAP4K3. FLAG immunoprecipitates were immunoblotted, and TFEB serine 211 phosphorylation as a fraction of total TFEB was quantified by densitometry. c TFEB serine 3 phosphorylation is required for interaction with 14-3-3. TFEB k.o. cells were transfected with no TFEB (--), inducible TFEB-WT-FLAG, or inducible TFEB-S3A-FLAG, and Torin1 treated, whereas all cells received doxycycline to induce TFEB-WT or TFEB-S3A expression. Cell lysates and TFEB immunoprecipitates were immunoblotted and immunoprecipitated 14-3-3 was quantified by densitometry. One-way ANOVA with post-hoc Tukey’s test; * P < 0.05, ** P < 0.01. d TFEB serine 3 phosphorylation regulates TFEB nuclear localization. TFEB k.o. cells were transfected with inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM or amino-acid starved (– AA). Under amino acid deprivation, TFEB localizes to nucleus, regardless of serine 3 status; however, upon amino acid satiety, mutation of TFEB serine 3 to phospho-resistant alanine prevents retention of TFEB in the cytosol. Quantification of TFEB nuclear localization to right. n > 100 cells per condition. One-way ANOVA with post-hoc Tukey’s test; *** P < 0.001. e TFEB serine 3 phosphorylation regulates autophagy activation. TFEB k.o. cells were transfected with GFP-mCherry-LC3 and either inducible TFEB-WT-FLAG or TFEB-S3A-FLAG, and cultured in CM and doxycycline, as indicated. Note the red puncta indicative of autolysosomes in cells expressing TFEB-S3A. Autophagosome number per cell was determined by counting yellow puncta per GFP-mCherry-LC3-expressing cell, and autolysosome number per cell was determined by counting red puncta per GFP-mCherry-LC3-expressing cell. n > 50 cells per condition. One-way ANOVA with post-hoc Tukey’s test; * P 0 < 0.05, ** P < 0.01. All experiments performed in triplicate. Error bars = SEM. Scale bars = 20 μm

Article Snippet: Membranes were incubated overnight with primary antibodies against the following: LC3 (Novus Biologicals, NB100-2220) 1/3000; β-actin (Abcam, #ab8226) 1/10,000; Map4k3 (Cell Signaling, 9613) 1/1000; RagA (Cell Signaling, 4357) 1/1000; RagC (Cell Signaling, 5466) 1/1000; Lamtor1 (Cell Signaling, 8975) 1/1000; TFEB (Cell Signaling, 4240) 1/1000; pan 14-3-3 (Santa Cruz Biotechnology, sc-629) 1/1000; FLAG (M2) (Sigma; F1804); Raptor (24C12) (Cell Signaling, 2280) 1/1000; phospho-(Ser) 14-3-3 Binding Motif (Cell Signaling, 9601) (for detection of TFEB phosphorylation at Ser211) 1/1000; GST (Santa Cruz Biotechnology, sc-138) 1/1000, in 5% bovine serum albumin in PBS-T. Species-specific secondary antibodies were goat anti-rabbit IgG-HRP (Santa Cruz, sc-2004) or goat anti-mouse IgG-HRP (Santa Cruz, sc-2005), diluted 1/10,000 in 5% PBS-T milk and incubated for 1 h at RT.

Techniques: Phospho-proteomics, Transfection, Expressing, Immunoprecipitation, Cell Culture, Mutagenesis, Activation Assay