Journal: PLoS ONE

Article Title: C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

doi: 10.1371/journal.pone.0028294

Figure Lengend Snippet: Cells were cultured as described in “ ”. Total cell lysate and nuclear extracts were subject to Western blot analysis to determine ERK1/2 abundance and phosphorylation. A representative Western blot image is shown in the upper panel of each graph. A. The total ERK1 expression. B. ERK1 nuclear abundance . C. Total ERK1/2 phosphorylation. D. Nuclear ERK1/2 phosphorylation . GAPDH and histone H3 proteins were used as loading controls for total cell lysate and nuclear extracts, respectively. Results are means ± SE for 6 independent experiments. * P<0.05 versus 5 mM glucose without C-peptide.

Article Snippet: Rabbit polyclonal anti-phospho PKC α/β, δ, ε and histone H3 were from Cell Signaling Technology, Inc. (Beverly, MA) Monoclonal anti-phospho-ERK1/2 (P-Thr 202 /Tyr 204 ) and polyclonal anti-ERK1 were from New England BioLabs (Beverly, MA).

Techniques: Cell Culture, Western Blot, Expressing

Journal: PLoS ONE

Article Title: C-Peptide Increases Na,K-ATPase Expression via PKC- and MAP Kinase-Dependent Activation of Transcription Factor ZEB in Human Renal Tubular Cells

doi: 10.1371/journal.pone.0028294

Figure Lengend Snippet: C- peptide binds specifically to a membrane structure, most likely a G-protein coupled receptor, with subsequent activation of PLC, isoforms of both classic and novel PKC, Rho A, MEK1/2 and ERK1/2. The latter elicits activation of ZEB (AREB6) and regulation of the gene expression for Na,K-ATPase α 1 -subunit.

Article Snippet: Rabbit polyclonal anti-phospho PKC α/β, δ, ε and histone H3 were from Cell Signaling Technology, Inc. (Beverly, MA) Monoclonal anti-phospho-ERK1/2 (P-Thr 202 /Tyr 204 ) and polyclonal anti-ERK1 were from New England BioLabs (Beverly, MA).

Techniques: Activation Assay, Expressing

Journal: PLoS ONE

Article Title: A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

doi: 10.1371/journal.pone.0091037

Figure Lengend Snippet: A) Chemical modification of berberine chloride produced 80 protoberberine derivatives. B) Scheme for screening protoberberine derivatives for inhibition of various MKKs (MKK4/MKK7, MKK3/MKK6, and MEK1/MEK2) and MAPKs (JNKs, p38, and ERKs). Each MKK and MAPK was immunoprecipitated from HEK293 or CHO cells. C) Chemical structure and simplified synthetic process of HWY336 from berberine chloride.

Article Snippet: The following antibodies were used: anti-MEK1 (rabbit polyclonal), anti-MEK2 (rabbit monoclonal), and anti-JNK (rabbit polyclonal) (Millipore, MA) at a 1∶200 dilution; anti-MKK3 (rabbit monoclonal), anti-MKK4 (rabbit polyclonal), anti-MKK6 (rabbit polyclonal), anti-MKK7 (rabbit polyclonal), anti-ERK1 (rabbit polyclonal), and anti-p38 (rabbit polyclonal) (New England Biolabs, Frankfurt, Germany) at a 1∶150 dilution.

Techniques: Modification, Produced, Inhibition, Immunoprecipitation

Journal: PLoS ONE

Article Title: A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

doi: 10.1371/journal.pone.0091037

Figure Lengend Snippet: A, B) MKK4 and MKK7 were immunoprecipitated from HEK293T cells following MAPK pathway activation as described in EXPERIMENTAL PROCEDURES. The same quantity of kinase was used in each assay. The activity of MKK4 (A) and MKK7 (B) was assayed by measuring γ-P 32 phosphorylation of a endogenous substrate, JNK, in the presence of increasing HWY336 concentration and quantified using ImageJ software to determine IC 50 . The average relative kinase activity compared to DMSO is plotted as a percentage. Error bars represent the standard deviations. C, D) HEK293T cells were treated with 600 mM D-sorbitol for 30 min before harvest. (C) HEK293T cells were treated with 12 µM of HWY336 for 0, 1, 2, 3, or 4 hrs, and their total lysates were analyzed by western blots. p-JNKs, p-p38, JNKs, and p38 were detected as described. (D) HEK293T cells were treated with various concentrations of HWY336 (3, 6, 9, 12 µM) for 3 hrs and each cell lysate was prepared. p-JNKs, p-p38, p-MKK4, JNKs, p38, and MKK4 were detected by western blot analysis of cell lysates.

Article Snippet: The following antibodies were used: anti-MEK1 (rabbit polyclonal), anti-MEK2 (rabbit monoclonal), and anti-JNK (rabbit polyclonal) (Millipore, MA) at a 1∶200 dilution; anti-MKK3 (rabbit monoclonal), anti-MKK4 (rabbit polyclonal), anti-MKK6 (rabbit polyclonal), anti-MKK7 (rabbit polyclonal), anti-ERK1 (rabbit polyclonal), and anti-p38 (rabbit polyclonal) (New England Biolabs, Frankfurt, Germany) at a 1∶150 dilution.

Techniques: Immunoprecipitation, Activation Assay, Activity Assay, Concentration Assay, Software, Western Blot

Journal: PLoS ONE

Article Title: A Protoberberine Derivative HWY336 Selectively Inhibits MKK4 and MKK7 in Mammalian Cells: The Importance of Activation Loop on Selectivity

doi: 10.1371/journal.pone.0091037

Figure Lengend Snippet: A) Phylogenetic tree for human MKK4, MKK7, MKK3, MKK6, MEK1, p38, JNK, and ERK. B) Amino acid sequence alignment starting from the hinge region to the activation loop are shown for MKK3 (UniProtKB accession code: P46734), MKK6 (UniProtKB accession code: P52564), MKK4 (UniProtKB accession code: P45985), and MKK7 (UniProtKB accession code: O14733). Green denotes a highly conserved region among these MKKs. Residues highlighted in yellow designate sequence variations in the activation loop. C, D) Three-dimensional structure of MKK4 suggests that HWY336 interacts with the activation loop through hydrogen bonding. C) (top) Amino acid sequence variations within the activation loop of MKKs. (bottom) Proposed docked pose of ATP in MKK4. The arrow designates different amino acids that may determine MKK selectivity. The MKK4 structure is shown in the background with the activation loop (white) containing the varying amino acids (Arg 262 ) at the respective positions (generated with the Pymol program; www.pymol.org ). D) Hydrophobic interactions between HWY336 and the MKK4 active site are shown. Hydrophobic residues within the active site are designated by the cap-stick model and HWY336 is displayed using transparent hydrophobic surfaces. The hydrophobicity index is displayed on the left, where brown and blue denote highly hydrophobic and hydrophilic areas, respectively. Pro 268 , Phe 305 , Pro 308 , and Val 313 interact with HWY336 side chains. HWY336 interacts with the activation loop of MKK4 through hydrogen bonding via the hydroxyl group of Thr 261 .

Article Snippet: The following antibodies were used: anti-MEK1 (rabbit polyclonal), anti-MEK2 (rabbit monoclonal), and anti-JNK (rabbit polyclonal) (Millipore, MA) at a 1∶200 dilution; anti-MKK3 (rabbit monoclonal), anti-MKK4 (rabbit polyclonal), anti-MKK6 (rabbit polyclonal), anti-MKK7 (rabbit polyclonal), anti-ERK1 (rabbit polyclonal), and anti-p38 (rabbit polyclonal) (New England Biolabs, Frankfurt, Germany) at a 1∶150 dilution.

Techniques: Sequencing, Activation Assay, Generated

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Insulin is distributed in dynamic punctate structures at the Golgi apparatus. (A) INS1 832/13 cells were immunostained for insulin (pan-insulin antibody; green), TGN38 (magenta) and counterstained with DAPI (blue). The middle image demonstrates 3D-rendering of the TGN38 volume mask which was used for image segmentation to specifically examine insulin staining within the TGN (right image). (B) A panel of electron micrographs from cells of pituitary gland and mouse islets demonstrating condensed structures within the Golgi lumen. (C) Representative images from a single slice from a confocal image of INS1 832/13 cells stained with the insulin processing enzyme protein convertase 2 (PC2; green) and insulin (pan-insulin antibody; red). Note the colocalization of proinsulin puncta with PC2 at the Golgi apparatus which is outlined in the merge image using dashed lines. (D) Images obtained from INS1 832/13 cells which were stained with TGN38 (red) and PC (green), treated with either 0.05% DMSO (control; top) or 5 µg/ml Brefeldin A (bottom). Shown here is a single slice from a confocal stack. Note the disassembly of TGN38 based on staining upon Brefeldin A treatment which is accompanied by a loss of PC2 puncta in the perinuclear region, marked using dashed line. (E) A schematic description of a pulse-chase assay in INS1 832/13 cells stably expressing SNAP-tagged proinsulin. Cells are initially incubated with a non-fluorescent blocking probe to mask the existing proteins in the cells. After 2 h, cells are labeled with SNAP-505 to mark the newly synthesized proteins (20 min). After two washes in growth medium either in presence or absence of concanamycin A (control), cells are fixed immediately to monitor the pulse of new synthesized proinsulin arriving at the Golgi apparatus. (F) Representative images at the top show single slice from a confocal image of INS1 832/13 cells labeled with SNAP-505 (green) to monitor newly arrived proinsulin at the TGN (red) in control (left) and concanamycin A (right) treatment. The insets below are zoomed in images from a single cell to highlight the punctate vs diffuse distribution of proinsulin at the TGN in control (left) and concanamycin A (right) treatment. (G) A panel of images extracted from a movie from live imaging of INS1 832/13 cells transiently transfected with RINS1, a fluorescent insulin reporter construct. Dynamics of the punctate structures (pink dashed line) are captured in the image sequence where structures undergo fission or fusion events. Images shown here are single confocal slices upon imaging in the conventional confocal mode (i) or in the airy scan confocal mode (ii). Images have been smoothened using the function in ImageJ for visual representation purposes. (H) A panel of images extracted from a movie from live imaging of HeLa cells expressing Halo-RUSH-CGB (magenta) and GalT-GFP (green) to monitor the budding of CGB granules from the Golgi. The arrowhead denotes a budding event from the Golgi.

Article Snippet: For purposes of western blotting, antibodies against GFP (11814460001; Roche/Sigma-Aldrich) SNAP-tag (P9310S; NEB), and β-actin (A5441; Sigma-Aldrich) were used.

Techniques: Staining, Pulse Chase, Stable Transfection, Expressing, Incubation, Blocking Assay, Labeling, Synthesized, Imaging, Transfection, Construct, Sequencing

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Ectopic expression of soluble secreted proteins in INS1 832/13 cells results in their routing to insulin granules. (A and B) Representative images from INS1 832/13 cells expressing LyzC-GFP (A; green) or EqSol-GFP (B; green) and stained with insulin antibody (red) to observe the localization of the ectopically expressed proteins with respect to insulin granules. Images are average projections from two slices from a confocal stack. Arrowheads point to cytoplasmic insulin granules which also shows the presence of LyzC-GFP and EqSOL-GFP respectively, in E and F. (C) Representative images from INS1 832/13 cells stably expressing CatD-GFP (green) and labeled with CGB antibody to observe the localization of ectopically expressed CatD-GFP with respect to SGs. Images are average projections from two slices from a confocal stack. Arrowheads point to some of the cytoplasmic SG, which shows colocalization of CGB and CatD-GFP. (D) Representative images from INS1 832/13 expressing HA-tagged version of the calcium ATPase, SPCA1 (green) and stained using CGB antibody (red). Images are a single slice from a confocal stack. Note that overexpressed SPCA1 remains localized at the Golgi apparatus with no signal seen from the CGB containing SGs. (E) Representative images from HeLa cells stably expressing CGA-GFP and transfected with LyzC-RFP. Images are a single slice from a confocal stack imaged in the airy-scan mode. The arrowheads point to some of the ectopic granule-like structures seen in HeLa cells upon expression of CGA-GFP. Note that LyzC-RFP gets routed to these ectopic granule-like structures. (F) Images extracted from live imaging of HeLa cells co-expressing Halo-RUSH-CGB (red) and LyzC-GFP (green) before and after addition of biotin for 52 min when CGB appears at the Golgi. (G) Images extracted from live imaging of HeLa cells co-expressing RUSH-CGB (red) and LyzC-GFP (green) after biotin addition and images after arrival of CGB at the Golgi. Arrow heads point to colocalizing structures at the Golgi and vesicles in the cytoplasm. (H) Western blot at the top shows bands for LyzC-GFP, probed using α-GFP antibody, in supernatant and lysates from INS1 832/13 cells stable expressing SNAP-tagged proinsulin. The basal condition represents cells grown in 3 mM glucose in serum-free medium and the stimulated condition represents cells grown in 15 mM glucose in serum-free medium, also containing 35 mM potassium chloride. Note the stronger band intensity in the supernatant in stimulated condition compared to the basal condition, although the levels in cell lysates are the same. The blot in the bottom left detects the presence of SNAP-tagged C-peptide, probed using α-SNAP-tag antibody, which is used as a proxy to measure insulin secretion. Again, the signal intensity of the band is stronger in stimulated condition as compared to the basal condition. The blot on the bottom right depicts actin bands in cell lysates obtained from basal and stimulated conditions. The graph quantifies secretion of LyzC-GFP normalized with levels in cell lysates in basal and stimulated conditions. Value of the band intensity in secreted compared to the band intensity in cell lysates was set to 1 for stimulated condition in each experiment. Data is represented as mean ± SD from three independent experiments. Statistical analysis was performed by two-tailed one-sample t test *P = 0.019. Source data are available for this figure: .

Article Snippet: For purposes of western blotting, antibodies against GFP (11814460001; Roche/Sigma-Aldrich) SNAP-tag (P9310S; NEB), and β-actin (A5441; Sigma-Aldrich) were used.

Techniques: Expressing, Staining, Stable Transfection, Labeling, Transfection, Imaging, Western Blot, Two Tailed Test

Journal: The Journal of Cell Biology

Article Title: Liquid–liquid phase separation facilitates the biogenesis of secretory storage granules

doi: 10.1083/jcb.202206132

Figure Lengend Snippet: Proinsulin co-traffics with CGB in vivo and is recruited to droplets in vitro. (A) Schematic depiction of dual pulse chase experiment in INS1 832/13 cells expressing SNAP tagged insulin (proCpepSNAP) and CLIP tagged CGB (CGB-CLIP). Cells are initially incubated with a non-fluorescent blocking probe to mask the existing proteins in the cells. After 2 h, cells are incubated with medium containing SNAP 505 and CLIP-TMR to label the newly synthesized proteins (20 min). After three washes in growth medium, cells are fixed immediately (0 h chase) when majority of the cargo is at the Golgi apparatus or after a chase of 2 h where most of the cargo has moved to the SG in the cytoplasm. (B) Top panel shows confocal images form INS1 832/13 cells expressing SNAP tagged insulin (proCpepSNAP; green) and CLIP tagged CGB in red and fixed immediately after labeling with fluorescent probes, SNAP-505 and CLIP-TMR to monitor the Golgi resident (peri-nuclear) pool of the proteins. Bottom panels show images after a 2 h chase and the arrows point to some of the colocalizing structures which are cytoplasmic SGs. (C) INS1 832/13 wild-type (top) and CGA/CGB dKO (bottom) cells fixed and labeled with antibodies to TGN38 (red) and PC2 (green). Left and the middle images are extracted from a 3D projection. The image on the right represents surfaces which were created using the TGN38 staining (red outline) on deconvolved images in Imaris. The TGN38 volume mask was then used to generate distinct surfaces in the PC2 channel. (D) A scatter plot (median) depicting differences in the numbers of PC2 surfaces between wild-type and CGA/CGB dKO cells from 22 wild-type and 24 dKO cells. Statistical analysis was performed using Mann–Whitney test. ***P < 0.001. (E) Graph showing normalized glucose stimulated insulin secretion (GSIS; stimulated/basal) in wild-type, CGA/CGB dKO cells. Data is represented as mean ± SD from six independent experiments. Statistical analysis was performed using unpaired two-tailed t test. ***P < 0.001. (F) CGB-GFP (16 µM; green) was mixed with Cy3 tagged proinsulin (1 µM; red) in (i). Tagged proinsulin gets recruited to the CGB-GFP droplets as evident from the colocalization image. When GFP (16 µM; green) is mixed with Cy3 tagged proinsulin (1 µM; red) in (ii), no droplets are seen either with GFP or proinsulin indicating that GFP or Cy3-proinsulin are incapable of forming droplets on their own at these concentrations.

Article Snippet: For purposes of western blotting, antibodies against GFP (11814460001; Roche/Sigma-Aldrich) SNAP-tag (P9310S; NEB), and β-actin (A5441; Sigma-Aldrich) were used.

Techniques: In Vivo, In Vitro, Pulse Chase, Expressing, Incubation, Blocking Assay, Synthesized, Labeling, Staining, MANN-WHITNEY, Two Tailed Test