Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: Genotypes of offspring from Csn2 WT/K70E intercrosses.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques:

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Body weight measurement of wild-type and Csn2 WT/K70E (Het) mice at the indicated age (*** p < 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect). Data are presented as mean ± SEM. b Feeding and fasting (20 h) glucose levels ( n = 5). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. c , d Feeding and fasting (16 h) insulin ( c ) and C-peptide ( d ) levels ( n = 5). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. e Intraperitoneal glucose tolerance test [ n = 5 (WT), or 6 (Het), * p < 0.05, ** p < 0.01, calculated by two-tailed Student’s t test.]. Data are presented as mean ± SD. f Intraperitoneal insulin tolerance test (ITT; n = 5 (WT), or 6 (Het), * p = 0.012, calculated by two-way repeated measures ANOVA testing genotype × time effect). Data are presented as mean ± SD. 100% blood glucose means 6.37 ± 0.6 mM for WT and 7.27 ± 0.8 mM for Het. g , h Effect of streptozocin (STZ) treatment (50 mg/kg, 4 days) on serum insulin levels ( g ) and insulin tolerance test ( h ) of wild-type and Csn2 WT/K70E (Het) mice, ( n = 5). Data are presented as mean ± SD. 100% blood glucose means 14.0 ± 5.4 mM for WT and 11.2 ± 2.2 mM for Het. P values were calculated by two-tailed Student’s t test. i Glucose-stimulated insulin secretion in mice [ n = 4 (WT), or 5 (Het), ** p < 0.01, calculated by two-tailed Student’s t test.]. Data are presented as mean ± SEM. j Glucose-stimulated insulin secretion in isolated pancreatic islets ( n = 3, *** p < 0.001, calculated by two-tailed Student’s t test.). Data are presented as mean ± SEM. k Basal and secretagogue-stimulated insulin secretion (1 h) in isolated islets ( n = 3). Data are presented as mean ± SD. Islets from Csn2 WT/K70E mice secrete more insulin with l -arginine stimulation, suggesting that altered membrane potential or Ca 2+ flux are not major causes of their enhanced insulin secretion. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Two Tailed Test, Isolation, Membrane

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a , b H&E staining-based morphometric analysis of pancreatic islets from wild-type and Csn2 WT/K70E mice. Number of islets per pancreas ( a ) and average islet area ( b ) was measured by examining 20 slides with every five slides in between, covering the whole pancreas ( n = 5, n.s. not significant). Data are presented as mean ± SEM. Scare bars, 1.5 μm ( a ) and 0.4 μm ( b ). c Immunofluorescence staining of insulin and glucagon in pancreatic islets from wild-type and Csn2 WT/K70E mice. Scare bars, 0.2 μm. Lower panel: percentage of α or β cells in an islet is measured based on the area of green (anti-glucagon, α cells) and red fluorescence (anti-insulin, β cells; n = 8, n.s. not significant). Data are presented as mean ± SEM. Right panel: quantification of the immunofluorescence intensity of insulin staining ( n = 8). P values were calculated by two-tailed Student’s t test. d , e Average islet cytoplasmic Ca 2+ responses to depolarization induced by 20 mM glucose ( d ) or 25 mM KCl ( e ), assayed with Fura2AM ( n = 5, n.s. not significant). Data are presented as mean ± SEM. f Total insulin content of pancreatic islet measured using ELISA assay ( n = 10). P values were calculated by two-tailed Student’s t test. g Levels of Ins1 , Ins2, Sytl3 , and Exoc6 transcripts from pancreas measured by real-time PCR ( n = 10). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test. h Representative electron micrographs of pancreatic islets showing β cell insulin granules. Dotted lines: plasma membrane (PM). Docked granules are marked by arrow heads. Scale bars, 0.5 μm ( n = 6). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test. i GSIS of wild-type and Het islets measured as percentage of insulin secretion. Islets were lysed after GSIS to determine total insulin content ( n = 5). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Staining, Immunofluorescence, Fluorescence, Two Tailed Test, Enzyme-linked Immunosorbent Assay, Real-time Polymerase Chain Reaction, Clinical Proteomics, Membrane

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Effect of MLN4924 treatment (2 μM, 2 h) on basal and glucose-stimulated insulin secretion by wild-type and Csn2 WT/K70E islets ( n = 6, *** p < 0.001, calculated by two-way ANOVA testing the effect of 2.8 or 16.8 mM glucose in the presence of MLN4924, the other p values were calculated by two-tailed Student’s t test). Data are presented as mean ± SEM. b , c Mice serum insulin levels with/without MLN4924 treatment (15 mg/kg, twice weekly for 6 weeks). Insulin levels were measured at the same time of the day to minimize fluctuation. ( n = 6, ** p = 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect for Het mice). Data are presented as mean ± SEM. c Mice body weight with/without MLN4924 treatment (15 mg/kg, twice weekly for 6 weeks; n = 6, *** p < 0.001, calculated by two-way repeated measures ANOVA testing genotype × time effect for Het mice). Data are presented as mean ± SEM. d Levels of total and neddylated Cullins, COP1, and CRL4 substrates in the pancreas after 6 h fasting to normalize variation in blood glucose levels. e Ex vivo, lentiviral shRNA-based knockdown screening for CRL4 adaptors whose depletion alters islet insulin secretion. Quantified values of insulin secretion were normalize by that of Ctrl islets and presented in Log 2 scale. f Effect of adenovirus-mediated in-islet COP1 knockdown on basal and glucose-stimulated insulin secretion ( n = 3, *** p < 0.001, calculated by two-way ANOVA testing the effect of 2.8/16.8 mM glucose in the presence of shCOP1, the other p values were calculated by two-tailed Student’s t test). Data are presented as mean ± SEM. Inset: western blot showing efficient COP1 knockdown. Arrow indicates COP1 band. An asterisk (*) indicates nonspecific band. g Levels of ETV4 and ETV5 in MIN6 cells with COP1 (left) or Det1 (right) knockdown using two independent shRNA. h Levels of ETV5 in MIN6 cells with Myc-Det1 and HA-COP1 single or double overexpression. i Levels of ETV5 in wild-type and Csn2 WT/K70E islets with/without MLN4924 treatment (2 μM, 2 h). j Levels of ubiquitylated ETV5 enriched by GST-TUBE pulldown. k Effect of CSN2 knockdown on ETV5 ubiquitylation in MIN6 cells. l Effect of adenovirus-mediated in-islet ETV5 knockdown on insulin secretion ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. m Effect of ETV5 overexpression on insulin secretion ( n = 4). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. n Effect of adenovirus-mediated in-islet ETV5 knockdown on mRNA levels of ETV5 transcriptional targets ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. o Effect of ETV5 overexpression on mRNA levels of ETV5 transcriptional targets ( n = 3). Data are presented as mean ± SD. P values were calculated by two-tailed Student’s t test. p Effect of Sytl3 and Exoc6 overexpression on insulin secretion ( n = 3). Data are presented as mean ± SEM. P values were calculated by two-tailed Student’s t test.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Two Tailed Test, Ex Vivo, shRNA, Knockdown, Western Blot, Over Expression

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: a Structural models illustrating steric incompatibility between COP1 and CSN. For modeling details, see “Methods”. b Effect of COP1 and DET1 overexpression, individually or combined, on CRL–CSN complex formation in MIN6 cells. c , d CRL4–COP1 complex formation detected by COP1 co-immunoprecipitation with Cul4A ( c ) and Cul4B ( d ) from WT and Csn2 WT/K70E pancreas lysates. e Effect of adding IP 6 (20 μM) into MIN6 cell lysates on CRL4B COP1 complex formation. f Glucose fasting (2 h) increases CSN3-Cul4B co-ip in MIN6 cells, which is reversed by replacement with glucose-proficient medium (1 h). g Glucose fasting (2 h) decreases Cul4B-CSN3 co-ip in MIN6 cells, which is reversed by replacement with glucose-proficient medium (1 h).

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Over Expression, Immunoprecipitation, Co-Immunoprecipitation Assay

Journal: Nature Communications

Article Title: IP 6 -assisted CSN-COP1 competition regulates a CRL4-ETV5 proteolytic checkpoint to safeguard glucose-induced insulin secretion

doi: 10.1038/s41467-021-22941-3

Figure Lengend Snippet: CRL4-sequestration by CSN is compromised by CSN2-K70E mutation, or hyperglycemia elicited by nutrient oversupply. This leads to the assembly of CRL4 COP1 E3 ligase to promote ETV5 degradation, which acts as a transcriptional “brake” suppressing congenital hyperinsulinemia and consequent obesity/diabetes.

Article Snippet: The primary antibodies used were: Cul1 (1:1000, CST, 4995), Cul3 (1:1000, CST, 2759), CSN5 (1:1000, CST, 6895), CDT1 (1:1000, Proteintech, 14382-1-AP); ETV1 (1:1000, Abcam, ab184120), ETV5 (1:1000, Abcam, ab102010, CHIP and WB), Cul4A (1:1000, Abcam, ab72548, WB and IP), CSN3 (1:1000, Abcam, ab79698), Cul2 (1:1000, Bethyl, A302–476A), Cul5 (1:1000, Bethyl, A302–173A), COP1 (1:1000, Bethyl, A302–173A), CSB (1:1000, Genetex, GTX104589), GAPDH (1:3000, Proteintech, 10494-1-AP), Exoc6 (1:1000, Proteintech, 12723-1-AP), Sytl3 (1:1000, Proteintech, 22076-1-AP), CSN2 (1:3000, Proteintech, 10969-2-AP), ETV4 (1:1000, Thermo, MA5-15424), Cul4B (1:1000, Sigma, C9995, WB and IP), GST (1:3000, Sigma, A7340), Det1 (1:1000, Santa Cruz, sc-514348), insulin (1:100, R&D Systems, I2018, for IF), and glucagon (1:100, Abcam, ab10988, for IF).

Techniques: Mutagenesis

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: CSN2 and CSN5 knockdowns result in Cul-1 hyperneddylation, loss of Skp-2 and accumulation of p27 . K562 cells were transiently transfected in the absence of plasmid DNA (mock), with pMACS K k .II plasmid alone (HKK) or transiently co-transfected with HKK plasmid together with either vector control (shVC), CSN2 knockdown (shCSN2) or CSN5 knockdown (shCSN5) plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested for analysis. (A) CSN2 (left) and CSN5 (right) protein knockdown was determined by western blot in three independent transfectants. (B) mRNA levels in vector control and knockdown cells were determined by QRT-PCR (CSN2, left, P = 0.0012; CSN5, right, P = 0.000031). Data represents 3 independent sets of triplicate transfections. (C) CSN5 and CSN2 protein levels were determined by western blot in n = 3 CSN2 (top) and CSN5 (bottom) knockdowns, respectively. (D) CSN5 mRNA levels in n = 3 vector control and CSN2 knockdown cells were determined by QRT-PCR (P = 0.011). (E) The level of Cul1 neddylation (top panels, † indicates neddylated Cul-1), Skp2 protein (second panels) and p27 protein (third panels) was determined in n = 3 shCSN2 (left) and shCSN5 (right) samples by western blot. Even gel loading was determined by β-actin signal. Graphical data indicates the mean ± s.e.m. * indicates significance with a p value of less than 0.05.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Transfection, Plasmid Preparation, Control, Knockdown, Cell Culture, Western Blot, Quantitative RT-PCR

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: Loss of CSN2 and CSN5 results in sequential loss of F-box proteins . K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) shVC and shCSN2 cells were harvested day 6 and 9 post transfection and the level of Skp2, Cdc4, β-TrCP and β-actin protein determined by western blot. (B) The level of Skp2, Cdc4 and β-TrCP proteins (normalised for loading using β-actin) in shCSN2 at each time point was normalised to expression in shVC cells and the data plotted as the mean ± s.e.m. (C) shVC, shCSN2 and shCSN5 cells were treated with DMSO (control) or the proteasome inhibitor MG132 (10 μM) for the final 18 hours of culturing and the level of Skp-2, Cdc4, β-TrCP and β-actin protein determined by western blot. (D, E) The level of Skp2, Cdc4 and β-TrCP mRNA in shCSN2 (D) and shCSN5 (E) cells was determined at each time point post transfection relative to expression in vector control scramble cells by QRT-PCR. Data shown is the mean ± s.e.m of n = 3 transfections. * indicates a significant difference to vector controls with a p value of less than 0.05.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Western Blot, Expressing, Control, Quantitative RT-PCR

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: Both CSN2 and CSN5 knockdowns result in reduced cell growth . K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and cells re-cultured. (A) Cell counts were taken daily and the cumulative growth calculated. The cumulative growth of shCSN2 and shCSN5 cells is shown relative to shVC. The insert graph has a different Y scale to highlight the differences between shCSN2 and shCSN5 cumulative growth profiles. Data shown are the mean ± s.e.m. of n = 3. * indicates a significant difference to shVC cell growth with a p value of less than 0.05. (B) Thymidine incorporation in shCSN2 cells relative to shVC cells was measured day 3, 5 and 7 post transfection. Data shown are the mean ± s.e.m. of n = 3. (C) Thymidine incorporation in shCSN5 cells relative to shVC cells was measured day 3 and 4 post transfection. Data shown are the mean ± s.e.m. of n = 3. * indicates significance with a p value of less than 0.05.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Transfection, Plasmid Preparation, Cell Culture

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: CSN5, but not CSN2, knockdown results in apoptotic cell death . K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Binding of Annexin V and uptake of propidium iodide were analysed by flow cytometry. The lower left quadrant encompasses the viable population of cells, the lower right quadrant contains early apoptotic cells, the upper right quadrant contains late apoptotic cells and the upper left quadrant contains the necrotic cell population. Dot plots shown are representative of n = 3 transfections. The mean of three data sets was taken and the values shown in the corresponding quadrant ± s.e.m. * indicates significance with a p value of less than 0.05. (B) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of caspase-9 cleavage determined by western blot. (C) Propidium iodide uptake was determined day 6 post transfection. The histogram shown is representative of n = 3 shVC (light grey in fill), shCSN2 (dark grey line) and shCSN5 (black line). The significant shift in propidium iodide staining in shCSN2 cells is shown ± s.e.m. * indicates significance with a p value of less than 0.05.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Knockdown, Transfection, Plasmid Preparation, Cell Culture, Binding Assay, Flow Cytometry, Western Blot, Staining

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: Cells lacking CSN2, but not CSN5, are associated with autophagy . K562 cells were transiently co-transfected with HKK plasmid together with either shVC, shCSN2 or shCSN5 plasmid. HKK positive cells were sorted 24 hours post-transfection and re-cultured. (A) Jenner-Giemsa staining of shVC and shCSN2 cytospins day 6 post transfection. (B) shVC and shCSN2 cells were stained with the autophagosome marker monodansylcadaverine day 6 post transfection. All images shown are representative of n = 3 transfections. (C) Cells were harvested day 6 (shCSN2, top panels) or day 4 (shCSN5, bottom panels) post transfection and the level of LC3-II protein determined by western blot. Even loading was determined by β-actin signal. (D) Cell counts were taken daily and the cumulative growth of shVC cells and shCSN2 cells +/- 3-MA calculated. The insert has had the untreated shVC data removed in order to demonstrate the similarity between the shVC +3-MA and shCSN2 cumulative growth profiles. Data shown is the mean of n = 3 ± s.e.m. (E) Jenner-Giemsa staining of shVC +/- 1 μM bafilomycin A1 cytospins day 7 post transfection. Images shown are representative of n = 3 transfections. (F) Cells were harvested day 7 post transfection and the level of LC3-II and β-actin protein determined by western blot.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Staining, Marker, Western Blot

Journal: BMC Cell Biology

Article Title: Analysis of the role of COP9 Signalosome (CSN) subunits in K562; the first link between CSN and autophagy

doi: 10.1186/1471-2121-10-31

Figure Lengend Snippet: Loss of CSN2 results in the formation of CSN5 containing CSN subcomplexes . K562 cells were transiently co-transfected with HKK plasmid together with either shVC or shCSN2 plasmid. HKK positive cells were sorted 24 hours post-transfection, re-cultured and harvested day 3 post transfection. Both CSN2 (A) and CSN5 (B, short exposure; C, long exposure) distribution in shVC and shCSN2 cells was determined by 2-Dimensional Native-PAGE/SDS-PAGE and western blot analysis. All data shown is representative of n = 3 transfections.

Article Snippet: For western blot analysis, the following antibodies were used at 1:1000 dilution: CSN2 (Bethyl), CSN5 (Bethyl), Cul-1 (Zymed), Skp2 (Zymed), p27 (Santa Cruz), caspase-9 (Cell Signalling), LC-3 (Novus Biologicals) and β-actin (Sigma).

Techniques: Transfection, Plasmid Preparation, Cell Culture, Clear Native PAGE, SDS Page, Western Blot

Journal: PLoS ONE

Article Title: Loss of Igfbp7 Causes Precocious Involution in Lactating Mouse Mammary Gland

doi: 10.1371/journal.pone.0087858

Figure Lengend Snippet: (A) The transcriptome profiles of the Wild-Type (WT) and the Igfbp7 −/− glands on the lactation day 3 were analyzed using the RNA-Seq technique. Differentially expressed transcripts were identified by imposing a minimum of 1.5 fold change in the transcript expression level and a P-value of 0.05 or lower and are depicted on a Volcano plot. (B and C) The differentially expressed transcripts in the Igfbp7 −/− mammary epithelial cells (MECs) are categorized into the different KEGG Signaling Pathways using the DAVID Bioinformatics Resources. The fold enrichment scores are calculated based on the number of differentially regulated genes that belong to a particular KEGG signaling pathway out of the total gene set. The Benjamini statistics is used to identify the statistically significant gene enrichment for specific KEGG signaling pathways. Using this analysis the down regulated transcripts were found to be enriched in extracellular matrix receptor interaction, focal adhesion and cell adhesion molecules. The up regulated transcripts in the Igfpb7 −/− MECs showed enrichment for the WNT, TGF beta and adherens junction proteins. (D) To examine if the differentially regulated transcripts in the Igfbp7 −/− MECs show enrichment for involution-related genes, we first obtained a common involution gene signature by comparing 3 publically available transcriptome profile of involuting glands. Set 1 is from , Set 2 is from , Set 3 is from . The Ven diagram depicts a 72 gene-set that is common among all 3 data sets.

Article Snippet: The protein gels were transferred onto PVDF membranes (Millipore, Cat#IPVH00010) and the expression of specific proteins were determined using rabbit anti-Stat5a (1∶1000; Santa Cruz Biotechnology, cat#sc-1081), anti Stat3 (1∶1000; Cell Signaling Technology, cat#9139), mouse anti pStat3 (1∶2000; Cell Signaling Technology, cat#9145), anti Igf-1R (1∶1000; Cell Signaling Technology, cat#3027), and AKT (1∶1000; Cell Signaling Technology, cat#4691), anti pAKT (1∶1000; Cell Signaling Technology, cat#4060), anti Igfbp5 (1∶500; Abcam, cat#ab4255), anti Igfbp7 (1∶1000; Abcam, cat#ab74169), anti beta casein (1∶1000 BETHYL laboratories Inc. cat#IHC-00179) and anti-beta actin (1∶10,000; Sigma, cat#WH0000060M1) antibodies using chemiluminescence as per standard protocols.

Techniques: RNA Sequencing, Expressing, Protein-Protein interactions

Journal: PLoS ONE

Article Title: Loss of Igfbp7 Causes Precocious Involution in Lactating Mouse Mammary Gland

doi: 10.1371/journal.pone.0087858

Figure Lengend Snippet: To determine if Igfbp7 −/− glands exhibit molecular changes that are the hallmark of involution process we prepared protein extracts from the Wild Type (WT) or the Igfbp7 −/− glands on lactation day 3 (WT LD3, KO LD3 respectively) or from WT lactating glands were weaned for 5 days (WT Inv D5) to induce post-lactational involution. The expression of Stat5a, Stat3, phospho Stat3 (pStat3), AKT, pAKT, Igfbp5, and IGF-1R proteins was determined by Western Blots. (A, C–D) Representative Western Blots are shown. The protein expression levels for Stat5a and Stat3, AKT, Igfbp5, and IGF-1R have been normalized to beta actin expression while the expression of pStat3, pAKT have been normalized to total corresponding protein expression in each sample. The bar graphs show the average expression obtained from of 3 independent protein extracts and the statistical significance was calculated based on two-tailed t-test (*P = <0.05, **p<0.005, ***P<0.0005). As shown, lactating KO LD3 glands show elevated Stat3, pSTat3, and Igfbp5, along with decreased Stat5a, IGF-1R, and pAKT; indicating that Igbp7 −/− glands are undergoing involution. (B) Immunohistochemical staining was used to detect the expression of Stat3 in tissue sections obtained from WT or Igbp7 −/− glands on lactation days 1 or 3. As shown, expression of Stat3 can be detected as early as the first day of lactation in the Igbp7 −/− glands while WT glands show detectable expression of Stat3 .

Article Snippet: The protein gels were transferred onto PVDF membranes (Millipore, Cat#IPVH00010) and the expression of specific proteins were determined using rabbit anti-Stat5a (1∶1000; Santa Cruz Biotechnology, cat#sc-1081), anti Stat3 (1∶1000; Cell Signaling Technology, cat#9139), mouse anti pStat3 (1∶2000; Cell Signaling Technology, cat#9145), anti Igf-1R (1∶1000; Cell Signaling Technology, cat#3027), and AKT (1∶1000; Cell Signaling Technology, cat#4691), anti pAKT (1∶1000; Cell Signaling Technology, cat#4060), anti Igfbp5 (1∶500; Abcam, cat#ab4255), anti Igfbp7 (1∶1000; Abcam, cat#ab74169), anti beta casein (1∶1000 BETHYL laboratories Inc. cat#IHC-00179) and anti-beta actin (1∶10,000; Sigma, cat#WH0000060M1) antibodies using chemiluminescence as per standard protocols.

Techniques: Expressing, Western Blot, Two Tailed Test, Immunohistochemical staining, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae inhibited milk fat and protein synthesis in BMECs. (A–C) mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (D) DEGs heat maps. (E) Enrichment analysis of differentially expressed genes KEGG. (F) The protein levels of SREBP1 and β-casein were detected in BMECs. (G, H) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (I, J) mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (K) Typical images of BODIPY 493/503 staining in BMECs. * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative RT-PCR, Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: K. pneumoniae caused mitochondrial dysfunction in mammary gland and dyssynthesis of milk fat and protein. (A) Representative images of H&E staining. (B) The severity of mastitis was assessed by differences in histological score ( n = 6 cows per group) between the control and K. pneumoniae infection groups. (C–G) mRNA levels of TNF-α , IL-1β , IL-6, SREBP1 , and β-casein were detected by RT-qPCR method in mammary gland tissues (mean ± SEM, n = 6). (H) The protein levels of SREBP1, β-casein OPA1, MFN1, COX I, DRP1, and FIS1 were detected in mammary gland tissues. (I–O) Relative protein abundance of OPA1, MFN1, COX I, DRP1, FIS1, SREBP1, and β-casein were normalized to β-actin (mean ± SEM, n = 3). (P) Relative ATP levels (mean ± SEM, n = 6). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Staining, Control, Infection, Quantitative RT-PCR, Quantitative Proteomics

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: Mdivi-1 recovered K. pneumoniae -induced mitochondrial damage and dyssynthesis of milk fat and protein in BMECs. (A) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of OPA1, MFN1, COX I, DRP1, and FIS1. (B–F) Relative protein abundance of OPA1, MFN1, COX I, DRP1, and FIS1 were normalized to β-actin (mean ± SEM, n = 3). (G) Relative ATP levels (mean ± SEM, n = 3). (H) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze the protein levels of SREBP1 and β-casein. (I, J) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (K, L) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of SREBP1 and β-casein (mean ± SEM, n = 3). (M) Typical images of BODIPY 493/503 staining in BMECs. (N–P) BMECs were treated with K. pneumoniae and/or 10 μM Mdivi-1 to analyze mRNA levels of TNF-α , IL-1β , and IL-6 (mean ± SEM, n = 3). (Q) Detection of the content of LDH (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Quantitative Proteomics, Staining

Journal: Journal of Animal Science

Article Title: Klebsiella pneumoniae causes mammary gland damage via FNIP1-mediated mitochondrial dysfunction

doi: 10.1093/jas/skaf384

Figure Lengend Snippet: FNIP1 silencing alleviated K. pneumoniae -induced milk fat and protein dyssynthesis. (A) After FNIP1 silence, BMECs were infected with K. pneumoniae for 6 h to analyze the protein levels of SREBP1 and β-casein. (B, C) Relative protein abundance of SREBP1 and β-casein were normalized to β-actin (mean ± SEM, n = 3). (D, E) After FNIP1 silence, mRNA levels of SREBP1 and β-casein were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). (F) Typical images of BODIPY 493/503 staining in BMECs. (G–I) After FNIP1 silence, mRNA levels of TNF-α , IL-1β , and IL-6 were detected by RT-qPCR method in BMECs (mean ± SEM, n = 3). * P < 0.05; ** P < 0.01.

Article Snippet: The following primary antibodies were used: SREBP1 (14088-1-AP, Proteintech, Hubei, China), β-casein (HY-P81091, MCE, Shanghai, China), OPA1 (DF8587, Affinity, Jiangsu, China), MFN1 (13798-1-AP, Proteintech, Hubei, China), cytochrome c oxidase I (COX I) (DF8920, Affinity, Jiangsu, China), DRP1 (12957-1-AP, Proteintech, Hubei, China), FIS1 (10956-1-AP, Proteintech, Hubei, China), FNIP1 (28380-1-AP, Proteintech, Hubei, China), β-actin (AF7018, Affinity, Jiangsu, China).

Techniques: Infection, Quantitative Proteomics, Quantitative RT-PCR, Staining