Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: HIF-1α is a negative regulator of interferon regulatory factors: Implications for interferon production by hypoxic monocytes

doi: 10.1073/pnas.2106017118

Figure Lengend Snippet: Schematic representation of HIF-1α’s role in hypoxic inflammation. HMGB1-exposed primary monocytes release proinflammatory cytokines and type I IFN through activating NF-κB and IRF3 and 5 ( Left ). In hypoxic conditions or following DMOG treatment only NF-κB is activated as HIF-1α represses IRF3 and IRF5 ( Right ). Thus, increased HIF-1α leads to production of proinflammatory cytokines but not IFN.

Article Snippet: Dual-reporter promoter clone for IRF3 promoter was purchased from GeneCopoeia (HPRM38174-pEZX-PG04).

Techniques:

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Stearic acid cooperative effect with TNF-α for MIP-1α/CCL3 production requires IRF3. ( A ) THP-1 monocytic cells were transfected with either control or IRF3 siRNA and incubated for 36 h. Real time PCR was done to measure IRF3 expression. ( B , C ) IRF3 deficient THP-1 cells were stimulated with stearic acid and TNF-α. MIP-1α/CCL3 expression was determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. ( D ) IRF3 activity reporter monocytic cells were treated with stearic acid (200 µM) or 0.1% BSA (control) or TNF-α (10 ng/mL) or in combination. Culture media were collected after 24 h. Cell culture media were assayed for luciferase activity representing the degree of IRF3/ISRE activation using Quanti-Luc medium. ( E ) Western blot analysis showed that stearic acid induced IRF3 phosphorylation in a time dependent manner in THP-1 monocytes, verifies the role of IRF3 in the cooperative effect of stearic acid in the TNF-α mediated production of MIP-1α/CCL3. ( F ) Expression of phosphorylated IRF3 is shown as determined by densitometry of western blot bands. * P < 0.05; ** P < 0.01; *** P < 0.001.

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Control, Incubation, Real-time Polymerase Chain Reaction, Expressing, Activity Assay, Cell Culture, Luciferase, Activation Assay, Western Blot, Phospho-proteomics

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Polyinosinic-polycytidylic acid (poly I:C) act as a substitute of stearic acid cooperative effect on MIP-1α/CCL3 production. ( A , B ) THP-1 cells were treated (via transfection) with poly I:C (5 µg) for 2 h and then incubated with BSA (control) or stearic acid or TNF-α for 24 h. MIP-1α/CCL3 mRNA and protein were determined. The results obtained from minimum three independent experiments with three replicates of each experiment are shown. All data are expressed as mean ± SEM ( n ≥ 3); * P < 0.05; ** P < 0.01, when compared with stearic acid or TNF-α alone. ( C , D ) Western blot analysis showed that individuals with obesity have significant higher levels of phospho-IRF3 in monocytes compared to lean individuals ( P = 0.0179).

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Transfection, Incubation, Control, Western Blot

Journal: Biomedicines

Article Title: Stearic Acid and TNF-α Co-Operatively Potentiate MIP-1α Production in Monocytic Cells via MyD88 Independent TLR4/TBK/IRF3 Signaling Pathway

doi: 10.3390/biomedicines8100403

Figure Lengend Snippet: Schematic illustration of signaling pathways underlying the cooperative relationship between stearic acid and TNF-α for MIP-1α/CCL3 production. Blocking TLR4/IRF3 signaling pathways significantly suppress the cooperative production of MIP-1α/CCL3 by stearic acid/TNF-α. TLR: Toll like receptor; TNF-α: Tumor necrosis factor α; MyD88: Myeloid differentiation factor 88; IRAK: Interleukin-1 receptor-associated kinase; TRIF: TIR domain–containing adapter-inducing IFN-β; IRF3: Interferon regulatory factor-3; TBK: TANK-binding kinase 1; TRAM: Toll-receptor-associated molecule; TRAF6: tumor-necrosis factor receptor-associated factor 6: IKK-Iκ-B kinase; NF-kB: Nuclear factor-κB; TRADD: Tumor necrosis factor receptor 1-associated death domain protein; RIP: receptor-interacting protein; NEMO: NF-kappa-B essential modulator. BioRender.com was used for .

Article Snippet: TLR4 (ID 7099), Trilencer-27 Human siRNA (SR322051), scrambled (control) siRNA (SR30004), and IRF3 (cat#: ID 3661) Trilencer-27 Human siRNA (SR320690) were obtained from OriGene (Rockville, MD, USA).

Techniques: Protein-Protein interactions, Blocking Assay, Binding Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 1. IRF3 expression decreases in the hepatocytes of obese mice. WT mice were fed an NC diet or HFD for 26 weeks and ob/ob mice were fed the NC diet for 9 weeks. (A) Real-time PCR analysis of IRF3 mRNA levels in the liver (n 5 12 in each group). (B) Protein expression of IRF3 in the liver as detected by western blotting. Protein expression levels were normalized to a glyceraldehyde-3-phosphate dehydrogenase (GAPDH) loading control. All values are shown as the mean 6 SEM (n 5 3 per group). The statistical significance is relative to the WT NC-fed group, **P < 0.01; or the lean group, ##P < 0.01.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Expressing, Real-time Polymerase Chain Reaction, Western Blot, Control

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 2. IRF3 deficiency impairs hepatic insulin sensitivity in mice fed an HFD. (A) Fasting blood glucose levels of 6-hour fasted WT or KO mice were measured every 4 weeks during the 26 weeks of feeding. (B) The serum fasting insulin levels were determined every 8 weeks by enzyme- linked immunosorbent assay (ELISA) (n 5 5-13 per group at each timepoint). (C) The HOMA-IR index was calculated as [FBG (mmol/l) 3 Fins (mIU/l)]/22.5 (n 5 4-10 per group at each timepoint). (D) A GTT (1 g/kg body weight, glucose intraperitoneal injection) was performed on WT and KO mice fed both the NC diet and the HFD for 24 weeks. The corresponding area under the curve (AUC) of the blood glucose levels in each group was calculated (n 5 12-16 per group). (E) An ITT (0.75 units/kg body weight, insulin i.p. injection) was performed on the WT and KO mice in both the NC and HFD groups during the 25th week of feeding. The corresponding AUC for the blood glucose levels in each group was calculated (n 5 13-17 per group). (F) An immunoblot analysis measured systemic insulin resistance in the KO mice and detected the marked activation of IRS1-ser636/ser639 phosphorylation and the inhibition of IRS1-Tyr608 and Akt phosphorylation in insulin target organs. Right panel: Phosphorylated protein expression levels were normalized to GAPDH (n 5 4 per group). All values are expressed as the mean 6 SEM. Statistical significance is relative to the WT HFD group, #P < 0.05, ##P < 0.01, or the WT NC group, *P < 0.05, **P < 0.01.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Enzyme-linked Immunosorbent Assay, Injection, Western Blot, Activation Assay, Phospho-proteomics, Inhibition, Expressing

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 3. IRF3 deficiency promotes HFD-induced hepatic steatosis. (A) Comparison of body weight gains in the WT or IRF3 KO mice fed an NC diet or HFD for 24 weeks (n 5 28-39 per group). (B) Macroscopic pictures of livers from WT or IRF3 KO mice fed the HFD. The liver weights and the liver-to-body weight ratio (n 5 28-39 per group) are shown in bar graphs. (C) Representative liver sections stained with H&E (upper panel) and Oil red O (lower panel) from WT or KO mice fed an NC diet or HFD for 26 weeks. The scale bar denotes 100 lm. (D) Liver function was evaluated by detecting serum levels of ALT, AST, and ALP (n 5 6-7 per group). (E) TG, cholesterol, and FFA levels were measured in liver tissues of WT and KO mice fed the HFD (n 5 5 per group). (F) TG, cholesterol, FFA, LDL-C, and HDL-C serum levels were also measured (n 5 5-16 per group). All values are expressed as the mean 6 SEM. The statistical significance is relative to the WT HFD group, #P < 0.05, ##P < 0.01, or the WT NC group, **P < 0.01.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Comparison, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 4. Hepatic IRF3 overexpression preserves glucose and lipid homeostasis. (A) The body weights, liver weights, and liver-to-body weight ratios of WT mice fed the HFD were examined. These mice were injected with an IRF3 adenovirus or a GFP adenovirus control (n 5 12 per group). (B) The fasting blood glucose levels were measured after the mice were fasted for 6 hours, and the serum fasting insulin levels were determined by ELISA. The HOMA-IR index was also calculated (n 5 5-6 per group). (C) A GTT (1 g/kg body weight, glucose intraperitoneal injec- tion) was performed on the HFD-fed WT mice injected with an IRF3 adenovirus or a vector control virus. The corresponding AUC of the blood glu- cose levels in each group was calculated (n 5 12 per group). (D) An ITT (0.75 units/kg body weight, insulin intraperitoneal injection) was also performed, and the corresponding AUC of the blood glucose levels in each group was calculated (n 5 10-12 per group). (E) Left panel: Immu- noblotting analysis indicated that insulin resistance was reversed in mice with IRF3 liver-specific overexpression; these animals also displayed marked upregulation of the phosphorylation of IRS1-Tyr608 and Akt in the liver. Right panel: Phosphorylated protein expression levels were nor- malized to GAPDH (n 5 4 per group). (F) Representative liver sections stained with H&E (top panel) and Oil red O (lower panel) from IRF3- overexpressing and vector control mice fed the HFD. (G) The levels of TG, cholesterol, and FFA in liver tissue were measured (n 5 5 per group). (H) Liver function was evaluated by measuring serum levels of ALT, AST, and ALP (n 5 6 per group). All values are expressed as the mean 6 SEM. The statistical significance is relative to the GFP adenovirus-injected HFD group, #P < 0.05, ##P < 0.01; or the GFP adenovirus-injected NC group, **P < 0.01.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Over Expression, Injection, Control, Enzyme-linked Immunosorbent Assay, Plasmid Preparation, Virus, Phospho-proteomics, Expressing, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 5. IRF3 downregulates IKKb/NF-jB signaling in hepatocytes. (A) The serum levels of cytokines of the IRF3 KO and WT mice fed the HFD for 24 weeks (n 5 8-16 per group) were compared. The mRNA levels of the proinflammatory and antiinflammatory markers in IRF3 KO and WT livers (B) and GFP virus-infected and IRF3 virus-infected livers (C) were measured by real-time PCR (n 5 6-12 for each group). In (A,B), data were compared with the results of the WT HFD group, #P < 0.05, ##P < 0.01, or the WT NC group, **P < 0.01. In (C), data were compared with the results of the GFP adenovirus-injected HFD group, *P < 0.05. (D,E) IKKb/NF-jB signaling in the liver was evaluated by western blotting for IKKb-ser181, IjBa, and p65 phosphorylation. Protein expression levels were normalized to GAPDH (n 5 4 per group). All values are expressed as the mean 6 SEM. In (D), the statistical significance is relative to the WT NC group, *P < 0.05, the IRF3 KO NC group, †P < 0.05, or the WT HFD group, #P < 0.05. In (E), the statistical significance is relative to the GFP adenovirus-injected NC group, *P < 0.05, the IRF3 adenovirus-injected NC group, †P < 0.05, or the GFP HFD group, #P < 0.05. (F) In the left panel, primary cultured hepatocytes were transfected with a jB-luciferase construct infected with shRNA or shIRF3 adenovirus and treated with PBS or high levels of glucose. In the right panel, GFP or IRF3 adenovirus was used to infect hepatocytes. Luciferase activity was assayed as a measure of NF-jB activity (n 5 4); ##P < 0.01 versus AdshRNA group (left panel) or AdGFP group (right panel).

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Virus, Infection, Real-time Polymerase Chain Reaction, Injection, Western Blot, Phospho-proteomics, Expressing, Cell Culture, Transfection, Luciferase, Construct, shRNA, Activity Assay

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 6. Hepatic IjBa overexpression reverses HFD-induced insulin resistance and steatosis in IRF3 KO mice. (A) Adenovirus-mediated hepatic IjBa overexpression was confirmed by western blotting. The protein levels of IjBa and phosphorylated p65 were quantified and normalized to GAPDH (n 5 4 per group). (B) The mRNA levels of proinflammatory and antiinflammatory markers in IjBa-overexpressing and GFP control livers were determined by real-time PCR (n 5 9-12 per group). (C) Fasting blood glucose levels were detected after the mice were fasted for 6 hours and serum fasting insulin levels were determined using ELISA kits. The HOMA-IR index was also calculated (n 5 6-7 per group). (D) A GTT (1 g/kg body weight, glucose intraperitoneal injection) was performed on HFD-fed WT mice injected with an IjBa adenovirus or GFP control virus, and the corresponding AUC of blood glucose levels in each group was calculated (n 5 10-11 per group). (E) An ITT (0.75 units/kg body weight, insulin intraperitoneal injection) was also performed, and the corresponding AUC of the blood glucose levels in each group was calculated (n 5 10 per group). (F) Immunoblotting analyses indicated reversed insulin resistance in mice with IjBa liver-specific overexpression and marked up- regulation of the phosphorylation of IRS1-Tyr608 and Akt in the liver. Phosphorylated protein expression levels were normalized to GAPDH (n 5 4 per group). (G) Liver weights and the liver-to-body weight ratios (n 5 10-11 per group). (H) Representative liver sections stained with H&E (upper panel) and Oil red O (lower panel) from IjBa-overexpressing and GFP control mice fed an NC diet or HFD. (I) TG, cholesterol and FFA lev- els in liver tissue (n 5 5 per group). (J) Liver function was evaluated by measuring serum levels of ALT, AST, and ALP (n 5 7 per group). All val- ues are expressed as the mean 6 SEM; *P < 0.05, **P < 0.01 versus the GFP adenovirus-injected NC group; #P < 0.05, ##P < 0.01 versus the AdGFP HFD group; †P < 0.05 versus the AdIjBa NC group.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Over Expression, Western Blot, Control, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Injection, Virus, Phospho-proteomics, Expressing, Staining

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 7. IRF3 interacts with IKKb to constrain IKKb/NF-jB signaling. (A) GST pull-down assays of FLAG-tagged IKKb with GST or GST-IRF3 are shown. (B) Co-IPs of IRF3 and IKKb are shown. The cell lysates from 293T cells transfected with EGFP-myc-tagged IRF3 and FLAG-tagged IKKb were prepared and subjected to IP with an myc antibody or IgG and analyzed by immunoblotting using myc or FLAG antibodies. These lysates were also subjected to IP using a FLAG antibody and immunoblotted using myc or FLAG antibodies. (C) Colocalization of IRF3 and IKKb in the cytoplasm is shown. pCherry-IKKb and pEGFP-IRF3 were transfected into 293T cells. The nuclei were stained with DAPI, and the cells were visualized by confocal microscopy. (D) Upper panel: a schematic representation of the four IRF3 deletion mutants is shown. Lower panel: map- ping of the IKKb-binding region of IRF3. The cell lysates from 293T cells transfected with FLAG-tagged IKKb and EGFP-myc-tagged IRF3 deletion mutants were immunoprecipitated with anti-FLAG followed by immunoblotting with anti-FLAG and anti-myc. (E) Upper panel: schematic represen- tation of the two IKKb deletion mutants. Lower panel: mapping of the IRF3 binding region of IKKb. 293T cells were transfected with EGFP-myc- tagged IRF3 and FLAG-tagged IKKb deletion mutants.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Transfection, Western Blot, Staining, Confocal Microscopy, Binding Assay, Immunoprecipitation

Journal: Hepatology (Baltimore, Md.)

Article Title: Interferon regulatory factor 3 constrains IKKβ/NF-κB signaling to alleviate hepatic steatosis and insulin resistance.

doi: 10.1002/hep.26751

Figure Lengend Snippet: Fig. 8. Schematic diagram: IRF3 constrains IKKb/NF-jB signaling to mitigate hepatic metabolic disorders. (A) In hepatocytes of normal livers, IRF3 interacts with the kinase domain of IKKb in the cytoplasm, blocking IKKb phosphorylation and its downstream signaling. The phosphoryla- tion and degradation of IjBa and the subsequent translocation of NF-jB into the nucleus are repressed. Thus, NF-jB-dependent inflammatory gene transcription is inhibited. (B) In hepatocytes of a fatty liver, which can be induced by a chronic HFD for instance, IRF3 levels are decreased in the cytoplasm. In the absence of IRF3 binding, IKKb is more readily phosphorylated and capable of relaying inflammatory signals to down- stream mediators. The degradation of IjBa frees NF-jB to translocate into the nucleus to induce inflammatory gene expression. (C) Inflamma- tion, insulin resistance, and nutrient overload form a vicious cycle. By constraining IKKb/NF-jB signaling, IRF3 preserves insulin sensitivity and prevents hepatic steatosis.

Article Snippet: Additional Supporting Information may be found in the online version of this article. used replication-defective adenoviral vectors containing all or a portion of the coding region of FLAG-tagged IRF3 (obtained from OriGene) and FLAG-tagged IjBa (S32A/S36A; ordered from Seajet Scientific) under the control of the cytomegalovirus promoter.

Techniques: Blocking Assay, Phospho-proteomics, Translocation Assay, Binding Assay, Gene Expression

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: Rab6a-mediated Golgi biogenesis after BFA-WO. ( A ) Giantin (top panel) and NMIIA (bottom panel) W-B of complexes pulled down with anti-Rab6a Ab from lysates of LNCaP cells treated with BFA for 60 min and then 30 min WO. Amounts of lysates used for IP were normalized to giantin or NMIIA, as described in . Non-specific rabbit IgG was used for control IP. Red and blue markers indicate BFA and BFA-WO samples, respectively. ( B ) Confocal immunofluorescence images of Rab6a colocalization with giantin and NMIIA in LNCaP BFA-WO cells. ( C ) Quantification of Pearson’s overlap coefficient for indicated pairs of stained proteins in cells after BFA-WO. All confocal images were acquired with the same imaging parameters; bars, 10 μm. Data collected from 90 cells of three independent experiments, results are expressed as a mean ± SD; * p < 0.001. ( D ) Giantin and Rab6a W-B of the postnuclear supernatant (PNS) and Golgi membranes collected from the HeLa cells: control and treated with BFA. ( E ) The representative 3D reconstruction of the SIM imaging of Golgi membranes isolated from HeLa cells: non-treated, and treated with BFA followed by incubation with or without Rab6a protein. The regenerated Golgi membranes were stained with giantin; bars, 2 µm. ( F ) Quantification of the average length of Golgi membranes ( n = 30 from each sample) presented in ( E ). ( G ) Top panel: the reconstructed 3D SIM imaging of Golgi in HeLa cells after 30 min of BFA-WO. Cells were stained with Rab6a (green) and giantin (red). The representative area of Golgi is presented in the bottom panel. Note the Rab6a punctae between giantin-positive emerging Golgi membranes. ( H ) PLA in HeLa cells treated with DMSO, BFA, and BFA-WO. The proximity of giantin and Rab6a was evaluated using mouse anti-giantin and rabbit anti-Rab6a Abs. Red punctae indicate PLA signal, nucleus is in blue, DAPI; bars, 10 μm. ( I ) Quantitation of proximity ligation for Rab6a and giantin is presented as the corrected total fluorescence intensity (a.u.). The results are measured as a mean ± SD; * p < 0.001.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: Immunofluorescence, Staining, Imaging, Isolation, Incubation, Quantitation Assay, Ligation, Fluorescence

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: The overlap of giantin and Rab6a during Golgi biogenesis. ( A ) Confocal immunofluorescence images of Rab6a in HeLa cells after 60 min of BFA-WO, pretreated with scramble, giantin, GM130, or GRASP65 siRNAs. All confocal images acquired with the same imaging parameters; bars, 10 μm. ( B ) Quantification of cells with membranous Rab6a in cells presented in ( A ); n = 90 cells from three independent experiments, results are expressed as a mean ± SD; * p < 0.001. ( C ) Giantin immunostaining in DMSO- and BFA-treated HeLa cells. ( D ) Giantin immunostaining in HeLa cells after 60 min of BFA-WO, transfected with scramble, Rab6a siRNAs, and dominant-negative (GDP-bound) Rab6a(T27N). ( E ) Rab6a W-B of lysates of HeLa cells treated with corresponding siRNAs; β-actin was a loading control. ( F ) Quantifications of cells with perinuclear Golgi in cells from ( C , D ); n = 90 cells from three independent experiments, results expressed as a mean ± SD; *, p < 0.001.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: Immunofluorescence, Imaging, Immunostaining, Transfection, Dominant Negative Mutation

Journal: Cells

Article Title: Post-ER Stress Biogenesis of Golgi Is Governed by Giantin

doi: 10.3390/cells8121631

Figure Lengend Snippet: Schematic illustrations of Golgi biogenesis. The fusion of Golgi membranes (highlighted below) is presumably initiated by tethering of giantin from the rim of one cisterna to the rim of opposite cisterna via disulfide bond in the luminal domain. Interaction of gaintin and Rab6a via their N-terminus may provide twisting of giantin monomers required for the coiled-coil dimeric structure. Such fusion requires the force presumably created by the action of F-actin based motor protein, non-muscle Myosin IIB.

Article Snippet: Rab6a–pCMV3–C–OFPSpark (RFP tag) was ordered from Sino Biological.

Techniques: