Journal: Journal of Cellular and Molecular Medicine

Article Title: Reticulocalbin 1 is required for proliferation and migration of non‐small cell lung cancer cells regulated by osteoblast‐conditioned medium

doi: 10.1111/jcmm.17040

Figure Lengend Snippet: Information of antibodies

Article Snippet: p‐PERK(phosphoT980) , Bioss Bs−3330R , 1:1000 , 4℃ overnight.

Techniques:

Journal: BMJ Open Diabetes Research & Care

Article Title: Nicorandil attenuates high glucose-induced insulin resistance by suppressing oxidative stress-mediated ER stress PERK signaling pathway

doi: 10.1136/bmjdrc-2020-001884

Figure Lengend Snippet: Effects of nicorandil on cytoplasmic free calcium, protein kinase C (PKC)/NAPDH oxidases (NOX) activities and oxidative stress-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling activation in skeletal muscle from IR rats with diabetes. (A) The calcium indicator Fura2/AM was used to stain the cytoplasmic free calcium ([Ca 2+ ] i ) on cryostat sections of skeletal muscle tissue. The captured fluorescent images were demonstrated. Columns indicated the detected mean fluorescent intensities of Fura2/AM staining of cryostat sections of skeletal muscle tissue harvested from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (B) and (C) Columns indicated the PKC and NOX activities of skeletal muscle tissue harvested from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (D) Generated reactive oxygen species (ROS) was stained with dihydroethidium (DHE) on cryostat sections of skeletal muscle tissue. Captured images of DHE staining of each group were demonstrated. Columns indicated mean fluorescent densities of DHE staining of cryostat sections of skeletal muscle tissue harvested from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (E) and (F) Columns indicated detected malondialdehyde (MDA) and total antioxidant capacity (TAC) of skeletal muscle tissue harvested from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (G) Immunoblots of GRP78 and GAPDH were demonstrated. Columns indicated the relative expression levels of GRP78, which is the hall marker of endoplasmic reticulum stress in skeletal muscle tissue harvested from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (H) Immunoblots of phosphorylated PERK (p-PERK) and PERK were demonstrated. Columns indicated the relative phosphorylation levels of PERK in skeletal muscle tissue from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (I) Immunoblots of phosphorylated EIF2α (p-EIF2α) and EIF2α were demonstrated. Columns indicated the relative phosphorylation levels of EIF2α in skeletal muscle tissue from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. (J) Immunoblots of ATF4, CHOP, TRB3 and GAPDH were demonstrated. Columns indicated the relative expression levels of ATF4, CHOP, TRB3 and GAPDH in skeletal muscle tissue from control, STZ, STZ+N5, STZ+N10 and STZ+N15 groups, respectively. *P<0.05 (n=10). DAPI, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride; STZ, streptozotocin.

Article Snippet: After incubation with blocking buffer (Abcam), the membranes were further incubated with primary antibodies against glucose regulated protein (GRP)78 (Cell Signaling Tech, 1:2000), PERK (Cell Signaling Tech, 1:2000), phosphorylated PERK (p-PERK, Cell Signaling Tech, 1:2000), eukaryotic initiation factor (EIF)2α (Abcam, 1:2500), phosphorylated EIF2α (p-eIF2α, Abcam, 1:2500), activating transcription factor (ATF)4 (Abcam, 1:2000), CHOP (Abcam, 1:2000), TRB3 (Abcam, 1:1000), Akt (Abcam, 1:2000), phosphorylated Akt (p-Akt, Abcam, 1:2000), AS160 (Abcam, 1:1000), phosphorylated AS160 (p-AS160, Abcam, 1:1000), GLUT4 (Cell Signaling Tech, 1:2000) and GAPDH (Abcam, 1:4000) at 4°C for 10 hours.

Techniques: Activation Assay, Staining, Generated, Western Blot, Expressing, Marker

Journal: BMJ Open Diabetes Research & Care

Article Title: Nicorandil attenuates high glucose-induced insulin resistance by suppressing oxidative stress-mediated ER stress PERK signaling pathway

doi: 10.1136/bmjdrc-2020-001884

Figure Lengend Snippet: Effects of nicorandil on cytoplasmic free calcium, protein kinase C (PKC)/NAPDH oxidases (NOX) activities and oxidative stress-mediated protein kinase RNA-like endoplasmic reticulum kinase (PERK) signaling activation in high glucose incubated L6 muscle cells. (A) Fura2/AM staining was used to evaluate cytoplasmic free calcium ([Ca 2+ ] i ) in cultured L6 cells. Captured fluorescent images of Fura2/AM, 2-(4-amidinophenyl)-6-indolecarbamidine dihydrochloride (DAPI) and their overlay were demonstrated. Columns indicated the mean fluorescent intensities of Fura2/AM in each group, respectively. (B) and (C) Columns indicated the PKC and NOX activities in L6 cells of each group, respectively. (D) Intracellular production of reactive oxygen species (ROS) was evaluated by dihydroethidium (DHE) staining. Captured fluorescent images of DHE staining of L6 cells were demonstrated. Columns indicated the mean fluorescent intensities of DHE staining in L6 cells from each group. (E) and (F) Columns indicated the measured malondialdehyde (MDA) and total antioxidant capacity (TAC) in L6 cells from each group. (G) Immunoblots of GRP78 and GAPDH were demonstrated. Columns indicated the relative expression levels of GRP78 in L6 cells from each group. (H) Immunoblots of phosphorylated PERK (p-PERK) and PERK were demonstrated. Columns indicated the relative phosphorylation levels of PERK in L6 cells from each group. (I) Immunoblots of phosphorylated EIF2α (p-EIF2α) and EIF2α were demonstrated. Columns indicated the relative phosphorylation levels of EIF2α in L6 cells from each group. (J) Immunoblots of ATF4, CHOP, TRB3 and GAPDH were demonstrated. Columns indicated the relative expression levels of ATF4, CHOP, TRB3 and GAPDH in L6 cells from each group. *P<0.05 (n=10).

Article Snippet: After incubation with blocking buffer (Abcam), the membranes were further incubated with primary antibodies against glucose regulated protein (GRP)78 (Cell Signaling Tech, 1:2000), PERK (Cell Signaling Tech, 1:2000), phosphorylated PERK (p-PERK, Cell Signaling Tech, 1:2000), eukaryotic initiation factor (EIF)2α (Abcam, 1:2500), phosphorylated EIF2α (p-eIF2α, Abcam, 1:2500), activating transcription factor (ATF)4 (Abcam, 1:2000), CHOP (Abcam, 1:2000), TRB3 (Abcam, 1:1000), Akt (Abcam, 1:2000), phosphorylated Akt (p-Akt, Abcam, 1:2000), AS160 (Abcam, 1:1000), phosphorylated AS160 (p-AS160, Abcam, 1:1000), GLUT4 (Cell Signaling Tech, 1:2000) and GAPDH (Abcam, 1:4000) at 4°C for 10 hours.

Techniques: Activation Assay, Incubation, Staining, Cell Culture, Western Blot, Expressing

Journal: BMJ Open Diabetes Research & Care

Article Title: Nicorandil attenuates high glucose-induced insulin resistance by suppressing oxidative stress-mediated ER stress PERK signaling pathway

doi: 10.1136/bmjdrc-2020-001884

Figure Lengend Snippet: Schematic diagram demonstrating speculated mechanisms of nicorandil’s activity on high glucose-induced insulin resistance. Under pathological condition of hyperglycemia, glucose is uptaken into skeletal muscle cells. Part of glucose was stored as glycogen. Redundant glucose experiences mitochondrial oxidation by nicotinamide adenine dinucleotide phosphate oxidases (NOX) and produces reactive oxygen species (ROS), which further mediates endoplasmic reticulum (ER) stress. As a sensor protein localized on ER membrane, protein kinase RNA-like endoplasmic reticulum kinase (PERK) is activated by self-phosphorylation up on ER stress. Phosphorylated PERK further phosphorylates EIF2α, which trigger transcription of ATF4. As a result, TRB3 expression is upregulated which is capable of inhibiting Akt phosphorylation. Akt phosphorylates its substrate AS160, which facilitates glucose transporter (GLUT)4 translocation to plasma membrane where glucose uptake takes place. Thus, the circumstance of hyperglycemia would abate the capability of glucose uptake in skeletal muscle cells, resulting in insulin resistance. Nicorandil was potent of reducing cytoplasmic free calcium by dual mechanisms. Nicorandil opens ATP-sensitive potassium (K ATP ) channel, causing membrane hyperpolarizaiton, resulting in deactivation of VOC. Moreover, nicorandil facilities NO formation and thus decrease cytoplasmic free calcium via cGMP pathway. Decreased cytoplasmic free calcium would cause reduction of protein kinase C (PKC)/NOX activities, leading to alleviated ROS-mediated ER stress. Consequently, via PERK pathway described above, nocorandil mitigates hyperglycemia-induced insulin resistance in skeletal muscles. cGMP, cyclic guanosine monophosphate; NO, nitric oxide; VOC, voltage-operated channels.

Article Snippet: After incubation with blocking buffer (Abcam), the membranes were further incubated with primary antibodies against glucose regulated protein (GRP)78 (Cell Signaling Tech, 1:2000), PERK (Cell Signaling Tech, 1:2000), phosphorylated PERK (p-PERK, Cell Signaling Tech, 1:2000), eukaryotic initiation factor (EIF)2α (Abcam, 1:2500), phosphorylated EIF2α (p-eIF2α, Abcam, 1:2500), activating transcription factor (ATF)4 (Abcam, 1:2000), CHOP (Abcam, 1:2000), TRB3 (Abcam, 1:1000), Akt (Abcam, 1:2000), phosphorylated Akt (p-Akt, Abcam, 1:2000), AS160 (Abcam, 1:1000), phosphorylated AS160 (p-AS160, Abcam, 1:1000), GLUT4 (Cell Signaling Tech, 1:2000) and GAPDH (Abcam, 1:4000) at 4°C for 10 hours.

Techniques: Activity Assay, Expressing, Translocation Assay