gsk2606414 (Selleck Chemicals)

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Selleck Chemicals gsk2606414

a BMMs were cultured with RANKL (50 ng/mL) and various doses of thapsigargin (0, 0.05, 0.1, 0.2 nM) for 4 days, TRAP staining was conducted and TRAP-positive osteoclasts (≥3 nuclei) were counted. b , c RANKL (100 ng/mL) was used to stimulated BMMs differentiating into osteoclasts. Total proteins were extracted at different time points from 0 to 3 days of induction and western blot was performed to detect the proteins of PERK pathway. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01 versus 0 day group. d – f Thapsigargin and <t>GSK2606414</t> were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. TRAP staining was performed 4 days later and osteoclasts were counted. Total protein was extracted on day 4 and detect the protein expression of p-PERK, PERK and osteoclast differentiation marker genes. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01. TG represents thapsigargin in the figure.

Gsk2606414, supplied by Selleck Chemicals, used in various techniques. Bioz Stars score: 95/100, based on 129 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/product/3d+interactive+microscopy+visualization+software/pmc07554039-187-0-8?v=Selleck+Chemicals
Average 95 stars, based on 129 article reviews

gsk2606414 - by Bioz Stars, 2026-08

95/100 stars

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1) Product Images from "PERK controls bone homeostasis through the regulation of osteoclast differentiation and function"

Article Title: PERK controls bone homeostasis through the regulation of osteoclast differentiation and function

Journal: Cell Death & Disease

doi: 10.1038/s41419-020-03046-z

Figure Legend Snippet: a BMMs were cultured with RANKL (50 ng/mL) and various doses of thapsigargin (0, 0.05, 0.1, 0.2 nM) for 4 days, TRAP staining was conducted and TRAP-positive osteoclasts (≥3 nuclei) were counted. b , c RANKL (100 ng/mL) was used to stimulated BMMs differentiating into osteoclasts. Total proteins were extracted at different time points from 0 to 3 days of induction and western blot was performed to detect the proteins of PERK pathway. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01 versus 0 day group. d – f Thapsigargin and GSK2606414 were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. TRAP staining was performed 4 days later and osteoclasts were counted. Total protein was extracted on day 4 and detect the protein expression of p-PERK, PERK and osteoclast differentiation marker genes. Densitometric analysis of an immunoblot from three independent experiments. * p < 0.05, ** p < 0.01. TG represents thapsigargin in the figure.

Techniques Used: Cell Culture, Staining, Western Blot, Expressing, Marker

a – d BMMs were treated with PERK siRNA or small molecule inhibitor GSK2606414 (0.01, 0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days. Then, TRAP assay was performed. TRAP-positive multinucleated osteoclasts (≥3 nuclei) were counted. e – h BMMs were seeded in Osteo Aaasy Surface plate at a density of 2 × 10 4 cells/well, and cultured with 100 ng/mL RANKL and 30 ng/mL M-CSF. After mature osteoclasts were formed in each group, PERK siRNA and GSK2606414 were applied for 3 days. Bone resorption area was quantified by using Image J, Scale bar = 400 μm. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Figure Legend Snippet: a – d BMMs were treated with PERK siRNA or small molecule inhibitor GSK2606414 (0.01, 0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days. Then, TRAP assay was performed. TRAP-positive multinucleated osteoclasts (≥3 nuclei) were counted. e – h BMMs were seeded in Osteo Aaasy Surface plate at a density of 2 × 10 4 cells/well, and cultured with 100 ng/mL RANKL and 30 ng/mL M-CSF. After mature osteoclasts were formed in each group, PERK siRNA and GSK2606414 were applied for 3 days. Bone resorption area was quantified by using Image J, Scale bar = 400 μm. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Techniques Used: TRAP Assay, Cell Culture

BMMs were cultured with RANKL (100 ng/mL) and various concentrations of GSK2606414 (0.05, 0.1 nM) for 4 days, and actin ring fluorescence staining was performed. a , b F-actin ring fluorescence images were acquired and quantified. Scale bar = 400 μm, Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Figure Legend Snippet: BMMs were cultured with RANKL (100 ng/mL) and various concentrations of GSK2606414 (0.05, 0.1 nM) for 4 days, and actin ring fluorescence staining was performed. a , b F-actin ring fluorescence images were acquired and quantified. Scale bar = 400 μm, Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Techniques Used: Cell Culture, Fluorescence, Staining

a – c BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 2 or 4 days. p-PERK, PERK and osteoclast-related marker genes proteins expression were measured by western blot at the indicated times and mRNA levels were determined by RT-PCR. d – g BMMs were starved with a-MEM in the absence of FBS for 12 h, pretreated with or without GSK2606414 for 2 h. After that, BMMs were treated with or without RANKL (100 ng/mL) for the indicated times. The total proteins were extracted and western blot was conducted to detect the expression of NF-κB and MAPK signaling pathways. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Figure Legend Snippet: a – c BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 2 or 4 days. p-PERK, PERK and osteoclast-related marker genes proteins expression were measured by western blot at the indicated times and mRNA levels were determined by RT-PCR. d – g BMMs were starved with a-MEM in the absence of FBS for 12 h, pretreated with or without GSK2606414 for 2 h. After that, BMMs were treated with or without RANKL (100 ng/mL) for the indicated times. The total proteins were extracted and western blot was conducted to detect the expression of NF-κB and MAPK signaling pathways. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Techniques Used: Marker, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Protein-Protein interactions

a , b Micro-CT images of the distal femoral metaphyseal region from the SHAM, SHAM + GSK2606414, OVX, and OVX + GSK2606414 groups. Histograms represent the trabecular structural parameters of the distal femur: BV/TV, Tb.N, Tb.Th and Tb.Sp. Data are presented as means ± SEM, n = 8–10 mice per group. * p < 0.05 versus the OVX group. c , d TRAP and H&E staining were performed on sections of distal femurs. N.Oc/BS of the TRAP staining were calculated. Scale bar (40×) = 1000 μm, Scale bar (100×) = 400 μm. Data are presented as means ± SEM, n = 8–10 mice per group. ** p < 0.01 versus the OVX group.

Figure Legend Snippet: a , b Micro-CT images of the distal femoral metaphyseal region from the SHAM, SHAM + GSK2606414, OVX, and OVX + GSK2606414 groups. Histograms represent the trabecular structural parameters of the distal femur: BV/TV, Tb.N, Tb.Th and Tb.Sp. Data are presented as means ± SEM, n = 8–10 mice per group. * p < 0.05 versus the OVX group. c , d TRAP and H&E staining were performed on sections of distal femurs. N.Oc/BS of the TRAP staining were calculated. Scale bar (40×) = 1000 μm, Scale bar (100×) = 400 μm. Data are presented as means ± SEM, n = 8–10 mice per group. ** p < 0.01 versus the OVX group.

Techniques Used: Micro-CT, Staining

a , b After BMMs were stimulated by RANKL (100 ng/mL) for 0, 1, 2 and 3 days, total proteins were extracted to detect the levels of Beclin1 and LC3B. * p < 0.05, ** p < 0.01 versus 0 day group. c , d Thapsigargin and GSK2606414 were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. Total proteins were extracted on the 4th day and western blot was performed to examine the expression of autophagy-related proteins Beclin1 and LC3B. TG represents thapsigargin in the figure. e , f BMMs were treated with GSK2606414 (0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days, total proteins were extracted and western blot was used to verify the expression of Beclin1 and LC3B. ( g ) C57BL/6 mice were given GSK2606414 (50 mg/kg) by intragastric gavage every 2 days after ovariectomy. Six weeks later, femurs of mice were collected and sectioned, subjected to LC3 immunohistochemical staining. Scale bar = 400 μm, Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Figure Legend Snippet: a , b After BMMs were stimulated by RANKL (100 ng/mL) for 0, 1, 2 and 3 days, total proteins were extracted to detect the levels of Beclin1 and LC3B. * p < 0.05, ** p < 0.01 versus 0 day group. c , d Thapsigargin and GSK2606414 were added to osteoclast-induced differentiation medium containing RANKL (50 ng/mL), respectively. Total proteins were extracted on the 4th day and western blot was performed to examine the expression of autophagy-related proteins Beclin1 and LC3B. TG represents thapsigargin in the figure. e , f BMMs were treated with GSK2606414 (0.05, 0.1 μM) in the presence of RANKL (100 ng/mL) and M-CSF (30 ng/mL) for 4 days, total proteins were extracted and western blot was used to verify the expression of Beclin1 and LC3B. ( g ) C57BL/6 mice were given GSK2606414 (50 mg/kg) by intragastric gavage every 2 days after ovariectomy. Six weeks later, femurs of mice were collected and sectioned, subjected to LC3 immunohistochemical staining. Scale bar = 400 μm, Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Techniques Used: Western Blot, Expressing, Immunohistochemical staining, Staining

a , b After infecting BMMs with mRFP-GFP-LC3 tandem fluorescent protein adenovirus, GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs. On the 4th day, the fluorescence was observed with a confocal microscope and quantitative analysis was performed. Scale bar = 10 μm. c GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs for 2 days, the formation of autophagosomes in BMMs was observed under transmission electron microscopy. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Figure Legend Snippet: a , b After infecting BMMs with mRFP-GFP-LC3 tandem fluorescent protein adenovirus, GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs. On the 4th day, the fluorescence was observed with a confocal microscope and quantitative analysis was performed. Scale bar = 10 μm. c GSK2606414 and RANKL (100 ng/mL) were used to treat BMMs for 2 days, the formation of autophagosomes in BMMs was observed under transmission electron microscopy. Data are presented as means ± SD of 3 independent experiments; ** p < 0.01.

Techniques Used: Fluorescence, Microscopy, Transmission Assay, Electron Microscopy

a BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) for 4 days, and DCFH-DA probe was used to detect intracellular ROS levels. Scale bar = 200 μm. b , c RANKL (100 ng/mL) and NAC were used to stimulate BMMs, after 4 days, the total proteins were extracted and western blot was conducted to test the expression of PERK and its phosphorylation level. d , e BMMs were treated with NAC and PERK activator CCT020312 in the presence of RANKL (100 ng/mL) for 4 days, total proteins were extracted and Western Blot was performed to examine the expression of osteoclast-related proteins NFATc1, c-fos and Autophagy-related proteins Beclin1, LC3B. f The schematic model of the hypothesized mechanism by which PERK inhibition affects osteoclast differentiation. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Figure Legend Snippet: a BMMs were treated with GSK2606414 (0.1 μM) in the presence of RANKL (100 ng/mL) for 4 days, and DCFH-DA probe was used to detect intracellular ROS levels. Scale bar = 200 μm. b , c RANKL (100 ng/mL) and NAC were used to stimulate BMMs, after 4 days, the total proteins were extracted and western blot was conducted to test the expression of PERK and its phosphorylation level. d , e BMMs were treated with NAC and PERK activator CCT020312 in the presence of RANKL (100 ng/mL) for 4 days, total proteins were extracted and Western Blot was performed to examine the expression of osteoclast-related proteins NFATc1, c-fos and Autophagy-related proteins Beclin1, LC3B. f The schematic model of the hypothesized mechanism by which PERK inhibition affects osteoclast differentiation. Densitometric analysis of an immunoblot from three independent experiments; * p < 0.05, ** p < 0.01.

Techniques Used: Western Blot, Expressing, Phospho-proteomics, Inhibition

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1) Product Images from "PERK controls bone homeostasis through the regulation of osteoclast differentiation and function"

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