Journal: Journal of Cellular and Molecular Medicine

Article Title: Progranulin Protects Against Osteoporosis by Regulating Osteoclast and Osteoblast Balance via TNFR Pathway

doi: 10.1111/jcmm.70385

Figure Lengend Snippet: PGRN promoted osteoblast differentiation via interacting with TNFR2. (A) Representative images of ALP staining of MC3T3‐E1 cells co‐cultured with PGRN for osteogenic induction for 14 days. Scale bars, 100 μm. (B) Quantification of ALP staining analysis ( n = 3 for each group). (C) Western blot analysis of RUNX2 of the indicated groups after cultured for 3 days. (D) Quantification of WB analysis ( n = 3 for each group). (E, F) Real‐time PCR of RUNX2 of the indicated groups ( n = 3 for each group) after cultured for 8 h. (G) Western blot (WB) analysis of p‐Erk1/2, Erk1/2, p‐JNK and JNK of MC3T3‐E1 cells cultured with PGRN. (H) WB analysis of TNFR2 to detect the knockdown efficacy of TNFR2 RNAi in MC3T3‐E1 cells after cultured for 3 days. (I) Representative images of ALP staining of MC3T3‐E1 cells co‐cultured with PGRN and TNFR2 RNAi for osteogenic induction for 14 days. Scale bars, 100 μm. (J) Quantification of ALP staining analysis ( n = 3 for each group). (K) Representative images of ALP staining of BMSCs co‐cultured with PGRN and TNFR2 RNAi for osteogenic induction for 14 days. Scale bars, 100 μm. (L) Quantification of ALP staining analysis ( n = 3 for each group). (M) Representative images of Alizarin Red S staining of MC3T3‐E1 cells co‐cultured with PGRN and TNFR2 RNAi for osteogenic induction for 21 days. (N) Quantitative analysis of Alizarin Red S staining was detected by relative amounts of Alizarin Red S ( n = 3 for each group). (O) Western blot analysis of RUNX2 of the indicated groups after stimulated for 3 days. (P) Quantification of WB analysis ( n = 3 for each group). (Q, R) Real‐time PCR of RUNX2 and Col‐1 of the indicated groups ( n = 3 for each group) after cultured for 8 h. (S) Immunoprecipitation (IP) with anti‐PGRN or control IgG followed by Western blotting for TNFR2 ( n = 3). IB: Immunoblot. (T) Western blot analysis of RUNX2, after MC3T3‐E1 cells cultured with SCH772984 (5 μM) and PGRN for 3 days. (U) Quantification of WB analysis ( n = 3 for each group). (V) Real‐time PCR of RUNX2 of the indicated groups ( n = 3 for each group) after cultured for 8 h. Concentration: TNFα (10 ng/mL), rhPGRN (500 ng/mL), SCH772984 (5 μM). Data were presented as the mean ± SD. ** p < 0.01.

Article Snippet: To further investigate the involvement of the ERK signalling pathway, ERK inhibitor SCH772984 (5 μM) (MCE, USA) [ ] was added to MC3T3‐E1 cells with or without rhPGRN during osteoblast differentiation.

Techniques: Staining, Cell Culture, Western Blot, Real-time Polymerase Chain Reaction, Knockdown, Immunoprecipitation, Control, Concentration Assay

Journal: Oncology Letters

Article Title: Induction of entosis in prostate cancer cells by nintedanib and its therapeutic implications

doi: 10.3892/ol.2019.9951

Figure Lengend Snippet: Decreased CDC42 induces entosis by promoting the ROCK1/2 signaling pathway and E-cadherin expression in Pca cells (×400 magnification). (A) No entosis morphology or increased ROCK1/2 or E-cadherin expression was observed in Pca cells treated with Akt inhibitor (MK2206; 10 µM) or ERK1/2 inhibitor (SCH772984; 3 µM) for 4 weeks, respectively. (B) IC 50 values of the CDC42 inhibitor ML141 in the Pca cell lines. (C) Entosis-like morphology of Pca cells under ML141 pressure (2 µM for 4 weeks). (D) siRNA knockdown of CDC42 expression at the mRNA and protein level. *P<0.05 vs. control (Student's t-test) (E) CDC42 siRNA and ML141 (2 µM) promote ROCK1/2 expression and E-cadherin expression (following treatment for 1 week). *P<0.05 and **P<0.01 vs. control (one-way analysis of variance followed by Fisher's least-significant difference test). CDC42, cell division cycle 42; ROCK, Rho kinase; Pca, prostate cancer; Akt, protein kinase B; ERK, extracellular-signal-regulated kinase; IC 50 , half-maximal inhibitory concentration; siRNA, short interfering RNA; E-cadherin, epithelial cadherin; C, control; T, treatment; M, ML141 treatment; S/Si, siRNA treatment; Sc, scrambled.

Article Snippet: All drugs were purchased from Selleck Chemicals (Houston, TX, USA), and Pca cells were treated at the following concentrations: Nintedanib (3 µM for 4 weeks to develop resistance), PI3K inhibitor buparlisib (1.5 µM for 4 weeks) , protein kinase B (Akt) inhibitor MK2206 (10 µM for 4 weeks) , extracellular-signal-regulated kinase (ERK)1/2 inhibitor SCH772984 (3 µM for 4 weeks) , CDC42 inhibitor ML141 (2 µM for 4 weeks) , or ROCK1/2 inhibitor Y-27632 (10 µM for 1 week) ( ).

Techniques: Expressing, Concentration Assay, Small Interfering RNA

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Analysis of BTZ effects on NRF1 by two different antibodies. The c-19 antibody was used for further experiments in this study because it readily revealed all three forms of NRF1 (glycosylated, full length, and processed). B . A phosphorylated peptide that included the predicted ERK1/2 motif PYSP was identified by mass spectrometry at the C terminal of NRF1 (see text). C . In HEK293-NRF1 3×FLAG cells, inhibition of P97/VCP ATPase by the P97 inhibitors DBeQ and NMS-873 results in accumulation of the glycosylated but not further processed forms of NRF1. By contrast, ERK1/2 inhibition with SCH772984 does not disrupt NRF1 processing. D . Western blot detection of FLAG-tagged NRF1 by NRF1 antibody in HEK293-NRF1 3×FLAG cells. E . Colloidal blue staining of the co-immunoprecipitation sample that was analyzed by mass spectrometry. F . Quantification of NRF1 nuclear localization in the experiments shown in and , *p<0.05.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Mass Spectrometry, Inhibition, Western Blot, Staining, Immunoprecipitation

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Interaction between NRF1 and ERK1/2. Co-immunoprecipitation from HEK293 cells revealed a physical interaction between ERK1/2 and NRF1 3XFLAG that is enhanced by BTZ and inhibited by SCH772984. B . Dependence of NRF1 serine phosphorylation on ERK1/2. Immunoprecipitation from HEK293 cells showing that phosphorylation of NRF1 3XFLAG was blocked by SCH772984. C . BTZ-induced nuclear localization of NRF1 is dependent upon ERK1/2. A western blot shows that treatment decreases representation of NRF1 in the nuclear fraction. D . Immunofluorescence staining showing that SCH772984 inhibits BTZ-induced accumulation of NRF1 3XFLAG protein in HEK293 cell nuclei, which are labeled with DAPI. Quantification is shown in Figure S4F. Scale bar, 10 µm. E . The phosphosite S734A is required for NRF1 nuclear localization. Immunofluorescence staining and analysis was performed as in D. Scale bar, 10 µm. F . Diagram indicating phosphosite S734 within NRF1.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Immunoprecipitation, Phospho-proteomics, Western Blot, Immunofluorescence, Staining, Labeling

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . SCH772984 treatment increases BTZ-induced cell death in the Melanoma cell line A2058, detected by CAS3 cleavage. B . Flow cytometry detection of A2058 cell apoptosis (monitored by propidium iodide and Annexin V staining) after treatment with BTZ or/and SCH772984. Quantification of the Annexin V (+) PI (-) fractions are shown in . C . SCH772984 treatment increases BTZ-induced apoptosis in MM.1s cells, detected by western blotting for CAS3 cleavage. D . Flow cytometry analysis of apoptosis (assayed by propidium iodide and Annexin V staining) in MM.1s cells that were treated with BTZ or/and SCH772984. Quantification of the Annexin V (+) PI (-) fraction is shown in . E . ERK1/2 phosphorylation, an indicator of activity, is diminished by treatment with the ERK1/2 inhibitors SCH772984 and SCH772984-HCL. F . MM.1s cell growth and viability is impaired by combined BTZ and SCH772984 treatment, indicated by the Cell Counting Kit-8 colorimetric assay of cell proliferation, ****p<0.0001.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Flow Cytometry, Staining, Western Blot, Phospho-proteomics, Activity Assay, Cell Counting, Colorimetric Assay

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Proteasome activity was reduced in SH-SY5Y Nrf1 shRNA cells compared to SH-SY5Y shRNA control cells, ****p<0.0001. B . SCH772984 treatment does not significantly reduce the background level of proteasome activity in HepG2 cells, ns, p>0.05. C . Screening of a commercial MAPK inhibitor library for small molecules that could increase the level of BTZ-induced apoptosis in HepG2 cells, as shown in D. Red and yellow indicate strong (robust CAS3 increase over control) and moderate (detectable CAS3 increase over control) hits, respectively. D . Induction of apoptosis in HepG2 cells that were treated with BTZ along with the indicated MAPK inhibitors from the library in C, with cell death detected by western blotting for cleavage of CAS3. E . Repeat analysis of “strong” hits from the screen shown in C and D.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Activity Assay, shRNA, Control, Western Blot

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . NRF1 knockdown sensitizes tumor cells to BTZ-induced cell death. Western blotting shows that NRF1 knockdown increases BTZ-induced cell death in HepG2 cells, indicated by CAS3 cleavage. B . NRF1 but not NRF2 knockdown sensitizes HepG2 cells to BTZ-induced apoptosis. C . Inhibition of ERK1/2 by SCH772984 sensitizes HepG2 cells to BTZ-induced apoptosis. D . Inhibition of ERK1/2 by SCH772984 sensitizes MM.1s multiple myeloma cells to BTZ-induced apoptosis. Propidium iodide and Annexin V staining were monitored by flow cytometry. *p<0.05. E . SCH772984 sensitizes A2058 melanoma cells to BTZ-induced apoptosis, detected by flow cytometry, *p<0.05, **p<0.01. F . Result of a small inhibitor screen to identify drugs that increase HepG2 cell death synergistically with BTZ, detected by CAS3 western blot. 16 of 18 MEK inhibitors and 4 of 6 ERK inhibitors showed a positive synergistic effect. G, H . BTZ-induced expression of psma7 ( G ) and pcmd1 ( H ) proteasome subunit genes is decreased by SCH772894 treatment, assayed by RT-PCR in HepG2 cells, *p<0.05.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Knockdown, Western Blot, Inhibition, Staining, Flow Cytometry, Expressing, Reverse Transcription Polymerase Chain Reaction

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . SCH772984 enhances BTZ-induced inhibition of A2058 tumor cell growth. Mice were treated with Control, SCH772984 (20 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ for 10 days, n=14 for each cohort. Tumor size was measured at day 0, 4, 6, 8 and 10, *p<0.05. B . Quantification of the tumor growth inhibitory (TGI) effect of BTZ alone (Cont) or BTZ combined with SCH772984, *p<0.05. C, D . Inhibition of tumor cell proliferation by combined BTZ and SCH772894 treatment. Representative images (C) and quantification (D) of Ki67 staining are shown for tumors collected from mice described in A (n=20 per cohort), Scale bar, 100 µm, ns, p>0.05, **p<0.01. E . Combined BTZ and SCH772894 treatment reduced tumor cell weight. A2058 tumors were weighed after a 10 day treatment with solvent control, SCH772984 (20 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ, ns, p>0.05, *p<0.05. F . Inhibition of the proteasome recovery response in vivo by ERK1/2 inhibition. An RT-PCR analysis shows the expression of 6 proteasome subunit genes in tumors collected in (A), *p<0.05, **p<0.01. G . Schematic depiction of the ERK1/2-NRF1 pathway and the proteasome recovery response. Inhibition of the ERK1/2-NRF1 pathway represents a promising strategy for pharmacologically enhancing the efficacy of proteasome inhibition in cancer therapy.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Inhibition, Control, Staining, Solvent, In Vivo, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: bioRxiv

Article Title: ERK signaling licenses SKN-1A/NRF1 for proteasome production and proteasomal stress resistance

doi: 10.1101/2021.01.04.425272

Figure Lengend Snippet: A . Inhibition of A2058 tumor growth by SCH772984 and BTZ treatment in vivo . A2058 cells were injected into nude mice, and treated with solvent control, SCH772984 210 mg/kg, i.p. daily), BTZ (0.5 mg/kg, i.p. every other day), or a combination of SCH772984 and BTZ for 10 days. All tumors of each cohort were collected and shown. B . Western blot showing the relative levels of MEK, pMEK, ERK and pERK in tumor samples from (A). SCH772984 inhibits ERK but not MEK activity.

Article Snippet: BTZ, SCH772984, and the MAPK inhibitor library were purchased from Selleck Chemicals.

Techniques: Inhibition, In Vivo, Injection, Solvent, Control, Western Blot, Activity Assay

Journal: Developmental cell

Article Title: EGFR/Pak Signaling Selectively Regulates Glutamine Deprivation-Induced Macropinocytosis

doi: 10.1016/j.devcel.2019.05.043

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: SCH772984 , Chemietek , Cat#CT-SCH772.

Techniques: Recombinant, Fluorescence, Reverse Transcription, DC Protein Assay, Stripping Membranes, Transfection, Plasmid Preparation, Negative Control, Software

Journal: Molecular Systems Biology

Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

doi: 10.15252/msb.20145450

Figure Lengend Snippet: 04.01 melanoma cells were transduced with the kinome shRNA library (which was divided in four pools each containing ˜1,000 hairpins) with a multiplicity of infection (MOI) of 0.5. After selection with puromycin, cell pools were divided into four samples: one as a reference control, one was treated with DMSO as a control, one was treated with 0.15 μM PLX4720 and one was treated with 0.015 μM SCH772984. After 4 and 7 days of treatment, cells were harvested. Genomic DNA was isolated and deep sequenced. shRNAs that dropped out in the treated sample are highlighted by dashed empty circles and indicated by red arrows compared to the control. Hairpins that dropped out in the untreated sample compared to the reference sample are highlighted by dashed empty circles and indicated by green arrows. Venn diagram depicting the overlap of identified hairpins in the PLX4720 and SCH772984 sensitizer screens. On the right, the BRAF pathway and the two inhibitors used are depicted.

Article Snippet: Inhibitors used were PLX4720 (Selleck), SCH772984 (Merck, via a MTA), GSK269962A (Axon Medchem) and metabolic poison phenyl arsine oxide (PAO) (Sigma).

Techniques: Transduction, shRNA, Infection, Selection, Isolation

Journal: Molecular Systems Biology

Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

doi: 10.15252/msb.20145450

Figure Lengend Snippet: Fold change of the hairpins identified in the PLX4720 and SCH772984 sensitizer screen.

Article Snippet: Inhibitors used were PLX4720 (Selleck), SCH772984 (Merck, via a MTA), GSK269962A (Axon Medchem) and metabolic poison phenyl arsine oxide (PAO) (Sigma).

Techniques:

Journal: Molecular Systems Biology

Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

doi: 10.15252/msb.20145450

Figure Lengend Snippet: A Cells were transduced with sh-SCR(ambled) as a control or with one of the three different shRNAs against ROCK1 and analyzed for ROCK1 levels by Western blot analysis. β-actin served as a loading control. B, C Cells described in (A) were treated with a dilution series of PLX4720 (B) or SCH772984 (C) for 3 days. Cell viability was determined with a cell titer blue assay. The y -axis represents the percentage of living cells, normalized to cells expressing sh-SCR. Error bars represent standard error of the mean of one representative experiment done in triplicate. Dashed lines represent the change in IC 50 . D Cells described in (A) were plated and treated with 0.15 μM PLX4720 on the next day. After 3 days, cells were harvested (apoptotic cells in the supernatant were included in the analysis) and analyzed by Western blot. β-actin served as a loading control.

Article Snippet: Inhibitors used were PLX4720 (Selleck), SCH772984 (Merck, via a MTA), GSK269962A (Axon Medchem) and metabolic poison phenyl arsine oxide (PAO) (Sigma).

Techniques: Transduction, Western Blot, Expressing

Journal: Molecular Systems Biology

Article Title: ROCK1 is a potential combinatorial drug target for BRAF mutant melanoma

doi: 10.15252/msb.20145450

Figure Lengend Snippet: Three independent melanoma cell lines (04.01, 93.03 and 04.07) were treated with dilution series of PLX4720 either alone or in combination with the ROCK inhibitor GSK269962A. After 3 days, cell viability was determined by a cell titer blue assay and represented in the y -axis. Cells were treated same as in (A), but with a dilution series of the ERK inhibitor SCH772984.

Article Snippet: Inhibitors used were PLX4720 (Selleck), SCH772984 (Merck, via a MTA), GSK269962A (Axon Medchem) and metabolic poison phenyl arsine oxide (PAO) (Sigma).

Techniques: