Journal: Cell Death Discovery

Article Title: Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

doi: 10.1038/s41420-022-00867-z

Figure Lengend Snippet: A Total intracellular ATP content of DE cells treated with dynasore ( n = 3). B Percentage of CXCR4 and SOX17-positive cells in DE differentiation treated with dynasore and 1 mM ATP measured by flow cytometry ( n = 3). C Flow cytometry analysis to measure the percentage of CXCR4 and SOX17-positive cells in DE differentiation treated with oligomycin (oligo) ( n = 3). D Flow cytometry analysis to measure the percentage of CXCR4 and SOX17-positive cells in DE differentiation treated with IACS ( n = 3). E mRNA expression of endoderm factors FOXA2 and CXCR4 in DE cells treated with IACS ( n = 3). F Immunofluorescence microscopy of definitive endoderm cells for FOXA2 (green) and OCT4 (red) treated with 2 μM FCCP or 20 nM IACS. DAPI, blue. Scale bar = 400 μm. G Flow cytometry analysis to measure the percentage of CXCR4 and SOX17-positive cells in DE differentiation treated with FCCP ( n = 3). H mRNA expression of endoderm factors FOXA2 and CXCR4 in DE cells treated with FCCP ( n = 3). I Flow cytometric analysis of CXCR4- and SOX17-positive cells in DE differentiation treated with 1 μM oligomycin plus 0.5 mM ATP ( n = 4). The data of RT-PCR are normalized to the mRNA level of untreated (control) DE cells. All data are shown as mean ± SD. ns, not significant. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Small molecules used in this study are listed as follows: XCT790 (XCT), 2–5 μM (MCE, Cat# HY-10426); ATN-224 (ATN), 5–10 μM (MCE, Cat# HY-16074); Mdivi-1, 10 μM (MCE, Cat# HY-15886); IACS-010759 (IACS), 0.1–20 nM (Selleck, Cat# S8731); Dynasore (DYNA), 1–3 μM (Selleck, Cat# S8047); FCCP, 1–2 μM (Selleck, Cat# S8276); Oligomycin A (Oligo), 0.1–10 μM (Apexbio, Cat# A5588); Doxycycline (DOX), 100 ng/ml (Sigma, Cat# 324385).

Techniques: Flow Cytometry, Expressing, Immunofluorescence, Microscopy, Reverse Transcription Polymerase Chain Reaction, Control

Journal: bioRxiv

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

doi: 10.1101/2020.02.07.938985

Figure Lengend Snippet: (A) Timeline, representative images, and quantified mitochondrial length following CCCP postmitotic treatment. Each data point represents an individual cell average mitochondrial size from three biological replicate experiments. Mann-Whitney test. (B) Quantification of each cell fate marker+ cells among PC cells (6 hours post-label) from three biological replicate experiments following CCCP postmitotic treatment. Data are shown as mean ± SEM. ****P < 0.0001; Bonferroni’s multiple comparisons test. (C) Quantification of number of daughter cells under CCCP treatment from three biological replicate experiments. Data are shown as mean ± SEM. (D) Quantification of number of daughter cells under Sirt1 inhibitor Ex-527 treatment from three biological replicate experiments. Data are shown as mean ± SEM. (E) Timeline and quantification of cell fate marker-positive cells among PC cells following CCCP and Ex-527 treatment (6 hours post-PC) from four biological replicate experiments. Data are shown as mean ± SEM, *P < 0.05; Dunnett’s multiple comparisons test. (F) Quantification of number of daughter cells under CCCP and Ex-527 treatments from four biological replicate experiments. Data are shown as mean ± SEM. (G) Quantification of number of daughter cells under M1 and SRT1720 treatments from four biological replicate experiments. Data are shown as mean ± SEM.

Article Snippet: After photoconversion, medium was changed to Nb/B27 (Mouse) or DDM/B27+Nb/B27 (Human) medium with following chemical compounds: M1 (50μM, Sigma-Aldrich, Cat#SML0629), Mdivi-1 (50μM, Sigma-Aldrich, Cat#M0199), Roscovitine (50μM, Sigma-Aldrich, Cat#R7772), CCCP (10μM, Abcam, Cat#ab141229), Ex-527 (50μM, Santa Cruz Biotechnology, Cat#sc-203044), or SRT1720 (2μM, Santa Cruz Biotechnology, Cat#sc-364624).

Techniques: MANN-WHITNEY, Marker

Journal: bioRxiv

Article Title: Mitochondria dynamics in postmitotic cells drives neurogenesis through Sirtuin-dependent chromatin remodeling

doi: 10.1101/2020.02.07.938985

Figure Lengend Snippet: (A) Timeline and quantification of cell fate marker-positive cells among PC cells using Sirt1 inhibitor Ex-527 from three biological replicate experiments. Data are shown as mean ± SEM. **P < 0.01, ***P < 0.001; Bonferroni’s multiple comparisons test. (B) Timeline and representative images of PC cell using M1 and Sirt1 activator SRT1720. Quantified mitochondrial length from three biological replicate experiments. Each data point represents an individual cell average mitochondrial size. ***P < 0.001; Dunn’s multiple comparisons test. (C) Quantification of each cell fate marker+ cells among PC cells from four biological replicate experiments. Data are shown as mean ± SEM. *P < 0.05, ***P < 0.001, ****P < 0.0001; Dunnett’s multiple comparisons test. (D) Timeline and representative images of H4K16ac signal in PC cells. Quantified H4K16ac signal from two biological replicate experiments. Each data point represents an individual cell average H4K16ac signal. **P < 0.01, ****P < 0.0001; Dunn’s multiple comparisons test.

Article Snippet: After photoconversion, medium was changed to Nb/B27 (Mouse) or DDM/B27+Nb/B27 (Human) medium with following chemical compounds: M1 (50μM, Sigma-Aldrich, Cat#SML0629), Mdivi-1 (50μM, Sigma-Aldrich, Cat#M0199), Roscovitine (50μM, Sigma-Aldrich, Cat#R7772), CCCP (10μM, Abcam, Cat#ab141229), Ex-527 (50μM, Santa Cruz Biotechnology, Cat#sc-203044), or SRT1720 (2μM, Santa Cruz Biotechnology, Cat#sc-364624).

Techniques: Marker

Journal: Frontiers in Cardiovascular Medicine

Article Title: Association of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 With Angiotensin II Type 1 Receptor Impacts Mitochondrial Quality Control, Offering Promise for the Treatment of Vascular Senescence

doi: 10.3389/fcvm.2021.788655

Figure Lengend Snippet: ox-LDL induced Drp1 activation through AT1R and the CRAF/MEK/ERK pathway. (A) Immunoblots and quantitative analysis for CRAF, p-CRAF, MEK, p-MEK, ERK, p-ERK, and β-actin in cells with ox-LDL or the control. * P < 0.05 vs. the control ( n = 3 per group). (B) Immunoblots and quantitative analysis for CRAF, p-CRAF, MEK, p-MEK, ERK, p-ERK, Drp1, p-Drp1 (Ser616), and β-actin in Dabrafenib-treated vs. control cells with ox-LDL. * P < 0.05 vs. ox-LDL with the control ( n = 3 per group). (C) Immunoblots and quantitative analysis for CRAF, p-CRAF, MEK, p-MEK, ERK, p-ERK, Drp1, p-Drp1 (Ser616), and β-actin in PD184352-treated vs. control cells with ox-LDL. * P < 0.05 vs. ox-LDL with the control ( n = 3 per group). (D) Immunoblots and quantitative analysis for CRAF, p-CRAF, MEK, p-MEK, ERK, p-ERK, Drp1, p-Drp1 (Ser616), and β-actin in SCH772984-treated vs. the control cells with ox-LDL. * P < 0.05 vs. ox-LDL without SCH772984 ( n = 3 per group). (E) Immunoblots and quantitative analysis for CRAF, p-CRAF, MEK, p-MEK, ERK, p-ERK, and β-actin in Candesartan-treated vs. control cells with ox-LDL. * P < 0.05 vs. ox-LDL with the control ( n = 3 per group). All data are presented as the mean ± SEM. Statistical analysis were conducted by Wilcoxon rank sum test (A-F) .

Article Snippet: The following inhibitors were used: 20 μmol/L Mdivi-1 (Sigma, Cat #M0199), Drp1 inhibitor; 10 μmol/L candesartan cilexetil (Candesartan) (Sigma-Aldrich, Cat #SML0245), ARB; 10 μmol/L Dabrafenib (Selleck, Cat #S2807), RAF inhibitor; 2 μmol/L PD184352 (Selleck, Cat #S1020), MEK inhibitor; 2 μmol/L SCH772984 (Selleck, Cat #S7101), ERK inhibitor.

Techniques: Activation Assay, Western Blot, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Association of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 With Angiotensin II Type 1 Receptor Impacts Mitochondrial Quality Control, Offering Promise for the Treatment of Vascular Senescence

doi: 10.3389/fcvm.2021.788655

Figure Lengend Snippet: Rab9-dependent mitochondrial autophagy was not affected by ERK inhibition but was affected by MEK inhibition. (A) Representative images and quantitative analysis of TOMM20 (green) and LAMP2 (red) immunohistochemistry in SCH772984-treated vs. control vehicle-treated cells with ox-LDL. Scale bar = 10 μm. ( n = 3 per group). (B) Representative images and quantitative analysis of TOMM20 (green) and LAMP2 (red) immunohistochemistry in PD184352-treated vs. control vehicle-treated cells with ox-LDL. Scale bar = 10 μm. * P < 0.05 when compared with ox-LDL with the control ( n = 3 per group). (C) Representative images and quantitative analysis of LAMP2 (green) and Rab9 (red) immunohistochemistry in PD184352-treated vs. control cells with ox-LDL. Scale bar = 10 μm. * P < 0.05 when compared with ox-LDL with the control ( n = 3 per group). All data are presented as the mean ± SEM. Statistical analysis were conducted by Wilcoxon rank sum test (A-C) .

Article Snippet: The following inhibitors were used: 20 μmol/L Mdivi-1 (Sigma, Cat #M0199), Drp1 inhibitor; 10 μmol/L candesartan cilexetil (Candesartan) (Sigma-Aldrich, Cat #SML0245), ARB; 10 μmol/L Dabrafenib (Selleck, Cat #S2807), RAF inhibitor; 2 μmol/L PD184352 (Selleck, Cat #S1020), MEK inhibitor; 2 μmol/L SCH772984 (Selleck, Cat #S7101), ERK inhibitor.

Techniques: Inhibition, Immunohistochemistry, Control

Journal: Frontiers in Cardiovascular Medicine

Article Title: Association of Lectin-Like Oxidized Low-Density Lipoprotein Receptor-1 With Angiotensin II Type 1 Receptor Impacts Mitochondrial Quality Control, Offering Promise for the Treatment of Vascular Senescence

doi: 10.3389/fcvm.2021.788655

Figure Lengend Snippet: The mechanism for the AT1R inhibition of anti-senescence through mitochondrial quality control. ox-LDL induced mitochondrial fission through the association of LOX-1 and AT1R and the CRAF/MEK/ERK pathway, resulting in mitochondrial dysfunction and senescence. AT1R inhibition attenuated senescence by suppression of this cascade and induced Rab9-dependent alternative autophagy through the CRAF/MEK axis and aspects of the mitochondrial quality control process in human VSMCs.

Article Snippet: The following inhibitors were used: 20 μmol/L Mdivi-1 (Sigma, Cat #M0199), Drp1 inhibitor; 10 μmol/L candesartan cilexetil (Candesartan) (Sigma-Aldrich, Cat #SML0245), ARB; 10 μmol/L Dabrafenib (Selleck, Cat #S2807), RAF inhibitor; 2 μmol/L PD184352 (Selleck, Cat #S1020), MEK inhibitor; 2 μmol/L SCH772984 (Selleck, Cat #S7101), ERK inhibitor.

Techniques: Inhibition, Control

Journal: Cell Death Discovery

Article Title: Mitochondrial homeostasis regulates definitive endoderm differentiation of human pluripotent stem cells

doi: 10.1038/s41420-022-00867-z

Figure Lengend Snippet: A The western blot of TFAM and β-actin protein along with the removal time of doxycycline. B Schematic illustration of the differentiation protocol for inducible TFAM knockout PSCs. C Flow cytometric analysis to determine the change in differentiation efficiency between induced knockout and wild-type iPSCs. SOX17 + and CXCR4 + cells represented differentiated DE cells. D Immunofluorescence analysis of DE cells marked with SOX17 (green) (nucleus, DAPI, blue). Scale bar = 200 μm. E mRNA expression of endoderm- or pluripotency-marker SOX17 , OCT4 in DE cells treated with or without 100 ng/ml doxycycline ( n = 3). F Flow cytometric analysis to measure the percentage of cells expressing both differentiation markers (SOX17 and CXCR4) treated with XCT790 (XCT) in DE differentiation ( n = 3). G mRNA expression of endoderm transcription factors FOXA2 , SOX17 , and CXCR4 in DE cells treated with XCT ( n = 3). H mRNA expression of pluripotent markers OCT4 and SOX2 in DE cells treated with XCT ( n = 12). I Flow cytometric analysis to measure the percentage of cells expressing both DE markers (SOX17 and CXCR4) treated with dynasore (DYNA) ( n = 3). J mRNA expression of endoderm transcription factors FOXA2 , SOX17 , and CXCR4 in DE cells treated with dynasore ( n = 3). K mRNA expression of pluripotent markers OCT4 and SOX2 in DE cells treated with dynasore ( n = 6). The data of RT-qPCR are normalized to the mRNA level of untreated (control) DE cells. All data are shown as mean ± SD. * P < 0.05, ** P < 0.01 and *** P < 0.001.

Article Snippet: Small molecules used in this study are listed as follows: XCT790 (XCT), 2–5 μM (MCE, Cat# HY-10426); ATN-224 (ATN), 5–10 μM (MCE, Cat# HY-16074); Mdivi-1, 10 μM (MCE, Cat# HY-15886); IACS-010759 (IACS), 0.1–20 nM (Selleck, Cat# S8731); Dynasore (DYNA), 1–3 μM (Selleck, Cat# S8047); FCCP, 1–2 μM (Selleck, Cat# S8276); Oligomycin A (Oligo), 0.1–10 μM (Apexbio, Cat# A5588); Doxycycline (DOX), 100 ng/ml (Sigma, Cat# 324385).

Techniques: Western Blot, Knock-Out, Immunofluorescence, Expressing, Marker, Quantitative RT-PCR, Control