Journal: Ecotoxicology and environmental safety

Article Title: NAC antagonizes arsenic-induced neurotoxicity through TMEM179 by inhibiting oxidative stress in Oli-neu cells.

doi: 10.1016/j.ecoenv.2021.112554

Figure Lengend Snippet: Fig. 5. TMEM179 is expressed in OPCs. (a) Tmem179 gene expression in different cell types in adult mouse brain tissues. The data were obtained from the MCA database. (b) Immunofluorescence staining of NG2 and TMEM179 in the cerebral cortices of mice. Scale bar, 25 µm. (c) The subcellular localization of Tmem179 was assessed with a Tmem179 antibody and MitoTracker staining. Scale bar, 20 µm. (d, e) Measurement of Tmem179 expression after arsenic and NAC treatment for 24 h. Means ± SEMs, n = 3. *p < 0.05 and **p < 0.01, compared with the control group; #p < 0.05, compared with the arsenic-treated group, One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The membranes were blocked for 1 h at room temperature with QuickBlockTM Blocking Buffer (Beyotime, China) and incubated with primary antibodies against TMEM179 (Proteintech, 24799-1-AP, 1:1000), cleaved CASPASE-3 (Cell Signaling Technology, 9664S, 1:1000), BCL-2 (Cell Signaling Technology, 3498T, 1:1000), BAX (Cell Signaling Technology, 2772 T, 1:1000), cleaved PARP (Cell Signaling Technology, 9544S, 1:1000), and ACTB (Sigma-Aldrich, A5441, 1:5000) at 4 ◦C overnight.

Techniques: Gene Expression, Immunofluorescence, Staining, Expressing, Control, Comparison

Journal: Ecotoxicology and environmental safety

Article Title: NAC antagonizes arsenic-induced neurotoxicity through TMEM179 by inhibiting oxidative stress in Oli-neu cells.

doi: 10.1016/j.ecoenv.2021.112554

Figure Lengend Snippet: Fig. 6. Tmem179 is a key factor mediating the protective effects of NAC. (a) Cell viability, (b) total ROS levels, and (c) mtROS levels measured after Tmem179 overexpression and silencing. (d, e) ATP levels and the mitochondrial membrane potential (ΔΨm) measured after Tmem179 overexpression and silencing. (f, g) Cell apoptosis evaluated after Tmem179 overexpression and silencing. Means ± SEMs, n = 3. ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the arsenic-treated group; $p < 0.05, $$p < 0.01, and $$$p < 0.001, compared with arsenic and NAC-treated group, One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The membranes were blocked for 1 h at room temperature with QuickBlockTM Blocking Buffer (Beyotime, China) and incubated with primary antibodies against TMEM179 (Proteintech, 24799-1-AP, 1:1000), cleaved CASPASE-3 (Cell Signaling Technology, 9664S, 1:1000), BCL-2 (Cell Signaling Technology, 3498T, 1:1000), BAX (Cell Signaling Technology, 2772 T, 1:1000), cleaved PARP (Cell Signaling Technology, 9544S, 1:1000), and ACTB (Sigma-Aldrich, A5441, 1:5000) at 4 ◦C overnight.

Techniques: Over Expression, Membrane, Control, Comparison

Journal: Ecotoxicology and environmental safety

Article Title: NAC antagonizes arsenic-induced neurotoxicity through TMEM179 by inhibiting oxidative stress in Oli-neu cells.

doi: 10.1016/j.ecoenv.2021.112554

Figure Lengend Snippet: Fig. 7. PKC-β is a downstream factor of TMEM179. (a-g) Expression of apoptosis-related proteins (cleaved-PARP, BCL-2, BAX, cleaved-CASPASE-3) after TMEM179 overexpression and silencing. Means ± SEMs, n = 3. *p < 0.05, ** p < 0.01, and ***p < 0.001, compared with the control group; #p < 0.05, ##p < 0.01, and ###p < 0.001, compared with the arsenic-treated group; $p < 0.05, $$p < 0.01, and $$$p < 0.001, compared with the arsenic and NAC-treated group, One-way ANOVA followed by Tukey’s multiple comparison test.

Article Snippet: The membranes were blocked for 1 h at room temperature with QuickBlockTM Blocking Buffer (Beyotime, China) and incubated with primary antibodies against TMEM179 (Proteintech, 24799-1-AP, 1:1000), cleaved CASPASE-3 (Cell Signaling Technology, 9664S, 1:1000), BCL-2 (Cell Signaling Technology, 3498T, 1:1000), BAX (Cell Signaling Technology, 2772 T, 1:1000), cleaved PARP (Cell Signaling Technology, 9544S, 1:1000), and ACTB (Sigma-Aldrich, A5441, 1:5000) at 4 ◦C overnight.

Techniques: Expressing, Over Expression, Control, Comparison

Journal: Scientific Reports

Article Title: Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation

doi: 10.1038/s41598-021-03832-5

Figure Lengend Snippet: Activated HIF-1α with its increased protein expression in the heart primordium after heartbeat initiation. ( A ) DNA-binding activity of HIF-1α in the nuclear fraction in the pre- and post-heartbeat groups (N = 6). ( B , C ) Representative images of Western blots ( B ) and densitometry ( C ) of HIF-1α, HIF-1β, PHD1, and pVHL in the pre- and post-heartbeat group (N = 5–6). ( D , E ) Representative images of Western blots ( D ) and densitometry ( E ) of PHD2 in the pre- and post-heartbeat groups (N = 6). ( F ) Relative gene expression levels of HIF-1α, VHL, and PHD2 that were obtained by microarray analysis in the pre- and post-heartbeat groups. *p < 0.05 with Welch's t-test. N.S.: not significant. Full-length blots/gels are presented in Supplementary Figure S1.

Article Snippet: After being blocked with Tris Buffer Saline containing 5% skimmed milk and 0.05% Tween 20, the membrane was incubated overnight with primary antibodies against HIF-1α (CST #14,179, Cell Signaling, Danvers, MA), HIF-1β/ARNT (CST #5537S, Cell Signaling), PHD1 (ab113077, Abcam, Cambridge, MA), pVHL (sc-135657, Santa Cruz, Dallas, TX), PHD2 (CST #3293, Cell Signaling), GLUT1 (ab115730, Abcam), HK2 (sc-374091, Santa Cruz), PFKM (ab154804, Abcam), and GAPDH (sc-25778, Santa Cruz) for the glycolytic pathway, G6PD (ab76598, Abcam) for the pentose phosphate pathway, phospho-PDH E1α subunit Ser 297 (ab177461, Abcam), total PDH E1α subunit (ab110330, Abcam), PDK1 (LS‐B1733, LSBio, Seattle, WA), and PDP1 (sc-398117, Santa Cruz) for the TCA cycle, and Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam) containing targets for NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A for mitochondrial respiratory chain subunits.

Techniques: Expressing, Binding Assay, Activity Assay, Western Blot, Gene Expression, Microarray

Journal: Scientific Reports

Article Title: Enhanced glucose metabolism through activation of HIF-1α covers the energy demand in a rat embryonic heart primordium after heartbeat initiation

doi: 10.1038/s41598-021-03832-5

Figure Lengend Snippet: Increased protein expression of HIF-1α downstream targets without changes in mitochondrial respiratory complexes in the heart primordium after heartbeat initiation. ( A , B ) Representative images of Western blots ( A ) and densitometry ( C ) of GLUT1, HK2, PFKM, GAPDH, and G6PD in the pre- and post-heartbeat groups (N = 6). ( C , D ) Representative images of Western blots ( C ) and densitometry ( D ) of Ser 297 phospho-PDH E1α (PDEA1), total PDEA1, PDK1, and PDP1 in the pre- and the post-heartbeat groups (N = 6). ( E , F ) Representative images of Western blots ( E ) and densitometry ( F ) of NDUFS1, SDHB, UQCRC2, MTCO2, and ATP5A in the pre-heartbeat group, the post-heartbeat group, and the heart primordium at E11 (N = 6). ( G ) Representative transmission electron microscopy images of the heart primordium in the pre-heartbeat group, the post-heartbeat group, and the heart primordium at E11. Black scale bar indicates 1.0 μm. *p < 0.05 with Welch's t-test for comparison of two groups and ANOVA with Tukey–Kramer’s post hoc test for comparison of three groups. N.S.: not significant. Full-length blots/gels are presented in Supplementary Figures S2-S4.

Article Snippet: After being blocked with Tris Buffer Saline containing 5% skimmed milk and 0.05% Tween 20, the membrane was incubated overnight with primary antibodies against HIF-1α (CST #14,179, Cell Signaling, Danvers, MA), HIF-1β/ARNT (CST #5537S, Cell Signaling), PHD1 (ab113077, Abcam, Cambridge, MA), pVHL (sc-135657, Santa Cruz, Dallas, TX), PHD2 (CST #3293, Cell Signaling), GLUT1 (ab115730, Abcam), HK2 (sc-374091, Santa Cruz), PFKM (ab154804, Abcam), and GAPDH (sc-25778, Santa Cruz) for the glycolytic pathway, G6PD (ab76598, Abcam) for the pentose phosphate pathway, phospho-PDH E1α subunit Ser 297 (ab177461, Abcam), total PDH E1α subunit (ab110330, Abcam), PDK1 (LS‐B1733, LSBio, Seattle, WA), and PDP1 (sc-398117, Santa Cruz) for the TCA cycle, and Total OXPHOS Rodent WB Antibody Cocktail (ab110413, Abcam) containing targets for NDUFB8, SDHB, UQCRC2, MTCO2, and ATP5A for mitochondrial respiratory chain subunits.

Techniques: Expressing, Western Blot, Transmission Assay, Electron Microscopy, Comparison