Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: MiR-183-5p promotes VSMC proliferation and migration by reducing FKBPL levels. (A) Venn diagram analysis of miRNAs that hsa_circ_0001402 might interact with. (B-C) Western blot analysis of FKBPL, p21, PCNA, MMP9, and MMP2 after 48 h of transfection of miR-Control or miR-183-5p mimic in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05, ** p < 0.01 vs. miR-Control group, two-tailed paired t -test). (D) The binding site between the CDS region of FKBPL or Fkbpl mRNA and miR-183-5p. (E) Dual-luciferase reporter gene analysis of the interaction between miR-183-5p and FKBPL . WT: wild-type; MUT: mutant. Data are shown as mean ± SD ( n = 6, *** p < 0.001, two-tailed unpaired t -test). (F) EdU incorporation analysis of DNA synthesis after 48 h of transfection of miR-Control or miR-183-5p mimic in HASMCs. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents HASMCs with DNA synthesis. The scale bar is 10 µm. (G) The proportion of EdU-positive HASMCs. Data are shown as mean ± SD ( n = 5, * p < 0.05 vs. miR-Control group, two-tailed unpaired t -test with Welch's correction). (H) CCK-8 analysis of cell viability of HASMCs transfected with miR-Control or miR-183-5p mimic. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. miR-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (I) Migration analysis of MOVAS cells transfected with miR-Control or miR-183-5p mimic. The scale bar is 500 µm. (J) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, *** p < 0.001, two-tailed unpaired t -test). (K-L) Western blot analysis of FKBPL, p21, PCNA, MMP9, and MMP2 after 48 h of transfection of miR-Control or miR-183-5p inhibitor in MOVAS cells. Data are shown as mean ± SD ( n = 4 or 3, ** p < 0.01 vs. inhibitor-miR-Control group, two-tailed paired t -test). (M) CCK-8 analysis of cell viability of MOVAS cells transfected with miR-Control or miR-183-5p inhibitor. Data are shown as mean ± SD ( n = 9, *** p < 0.001 vs. inhibitor-miR-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (N) EdU incorporation analysis of DNA synthesis after 48 h of transfection of miR-Control or miR-183-5p inhibitor in MOVAS cells. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (O) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 5, *** p < 0.001 vs. inhibitor-miR-Control group, two-tailed unpaired t -test). (P) Migration analysis of MOVAS cells transfected with miR-Control or miR-183-5p inhibitor. The scale bar is 500 µm. (Q) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, ** p < 0.01, two-tailed unpaired t -test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Migration, Western Blot, Transfection, Control, Two Tailed Test, Binding Assay, Luciferase, Mutagenesis, DNA Synthesis, Fluorescence, CCK-8 Assay

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of miR-183-5p aggravates neointimal hyperplasia by decreasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, miR-Control agomir, or miR-183-5p agomir. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (*** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control agomir group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are presented as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of miR-Control or miR-183-5p agomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control agomir group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Silencing of miR-183-5p alleviates neointimal hyperplasia by increasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, miR-Control antagomir, or miR-183-5p antagomir. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (*** p < 0.001 vs. miR-Control antagomir group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. miR-Control antagomir group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 6 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of miR-Control or miR-183-5p antagomir in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 6 mice). Data are shown as mean ± SD (* p < 0.05 vs. miR-Control antagomir group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of hsa_circ_0001402 increases FKBPL and BECN1 levels by sponging miR-183-5p. (A) qRT-PCR analysis of miR-183-5p expression in HASMCs transfected with the expression vector of hsa_circ_Control or hsa_circ_0001402 , normalized to U6 . Data are shown as mean ± SD ( n = 12, *** p < 0.001 vs. hsa_circ_Control group, two-tailed unpaired t -test). (B) The binding site between hsa_circ_0001402 and miR-183-5p. (C) Dual-luciferase reporter gene analysis of the interaction between hsa_circ_0001402 and miR-183-5p. Data are shown as mean ± SD [ n = 6, ** p < 0.01, two-tailed unpaired t -test (left), two-tailed Mann-Whitney U test (right)]. (D-E) Western blot analysis of PCNA, MMP9, and MMP2 in HASMCs co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Data are shown as mean ± SD ( n = 4, * p < 0.05, ** p < 0.01 vs. control group, two-tailed paired t -test). (F) EdU incorporation analysis of DNA synthesis in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (G) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. control group, two-tailed unpaired t -test). (H) CCK-8 analysis of cell viability of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic. Data are shown as mean ± SD ( n = 9, ** p < 0.01, *** p < 0.001 vs. control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (I) Migration analysis of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic. The scale bar is 500 µm. (J) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, * p < 0.05, ** p < 0.01, two-tailed unpaired t -test). (K-L) Western blot analysis of FKBPL and p21 after 48 h of transfection of the expression vector of hsa_circ_Control or hsa_circ_0001402 in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05 vs. hsa_circ_Control group, two-tailed paired t -test). (M) Overexpression of hsa_circ_0001402 competitively binds to miR-183-5p to reduce the latter's inhibition of FKBPL -WT luciferase activity. Data are shown as mean ± SD ( n = 6, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (N) EdU incorporation analysis of DNA synthesis in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA for 48 h. Blue fluorescence (Hoechst 33342) represents the cell nuclei, while red fluorescence (EdU) represents MOVAS cells with DNA synthesis. The scale bar is 10 µm. (O) The proportion of EdU-positive MOVAS cells. Data are shown as mean ± SD ( n = 6, *** p < 0.001 vs. siRNA-Control group, two-tailed unpaired t -test). (P) CCK-8 analysis of cell viability of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA. Data are shown as mean ± SD ( n = 9, *** p < 0.001 vs. siRNA-Control group at the corresponding point in time, two-way ANOVA followed by Sidak's multiple comparisons test). (Q) Migration analysis of MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either Control or Fkbpl siRNA. The scale bar is 500 µm. (R) Quantify the migration area using ImageJ. Data are shown as mean ± SD ( n = 4, ** p < 0.01, two-tailed unpaired t -test). (S-T) Western blot analysis of LC3 in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. Data are shown as mean ± SD ( n = 4, * p < 0.05, *** p < 0.001 vs. control group, two-tailed paired t -test). (U) TEM analysis of autolysosomes in MOVAS cells co-transfected with hsa_circ_0001402 expression vector and either miRNA-Control or miR-183-5p mimic for 48 h. The scale bar is 1 μm. (V) Quantify the number of autolysosomes. Data are shown as mean ± SD ( n = 6, * p < 0.05 vs. control group, two-tailed paired t -test). (W) Western blot analysis of BECN1 after 48 h of transfection of the expression vector of hsa_circ_Control or hsa_circ_0001402 in HASMCs. Data are shown as mean ± SD ( n = 3, * p < 0.05 vs. hsa_circ_Control group, two-tailed paired t -test). (X) Overexpression of hsa_circ_0001402 competitively binds to miR-183-5p to reduce the latter's inhibition of BECN1 -WT luciferase activity. Data are shown as mean ± SD ( n = 6, * p < 0.05, ** p < 0.01, Kruskal-Wallis test followed by Dunn's multiple comparisons test). (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Quantitative RT-PCR, Expressing, Transfection, Plasmid Preparation, Control, Two Tailed Test, Binding Assay, Luciferase, MANN-WHITNEY, Western Blot, DNA Synthesis, Fluorescence, CCK-8 Assay, Migration, Inhibition, Activity Assay

Journal: Journal of Advanced Research

Article Title: Hsa_circ_0001402 alleviates vascular neointimal hyperplasia through a miR-183-5p-dependent regulation of vascular smooth muscle cell proliferation, migration, and autophagy

doi: 10.1016/j.jare.2023.07.010

Figure Lengend Snippet: Overexpression of hsa_circ_0001402 alleviates neointimal hyperplasia by increasing FKBPL and BECN1 levels. (A) The cross-sectional H&E staining images of the ligated mouse common carotid arteries after 21 days of treatment with Sham Control, hsa_circ_Control lentivirus, or hsa_circ_0001402 lentivirus. The scale bar is 100 µm. (B) Morphometric measurement of the intimal/media ratio in the mouse common carotid arterial sections ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01, *** p < 0.001, one-way ANOVA followed by Tukey's multiple comparisons test). (C) Immunofluorescence of FKBPL after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (D) The quantification of FKBPL levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (E) Immunofluorescence of p21 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (F) The quantification of p21 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (G) Immunofluorescence of PCNA after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (H) The quantification of PCNA levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (I) Immunofluorescence of MMP9 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (J) The quantification of MMP9 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (** p < 0.01 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (K) Immunofluorescence of MMP2 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (L) The quantification of MMP2 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (M) Immunofluorescence of BECN1 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (N) The quantification of BECN1 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (O) Immunofluorescence of p62 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (P) The quantification of p62 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). (Q) Immunofluorescence of total LC3 after 21 days of in situ delivery of hsa_circ_Control or hsa_circ_0001402 lentivirus in ligated mouse common carotid arteries. The scale bar is 100 µm. (R) The quantification of total LC3 levels in the ligated mouse common carotid arteries ( n = 4 mice). Data are shown as mean ± SD (* p < 0.05 vs. hsa_circ_Control lentivirus group, two-tailed unpaired t -test). Normal mouse IgG was used as a negative control. MFI: mean fluorescent intensity.

Article Snippet: Briefly, the samples were incubated with antibodies against FKBPL (1:50, Proteintech), p21 (1:50, Proteintech), PCNA (1:50, Proteintech), MMP9 (1:50, Proteintech), MMP2 (1:50, Proteintech), BECN1 (1:50, Proteintech), p62 (1:50, Proteintech), and LC3 (1:50, Proteintech).

Techniques: Over Expression, Staining, Control, Immunofluorescence, In Situ, Two Tailed Test, Negative Control

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) PLA utilizes a pair of oligonucleotide-conjugated secondary antibodies that bind corresponding antibodies targeting each interacting protein partner. The proximity of these two protein partners allows circular DNA amplification of the oligonucleotides template upon in situ ligation. Fluorescent probes complementary to this amplicon engender a detectable signal via fluorescence microscopy. Plasma membrane permeabilization was used for intracellular protein interactions. (B and C) PLA indicating protein-protein interactions of PAI-1 and low-density LRP1 on the cell surface (B) or intracellular (C) due to ASC-CM exposure in the presence or absence of the LRP1 inhibitor RAP. Representative micrograph images showing positive signal (red) for PAI-1/LRP1 interactions. DAPI was used to stain nuclei (blue). (D) ChIP-qPCR analysis of LRP1-ICD binding to two promoter regions of the SMAD4 gene in control (Ctrl) cells and CM-exposed cells treated and untreated with RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; **p < 0.01, ***p < 0.001. (E) qRT-PCR analysis of mRNA expression levels of SMAD4 gene in control cells and CM-exposed cells treated and untreated with Tip (50 μM) or RAP (5 μg/mL). Data represent mean ± SD, n = 3 technical replicates; *p < 0.05. (F) PLA demonstrating protein-protein interactions of SMAD4 and ubiquitin due to ASC-CM exposure in the presence or absence of the following inhibitors: Tip for PAI-1, RAP for LRP1, and protease inhibitor MG132. Micrograph images showing positive PLA signal (green) for SMAD4-ubiquitin interactions. DAPI was used to stain nuclei (blue). (G) Protein expression of LRP1 and SMAD4 by capillary western immunoassays. Endometrial epithelial EME6/7t cells were treated with control media, ASC-CM, and ASC-CM plus Tip, or with pre-treatment with LRP1 antagonist RAP or protease inhibitor MG132. The result shown is a representative of three independent experiments. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: DNA Amplification, In Situ, Ligation, Amplification, Fluorescence, Microscopy, Clinical Proteomics, Membrane, Protein-Protein interactions, Staining, ChIP-qPCR, Binding Assay, Control, Quantitative RT-PCR, Expressing, Ubiquitin Proteomics, Protease Inhibitor, Western Blot

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A) Bar plots showing mRNA expression levels of selected PAI1-regulated JAC genes by qRT-PCR in the two sh SMAD4 knockdown clones, compared to shCtrl in the presence or absence of ASC-CM. (B) ChIP-qPCR analysis of SMAD4 binding to promoter regions of the JAC genes in control (Ctrl) cells and ASC-CM-exposed cells. Data represent mean ± SD, n = 3 technical replicates; *p < 0.05, **p < 0.01, ***p < 0.001, Student’s t test. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing, Quantitative RT-PCR, Knockdown, Clone Assay, ChIP-qPCR, Binding Assay, Control

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A and B) tSNE plots of mass cytometry (CyTOF) analysis visualizing 74,048 epithelial cells, which were divided into clusters (n = 27) based on their proteomic profiles (A). These cells included immortalized EEC line EME6/7t (exposed to ASC-CM ± Tip) and primary endometrial tumors of patients with known BMIs (B). (C) tSNE-based phenographs showing abundance and cellular distributions of JAC proteins. (D) tSNE-based phenographs showing EpCAM and CK8/18 as epithelial cell markers and TGFR-2, SMAD2, and SMAD4 as TGF-β factors. See also and and .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Mass Cytometry

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: (A–C) Expression profiles of SMAD4 and JAC based on CyTOF were classified into five categories: I, II, III, IV, and V. Violin plots show ordered SMAD4 expression level (low to high) and distribution in the individual subpopulations (A). Expression heatmaps of JAC were aligned according to SMAD4 expression levels (in descending order) in different cellular subpopulations (B). Area of circle denotes relative cell proportion of a subpopulation within each sample and ordered in the aforementioned five categories in cellular subpopulations of EME6/7t lines and primary tumors (C). Tumors were ordered from top to bottom based on increased patients’ BMIs and histology classes. (D) Balloon plots of subpopulation distributions of endometrial tumors in the five categories based on histology and BMI. Circle area indicates the size of each subpopulation. Categories IV and V were associated with relatively high expression levels of SMAD4 and JAC, whereas low levels of SMAD4 and JAC were noted in categories I, II, and III. (E) Violin plots showing the average expression levels of four JAC markers—ADAM15, ROCK1, Cx43, and Gas1—based on patients’ tumor histology: papillary serous and endometrioid. (F) Balloon plots of subpopulation distributions in the five categories stratified by BMI into three groups: normal weight (NW), obesity (OB), and morbid obesity (MOB). (G) Violin plots showing the average expression levels of ADAM15, ROCK1, Cx43, and Gas1, based on three BMI groups: NW, OB, and MOB. Lower JAC expressions corresponded with patients with higher BMIs. ****p < 0.0001. See also .

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Expressing

Journal: Cell reports

Article Title: PAI-1-Dependent Inactivation of SMAD4-Modulated Junction and Adhesion Complex in Obese Endometrial Cancer

doi: 10.1016/j.celrep.2020.108253

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: For the association assay between SMAD4 and ubiquitin, cells were permeabilized with 0.2% (v/v) Triton X-100 for 10 min after fixing and incubated with goat polyclonal anti-SMAD4 antibody (1:200, R&D Systems, Cat# AF2097) and rabbit polyclonal anti-ubiquitin antibody (1:200, Novus Biologicals, Cat# NB300129) which were tagged with anti-goat PLUS and anti-rabbit MINUS.

Techniques: Ubiquitin Proteomics, Recombinant, Multiplex Assay, Reverse Transcription, SYBR Green Assay, In Situ, shRNA, Software

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A) Co-cultured conditions of U937-derived macrophages and A549 lung cancer cells or MRC-5 lung fibroblasts with cytometry by time-of-flight (CyTOF) workflow. (B) Violin plots showing the expression levels of AXL and pSTAT3 of macrophages in condition 1, 3, and 5. Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (C) AXL and pSTAT3 correlation scatterplot of macrophages showing the four categories based on mean values of AXL and pSTAT3 and bar graph of category proportions in condition 1, 3, and 5. (D and E) Violin plots of expression levels of the seven AXL-pSTAT3-related markers in macrophages of condition 1, 3, and 5. Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (F) CD86 and CD163 correlation scatterplot of macrophages showing the four subtypes based on mean values of CD86 and CD163 and bar graph of subtype proportions in condition 1, 3, and 5. (G) Circle plots showing the proportions and expression levels of AXL, pSTAT3, and the seven markers of the four AXL-pSTAT3 categories in condition 1, 3, and 5. (H) Boxplot of Shannon indices in condition 1, 3, and 5 of 32 PhenoGraph clusters (n = 32). Data are mean ± SD; ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests.

Article Snippet: U937 , ATCC , ATCC CRL-1593.2 TM.

Techniques: Cell Culture, Derivative Assay, Cytometry, Expressing

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A and B) IL-11 secretion was attenuated by dubermatinib (40 nmol/L) from A549 lung cancer cells (n = 2) and MRC-5 lung fibroblasts (n = 4). Data are mean ± SD; *p < 0.05, **p < 0.01, ***p < 0.001; Student’s t test for each time point. (C and D) Kaplan-Meier curves depict overall and disease-free survival probability in TCGA lung adenocarcinoma cohort based on high ( Z score > 1) and low ( Z score < 1) IL11 expression of lung tumors. (E) Flow chart of induction of U937-derived macrophages and IL-11 treatment for CyTOF analysis. (F) AXL and STAT3 correlation scatterplot of U937-derived macrophages. (G) Violin plots showing the expression levels of AXL and STAT3 without and with IL-11 treatment (25 ng/mL). Data are mean ± SD; ***p < 0.001; Student’s t test. (H) Western blot analysis of IL-11-activated AXL-STAT3 signaling, i.e., phosphorylation of AXL and STAT3 (pSTAT3) in U937-derived macrophages. The cleavage product, phosphorylated AXL intracellular domain (pAXL-ICD), was observed. (I and J) Violin plots of expression levels of the seven AXL-STAT3-related markers without and with IL-11 treatment (25 ng/mL). Data are mean ± SD; ***p < 0.001; Student’s t test. (K) CD86 and CD163 correlation scatterplot of macrophages showing the four subtypes based on mean values of CD86 and CD163 and bar graph of subtype proportions of macrophages without and with IL-11 treatment (25 ng/mL).

Article Snippet: U937 , ATCC , ATCC CRL-1593.2 TM.

Techniques: Expressing, Derivative Assay, Western Blot, Phospho-proteomics

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet: (A) Capillary western immunoassay (WES) of a stemness marker, CD44, in U937-derived macrophages untreated and treated with IL-11. Raw data of WES shown in . (B) Diagram of proximity ligation assay (PLA) (left) and PLA images of protein-protein interactions between IL-11/GP130 and pGP130/pSTAT3 in macrophages untreated and treated with IL-11 at 1, 3, and 72 h (right). PLA utilizes a pair of oligonucleotide-conjugated secondary antibodies that correspond to antibodies targeting each interacting protein partner. The proximity of protein partners allows circular DNA amplification of oligonucleotide templates and detectable fluorescence signals upon in situ ligation. (C and D) Quantitative analysis of PLA signals per cell indicating protein-protein interactions of IL-11 and the extracellular domain of GP130 (C) and pGP130 and pSTAT3 (D) in macrophages untreated and treated with IL-11 at 1, 3, and 72 h (n = 7). Data are mean ± SD; *p < 0.05, ***p < 0.001; one-way ANOVA followed by Duncan’s multiple range tests. (E) Chromatin immunoprecipitation (ChIP)-qPCR analysis of pSTAT3 binding to promoter regions of CD44 in untreated and IL-11-treated U937-derived macrophages (n = 3). Data are mean ± SD; ***p < 0.001; Student’s t test for each treatment. (F) qPCR analysis of CD44 expression in untreated and IL-11-treated U937-derived macrophages (n = 9). Data are mean ± SD; ***p < 0.001; Student’s t test. (G) Images of vasculogenic mesh formation and bar graph of mesh number in human umbilical vein endothelial cells (HUVECs) co-cultured without or with U937-derived macrophages (n = 8). Scale bar, 200 μm. Data are mean ± SD; Student’s t test. (H) Bar graph of mesh number in HUVECs co-cultured with U937-derived macrophages pre-treated without or with IL-11 (25 ng/mL) (n = 7). Data are mean ± SD; **p < 0.01; Student’s t test. (I) Bar graph of mesh number in HUVECs co-cultured with U937-derived macrophages pre-treated with IL-11 (25 ng/mL) and without or with CD44 inhibitor (n = 6). Data are mean ± SD; **p < 0.01; Student’s t test.

Article Snippet: U937 , ATCC , ATCC CRL-1593.2 TM.

Techniques: Western Blot, Marker, Derivative Assay, Proximity Ligation Assay, Protein-Protein interactions, DNA Amplification, Fluorescence, In Situ, Ligation, Chromatin Immunoprecipitation, ChIP-qPCR, Binding Assay, Expressing, Cell Culture

Journal: Cell reports

Article Title: AXL-initiated paracrine activation of pSTAT3 enhances mesenchymal and vasculogenic supportive features of tumor-associated macrophages

doi: 10.1016/j.celrep.2023.113067

Figure Lengend Snippet:

Article Snippet: U937 , ATCC , ATCC CRL-1593.2 TM.

Techniques: Recombinant, Reverse Transcription, Cell Viability Assay, In Situ, SYBR Green Assay, RNA Sequencing, Quantitative RT-PCR, Luciferase, Software

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: Lactate (Lac) induces the expression of early growth response protein 1 (EGR1) (A) BMDCs from WT mice were stimulated with LPS (100 ng/mL) and/or Lac (20 mM) for 4 hr, and the total RNA from these cells was subjected to microarray analysis. The log2 ratios for the indicated genes in BMDCs stimulated with LPS plus Lac are arranged in descending order. (B) Western blot analysis of EGR1 expression in BMDCs stimulated with LPS (100 ng/mL) and/or Lac (20 mM) for 24 hr. (C) EGR1 expression (green) in RAW 264.7 cells stimulated with Lac (20 mM) or control medium (Ctrl) for 24 hr. Nuclei were stained with Hoechst 33,342 (blue). Scale bars = 10 μm. (D) Tumor tissues or control normal skin (Ctrl) from WT mice at two weeks after B16-F1 cell implantation were examined for EGR1 expression (green). Scale bars = 100 μm. (E) Median fluorescence intensity (MFI) of EGR1 in leukocyte subpopulations in tumor tissues of WT mice 3 weeks post-B16-F1 cell implantation was analyzed using flow cytometry. On days 6, 9 and 12 after injection of B16-F1 cells, Lac (5 mM, 200 μL) or PBS control (200 μL) was intratumorally administered. Data are expressed as mean ± SD. (F) The relationship between EGR1 MFI of tumor-infiltrating DCs and tumor weight (left) or lactate level (right) in the tumor tissues of WT mice 3–4 weeks post-B16-F1 cell implantation. Individual findings are plotted and each data point represents one mouse. Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. Pearson's correlation coefficient was used to evaluate the correlations. ∗ p < 0.05; ∗∗ p < 0.01; n.s., not significant ( p > 0.05). See also and .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Expressing, Microarray, Western Blot, Control, Staining, Fluorescence, Flow Cytometry, Injection, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: EGR1 is expressed in DCs and correlates with LDHA in human melanoma tissues (A) Tumor tissues from patients with melanoma in situ and invasive melanoma were examined by fluorescent staining (Hoechst 33,342, blue), as well as by immunofluorescence analysis for EGR1 (green). Nuclei (enclosed within light blue lines) and EGR1-positive cells (enclosed within magenta lines) were counted automatically using the In Cell Analyzer software. Scale bars = 50 μm. (B) EGR1-positive ratios in tumor tissues of melanoma in situ and invasive melanoma are plotted (see also ). Each data point represents one patient. Bars indicate the mean. (C) Relationship between EGR1-positive ratio and tumor thickness (left) or serum 5-S-cysteinyl-DOPA (5-S-CD) levels (right). Individual findings are plotted and each data point represents one patient. (D) Primary tumor tissues from patients with invasive melanoma were examined by fluorescent staining as indicated. Scale bars = 50 μm. (E) Correlation analysis of EGR1 and LDHA expression levels using RNA-seq data set acquired from primary tumors from 45 skin cutaneous melanoma patients in TCGA (TCGA-SKCM). Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. Pearson's correlation coefficient was used to evaluate the correlations. ∗ p < 0.05; ∗∗ p < 0.01.

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: In Situ, Staining, Immunofluorescence, Software, Expressing, RNA Sequencing, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: CD80 expression level is increased EGR1-deficient DCs (A) Levels of IL-12 p40, TNF-α, and IL-6 secreted by BMDCs stimulated with LPS (100 ng/mL) for 24 hr were measured by ELISA. Data are expressed as mean ± SD from two independent experiments (n = 3–5). (B) RNA from BMDCs from WT and Egr1 −/− mice was subjected to microarray analysis. The log2 ratio was determined for the corresponding genes categorized in cell adhesion molecules within KEGG pathways and the results were arranged in descending order. Genes that overlapped with TLR signaling pathways are in red boxes. (C) Flow cytometric analyses of CD80 and CD86 in CD11c + DCs within splenocytes from WT and Egr1 −/− mice. Representative plots are shown (left). Data are expressed as mean ± SD from two independent experiments (right, n = 3). MFI, median fluorescence intensity. (D) BMDCs from WT or Egr1 −/− mice were stimulated with LPS (100 ng/mL) and/or lactate (Lac, 20 mM) for 24 hr and the MFI of CD80 was assessed (n = 3). Data are expressed as mean ± SD. (E) CFSE-labeled CD8 + T cells from OT-I transgenic mice were co-cultured with BMDCs from WT or Egr1 −/− mice pulsed with Ova 257–264 peptide. Proliferation of OT-I CD8 + T cells was assessed after 72 hr by flow cytometry. (F and G) Tumor growth kinetics in WT or Egr1 −/− mice subcutaneously injected with 1 × 10 6 B16-F1 melanoma cells and treated with anti-PD-L1 antibody or control antibody (Ctrl), Lac and/or PBS as indicated. On days 6, 9, and 12 after injection of B16-F1 cells, 200 μg of control IgG Ab or anti-PD-L1 Ab were injected intraperitoneally (F), and Lac (5 mM, 200 μL) or PBS control (200 μL) was intratumorally administered (G). Data are shown as mean ± SD of 3–4 mice per group. (H) Tumor growth kinetics in diphtheria toxin (DTX)-treated CD11c-DTR bone marrow chimeric mice with subcutaneous injection with 1 × 10 6 B16-F1 melanoma cells and treated intratumorally with 1×10 6 WT or Egr1 −/− BMDCs. Data are shown as mean ± SD (n = 4 mice per group). Significance was analyzed using a two-tailed Student's t-test, Welch's t test, or Mann–Whitney's U test. ∗ p < 0.05; ∗∗ p < 0.01; n.s., not significant ( p > 0.05). See also and .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Microarray, Protein-Protein interactions, Fluorescence, Labeling, Transgenic Assay, Cell Culture, Flow Cytometry, Injection, Control, Two Tailed Test, MANN-WHITNEY

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet: EGR1 interacts with SRF and may regulate its activation on the Cd80 promoter (A) ChIP-seq enrichment profiles for H3K27Ac at the Cd80 locus were generated using WT or Egr1 −/− BMDCs stimulated with LPS (100 ng/mL) and lactate (Lac, 20 mM) for 24 hr. (B) ChIP-seq enrichment profiles for EGR1 at the transcription start site (TSS) at the Cd80 locus were generated using WT BMDCs stimulated by LPS (100 ng/mL) and Lac (20 mM) for 24 hr. (C) The 3D protein structure of EGR1 and the EGR1 binding sequence from Protein DataBank were analyzed by CLC Genomics Workbench. (D) SRF was predicted as a potential transcription factor that binds to the region identified in (B) using TFBIND software. SRF was previously reported to interact with EGR1. TF, transcription factor. (E) ChIP-PCR analysis was performed using RAW 264.7 cells overexpressing Srf . ChIP was performed using control IgG (Ctrl) or anti-SRF antibody. PCR was performed using primers that detect sequences in the Cd80 promoter. (F) Co-immunoprecipitation experiments assessing EGR1 binding to SRF. Cell extracts from RAW 264.7 cells overexpressing both Srf and Myc-tagged Egr1 were immunoprecipitated with anti-SRF antibody or control IgG (Ctrl) and analyzed with anti-c-Myc and anti-SRF antibody by Western blotting. (G) Proposed schematic model of how lactate and its induction of EGR1 switches a hot tumor to a cold tumor. Lactate induces a shift from a hot tumor (presence of inflammation) to a cold (non-inflamed) tumor, while TLR4 ligands cause the opposite shift. Lactate upregulates the expression of EGR1, which may downregulate the expression of CD80. See also .

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Activation Assay, ChIP-sequencing, Generated, Binding Assay, Sequencing, Software, Control, Immunoprecipitation, Western Blot, Expressing

Journal: iScience

Article Title: A mechanism of cooling hot tumors: Lactate attenuates inflammation in dendritic cells

doi: 10.1016/j.isci.2021.103067

Figure Lengend Snippet:

Article Snippet: Immunofluorescent staining was performed using the following antibodies and reagents: anti-mouse EGR1 (ab174509, Abcam), anti-mouse EGR1 (ab133695, Abcam), anti-human EGR1 (ab194357, Abcam), Alexa Fluor 647-goat anti-rabbit IgG (H+L) (A-21245; Thermo Fisher Scientific), Alexa Fluor 405-goat anti-rabbit IgG H&L (Abcam), Hoechst 33342 solution (Dojindo, Kumamoto, Japan), APC anti-human CD20 Antibody (BioLegend), Alexa Fluor 594 anti-MART-1 antibody (BioLegend), APC anti-human CD56 (NCAM) antibody (BioLegend), APC anti-human CD68 antibody (BioLegend), and APC anti-human CD11c antibody (BioLegend).

Techniques: Control, Recombinant, Isolation, Real-time Polymerase Chain Reaction, Enzyme-linked Immunosorbent Assay, Cell Isolation, Reporter Assay, Mutagenesis, Chromatin Immunoprecipitation, DNA Purification, Staining, Lactate Assay, Microarray, Negative Control, Plasmid Preparation, Software, Transfection

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 1. Nucleolar SIRT7 participates in multiple pathways in ribosomal biogenesis. A, B, HeLa cells treated with RNase A or buffer alone (Ctrl). Localizations of endogenous MYBBP1A, SIRT7, and RPA194 were detected via immunofluorescence microscopy. C, SIRT7 interacts with proteins involved in ribosome biogenesis. Rela- tive abundances of SIRT7-interacting proteins specific to ribosome biogenesis are indicated by the logarithm of the ratio of NSAF/PAX values. Node color and size correlate with NSAF/PAX ratios. Protein networks were assembled by STRING and visualized by Cytoscape. D, E, representative collision-induced dissociation MS/MS spectra of doubly charged tryptic peptides from mTOR (D) and GTF3C1 (E). Assigned b and y ion sequence fragments and prominent neutral losses from the precursor are labeled. BPI, base peak intensity.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Immunofluorescence, Microscopy, Tandem Mass Spectroscopy, Sequencing, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 2. SIRT7 knockdown negatively regulates synthesis rate of rRNA. A, transcription inhibition in mitosis phase. Fluorescence mi- croscopy shows EU incorporation (EU-Alexan 488) into RNA 1 h after pulse-labeling. Cells undergoing mitosis exhibit chromosome con- densation (arrows). B, protein levels monitored via Western blotting following siRNA treatment (2 days). C, reduced synthesis rates of nascent RNA upon SIRT7 knockdown. Top: selected fluorescent images from EU-labeled HeLa cells treated with siRNA for 2 days. Relative EU levels, shown in parentheses, were calculated by normal- ization to the mean fluorescence value of control siRNA-treated cells. “Cell #” indicates the number of cells measured for each experiment. Bottom: normalized total cell fluorescence output from cells treated with siRNA targeting SIRT7. Mean fluorescence value (m) and stand- ard deviations (S.D.) for each condition are as follows: ctrl, m 69k, S.D. 44k; SIRT7, m 34k, S.D. 21k. p values were calculated using Student’s t test. Images were acquired using a 60 oil immer- sion lens; bar, 10 m. D, EU fluorescence distribution following pulse- labeling in cells treated with siRNA (si), with or without RNaseA, as analyzed via flow cytometry. E, pulse-and-chase experiment for measuring RNA stability. HeLa cells were treated with control or SIRT7 siRNA for 2 days and then EU labeled for 1.5 h. Then the cells were chased with different time points.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Fluorescence, Labeling, Western Blot, Control, Flow Cytometry

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 3. SIRT7 associates with ribosome units and regulates protein synthesis rate. A, co-localization of SIRT7-EGFP and RPL11 within nucleoli. A cell line stably expressing SIRT7-EGFP fusion pro- tein was transfected with RPL11 cDNA and monitored via immuno- fluorescence microscopy with a 60 oil immersion lens; bar, 10 m. B, co-fractionation of RPL5, RPL11, and SIRT7. Cells were treated with control and SIRT7 siRNA for 2 days. The distributions of ribo- some proteins and SIRT7 were analyzed by sucrose gradient. Pro- teins from individual fractions were precipitated and analyzed via Western blotting. C, SIRT7 knockdown inhibits protein synthesis. Protein synthesis rates after siRNA treatment for 3 days were moni- tored via pulse L-homopropargylglycine labeling followed by Click-iT reaction with a fluorescent dye.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Stable Transfection, Expressing, Transfection, Fluorescence, Microscopy, Fractionation, Control, Western Blot, Knockdown, Labeling

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 4. SIRT7 knockdown preferentially suppresses protein syn- thesis rates and inhibits proliferation. A, preferential inhibition of protein synthesis by SIRT7. Top: comparison of RNA and protein synthesis rates following siRNA treatment for 2 days. Bottom: knock- down efficiencies of individual siRNA were monitored via Western blotting. B, HeLa cells were transfected with SIRT7 siRNA and differ- ent plasmids (GFP, wild type, 111) for 2 days. The protein synthesis rates were measured using an HPG labeling method (top panel). The levels of SIRT7 were confirmed by Western blotting (bottom panel). WT: SIRT7 wild type; S111A: SIRT7 deacetylation-impacted mutant. GFP served as a control for overexpression. C, after siRNA treatment for 2 days, HeLa cells were treated with nucleotide releasing buffer for the detection of ATP levels. ADP levels were sequentially measured by adding ADP converting enzyme. The ratio of ADP to ATP in each condition was calculated. Camptothecin (CPT) was used as a control. D, E, cell proliferation rates following siRNA treatments for 2 and 3 days, respectively. p values were calculated using Student’s t test.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Knockdown, Inhibition, Comparison, Western Blot, Transfection, Labeling, Mutagenesis, Control, Over Expression

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 5. SIRT7 regulates the functions of mTOR and Pol III com- plexes. A, validation of SIRT7 interaction by reciprocal immunopre- cipitation using antibodies against GTF3C1 and mTOR. B, C, after SIRT7 siRNA treatment for 2 days, GTF3C1 and mTOR were immu- nopurified and analyzed via Western blotting using anti-acetyl-Lys antibody. D, HeLa cells were treated with SIRT7 siRNA for 2 days. The nucleus and nucleolus were enriched and analyzed via Western blot- ting using antibody specific to acetyl-lysine (Acetyl-K). E, SIRT7 knockdown affects mTOR pathway. Phosphorylated S6 (top panel) and LC3B protein (bottom panel) abundances were monitored via Western blotting in cells treated with siRNA. Numbers represent S6-P/S6 total (top panel) and LC3B/tubulin (bottom panel) ratios. F, STRING network of protein–protein interactions between RNA Pol III subunits and TFIIIC2 complex members. Colored nodes, reflecting relative abundance (NSAF, %), represent proteins co-isolated with SIRT7-EGFP from purified nuclei. “White nodes” added by STRING were colored gray, depicting the connectivity between RNA Pol III and

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques: Biomarker Discovery, Western Blot, Knockdown, Protein-Protein interactions, Isolation, Purification

Journal: Molecular & Cellular Proteomics

Article Title: Sirtuin 7 Plays a Role in Ribosome Biogenesis and Protein Synthesis

doi: 10.1074/mcp.m113.031377

Figure Lengend Snippet: FIG. 6. Model for potential roles of SIRT7 in ribosome biogen- esis. Role of SIRT7 in Pol I, Pol III, and mTOR. SIRT7 majorly localizes within nucleoli and forms multiple complexes with ribosomes, mTOR, and TFIIIC2. Both mTOR and SIRT7 can interact with TFIIIC2 to regulate Pol III transcription. The SIRT7–mTOR complex may regulate an autophagy pathway that affects cell growth and proliferation.

Article Snippet: Isolation of EGFP and SIRT7-EGFP from the nuclear-enriched fraction was performed under the same conditions used for whole cell lysates; the samples were suspended in a buffer optimized for SIRT7 immunoaffinity purification (20 mM HEPES-KOH, pH 7.4, 0.1 M potassium acetate, 2 mM MgCl2, 0.1% Tween 20, 1 M ZnCl2, 1 M CaCl2, 0.5% Triton X-100, 250 mM NaCl, 4 g/ml DNase, 1/100 (v/v) protease inhibitor cocktail (Sigma)), and the resulting lysate was incubated with magnetic beads (M270 Epoxy Dynabeads, Invitrogen) conjugated to in-house-developed rabbit polyclonal anti-GFP antibodies, as described (17).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 1. The Expression Level of Na/K-ATPase a1 Subunit in Liver Tissues, HCC Tissues and HCC Cell Line (100)

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques: Expressing

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 2. Tansfection Efficiency of HepG2 Cell with siRNA

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 3. Inhibitory Effects of Ouabain and Na/K-ATPase a1 siRNA on HepG2 Cell Proliferation by MTT

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 5. Effect of Ouabain and siRNA-Induced Apoptosis in HepG2 Cells for 24 h by Fluorescence Microscope (200)

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques: Fluorescence, Microscopy

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 8. Na/K-ATPase a1 Subunit, Mapk1, CyclinA1, CDK2 and PCNA and P21CIP1 mRNA Were Detected in HepG2 Cells under Different Treat- ments by RT-Real Time PCR

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques: Real-time Polymerase Chain Reaction

Journal: Biological & pharmaceutical bulletin

Article Title: Targeting the Na(+)/K(+)-ATPase alpha1 subunit of hepatoma HepG2 cell line to induce apoptosis and cell cycle arresting.

doi: 10.1248/bpb.33.743

Figure Lengend Snippet: Fig. 9. Na/K-ATPase a1 Subunit, Mapk1, CyclinA1, CDK2 and PCNA and P21CIP1 Protein Were Detected in HepG2 Cells under Different Treat- ments by Western Blot

Article Snippet: Na /K -ATPase a1 siRNA (sc-360010, Santa Cruz, U.S.A.), fluorescein isothiocyanate (FITC)-Control siRNA (sc-36869, Santa Cruz, U.S.A.), siRNA Transfection Reagent (sc-29528, Santa Cruz, U.S.A.); primary antibody: Na /K -ATPase a1 subunit (sc-16043, Santa Cruz, U.S.A.), Mapk1 (sc-73369, Santa Cruz, U.S.A.), PCNA (sc-53409, Santa Cruz, U.S.A.), CyclinA (sc-53229, Santa Cruz, U.S.A.), CDK2 (sc-53220, Santa Cruz, U.S.A.), P21CIP1 (sc-53393, Santa Cruz, U.S.A.) and b-actin (sc47778, Santa Cruz, U.S.A.); Secondary antibody (KPL, U.S.A.); PrimeScriptTM RT and all primers (TaKaRa, Japan); 2 SYBR green PCR master mix (ABI, U.S.A.); Trizol (Promega, U.S.A.); Fura-3/AM, H2DCF-DA (Invitrogen, U.S.A.).

Techniques: Western Blot

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Malic acid induced cells’ morphological changes and decreased the number of total viable human keratinocytes (HaCaT cells). Cells were incubated with or without 15 mM of malic acid for 6, 12, 24, and 48 h, and then were examined and photographed by a phase-contrast microscope ( A ) and were harvested for determination the percentage of viable cells by flow cytometry ( B ). Data are presented as means ± S.D. of the results from three independent experiments ( * p < 0.05 vs. the indicated group).

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Incubation, Microscopy, Flow Cytometry

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Malic acid changed the DNA content and induced apoptosis in HaCaT cells ( A ); and normal human epidermal keratinocytes (NHEKs) ( B ). Cells were treated with 15 mM of MA for 0, 12, and 24 h. The cell cycle distribution and sub-G 1 group (apoptosis phase) were determined using flow cytometric analysis and obtained from three independent experiments with similar results. Data are presented as means ± S.D. of the results from three independent experiments ( ** p < 0.01 vs. the indicated group).

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques:

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Effects of malic acid on DNA damage of HaCaT cells. Cells were treated with 15 mM of malic acid for various time periods. Cells were harvested individually and then were measured with DAPI staining ( A ); and DNA gel electrophoresis ( B ) as described in Materials and Methods. Data are presented from three independent experiments.

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Staining, DNA Gel Electrophoresis

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Increase of oxidative stress and anaerobic glycolysis in HaCaT cells treated with malic acid. ( A ) Malic acid increased mitochondrial ROS production in HaCaT cells detected by flow cytometry; ( B ) malic acid increased ROS production in terms of increasing 2',7'-dichlorodihydrofluorescein (DCF) fluorescence intensity in HaCaT cells detected by flow cytometry; ( C ) The mean values of oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were measured in real-time by a Seahorse XF24 Analyzer. Data are presented as means ± S.D. of the results from three independent experiments ( ** p < 0.01 vs. the indicated group, * ).

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Flow Cytometry, Fluorescence

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Malic acid stimulated ROS, caspase-3, -8 and -9 activities and altered viability after pre-incubation with specific inhibitors in HaCaT cells. ( A ) Cells were incubated with 15 mM malic acid for 24 h before exposure in presence and absence of the specific inhibitors of caspase-3 (Z-DEVD-FMK) for 3 h to measure the viability in HaCaT cells; ( B ) Malic acid-treated HaCaT cells were in the presence and absence of N-acetyl-L-cysteine (NAC) at a concentration of 10 mM. Cell viabilities were then determined as described above. Data are presented as means ± S.D. of the results from three independent experiments ( * p < 0.05 vs. the indicated group).

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Incubation, Concentration Assay

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: Western blot demonstrated the increase of Fas, FasL, Bax, Bid, caspase-3, -8, -9, cytochrome c , GADD153, GRP78, ATF6α, and decrease of Bcl-2 in HaCaT cells exposed to malic acid at 15 mM for different incubation periods. Data are presented as means ± S.D. of the results from three independent experiments ( * p < 0.05 vs. the control group).

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Western Blot, Incubation, Control

Journal: Toxins

Article Title: Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

doi: 10.3390/toxins7010081

Figure Lengend Snippet: The proposed signaling pathways of malic acid-induced G0/G1 phase arrest and apoptosis in human epidermal keratinocytes HaCaT cells.

Article Snippet: HaCaT cells (Cell Lines Service, Eppelheim, Germany) were cultured on Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 1% L-glutamine, 25 mM HEPES, 10% fetal bovine serum and 1% penicillin-streptomycin (Gibco, Carlsbad, CA, USA) at 37 °C in a humidified incubator with 5% CO 2 atmosphere [ ].

Techniques: Protein-Protein interactions

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Mating strategy to obtain Sst-Cre/tdTomato (control) and Sst-Cre/tdTomato/Dnmt1 loxP (KO) mice. Sst tm2.l(cre)Zjh /J mice were mated with tdTomato reporter mice ( B6.CgGt(ROSA)26Sor tml4(CAG-tdTomato)Hze ) resulting in Sst-Cre/tdTomato mice with an internal ribosomal entry site ( IRES ), a Cre-recombinase sequence, a polyA sequence, and a frt -flanked neo cassette located in the 3’ untranslated region (UTR) of the Somatostatin ( Sst ) locus on chromosome 16, limiting the respective Cre expression to Sst -positive neurons. The tdTomato sequence and a loxP-flanked stop cassette are located in the Rosa26 locus. Triple transgenic Sst-Cre/tdTomato/Dnmt1 loxP mice were obtained by crossing control mice with a Dnmt1 loxP strain ( B6; 129Sv-Dnmt1tm4Jae/J ; exon 4 and 5 of the Dnmt1 gene are loxP -flanked, resulting in a null allele of these loci and a subsequent DNMT1 deficiency. (b) Exemplary microphotographs depicting SST expression in tdTomato-cells in coronal brain sections of a four-month-old male Sst-Cre/tdTomato mouse (Bregma 2.76). (c) Magnified images of the areas outlined in b. Scale bars: 100 µm in b and 20 µm c. (d-g) Quantification of DNMT1 expression by immunohistochemistry in Dnmt1 -control (d, e) and Dnmt1 -KO (f, g) male mice (six-month-old individuals) in 30 µm, fixated coronal cryosections. Scale bars (d, f) 100 µm and 10 µm (e,g) . (h) Quantification of the ratio of DNMT1-positive to DNMT1-negative tdTomato -expressing cells (Bregma 1.18, 1.10, 0.14, –0.22, –2.92. –3.08, –3.28 were used for quantification). Nested t -test , (p < 0.001 ***, N = 3 for Sst-Cre/tdTomato ( n = 18) and Sst-Cre/tdTomato/Dnmt1 loxP ( n = 17), respectively.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Control, Sequencing, Expressing, Transgenic Assay, Immunohistochemistry

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: Differential gene expression and methylation analysis of Sst-Cre/tdTomato control (ctrl) and Sst-Cre/tdTomato/Dnmt1 loxP ( knockout; KO) mice based on total RNA and enzymatic methyl-sequencing of FAC-sorted tdTomato + cells isolated from the E14.5 basal telencephalon. For RNA– and methyl-sequencing, we separately processed two samples per genotype, each consisting of cells pooled from multiple embryos (RNA sequencing: Ctrl n = 23 embryos; KO n = 14 embryos; methyl-sequencing: Ctrl n = 15 embryos; KO n = 11 embryos). (a) Heatmap depicting the 50 genes with the lowest adjusted p -values, color-coded based on relative expression. (b) Volcano plot depicting the differentially expressed genes (DEGs) between control and KO samples. Genes annotated as significantly changed (adjusted p -value < 0.05) are colored based on the fold change of the expression in the KO group. ( c ) Volcano plot of differentially methylated sites (DMS) identified in KO cells compared to control samples ( p < 0.001 for color-labeled genes). (d) Venn diagram illustrating the overlap between up-or downregulated genes and genes associated with or containing a differentially methylated region (DMR). The intersection of upregulated and differentially methylated genes after knockout was tested for enrichment of the DNMT1 binding motif identified by DNMT1-ChIP-sequencing, shown in g. (e) DEGs identified in Dnmt1 KO samples were compared to DEGs determined in embryonic stem cell-derived murine neurons overexpressing DNMT1 (f) Selection of brain development-related gene ontology (GO) terms enriched in genes that were both upregulated and differentially methylated in E14.5 Sst-Cre/tdTomato/Dnmt1 KO cells. (g) DNA motif enriched in DNMT1-ChIP-sequencing experiments. ( h ) Scatter plot combining DNA methylation changes and differential gene expression highlighting Arx as a potential target of DNA methylation-dependent transcriptional control by DNMT1. Bismark Methylation calls were filtered for a minimum of 3 reads per CpG and averaged for the promoter of each gene that was associated with a DMR and was either up or downregulated. Promoter regions were defined as 1kb upstream of the TSS plus 100 bp into the gene body. (i) Position of differentially methylated regions relative to the transcription start sites of genes related to cIN development. Horizontal bars indicate the region’s size, and color-coded is the mean methylation change for the respective region. (j) RNA sequencing tracks combined with the methylation profile of the Arx gene locus obtained from ctrl and KO samples.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Gene Expression, Methylation, Control, Knock-Out, Sequencing, Isolation, RNA Sequencing, Expressing, Labeling, Binding Assay, ChIP-sequencing, Derivative Assay, Selection, DNA Methylation Assay

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a-c) ErbB4 level is increased after Dnmt1 knockdown (KD) in MGE cells (E14.5 + 1div). Exemplary microphotographs showing control siRNA-treated (a) and Dnmt1 -KD cells (b) stained for Phalloidin-647 and ErbB4. On the very right of each example, thermal-color-coded panels represent the respective fluorescent intensity of ErbB4 (therm. (thermal) LUT). A dark blue color indicates 0 and a red color represents 5000 fluorescent units. Scale bars: 10 µm. (c) Quantification of (a) and (b) comparing the integrated density (IntDens) of ErbB4 fluorescence signals of control and Dnmt1 -KD MGEs. Student’s t -test with subsequent Welch’s correction, p < 0.001 *** ( n = 88 cells for control siRNA and 78 cells for Dnmt1 siRNA, N = 4 experiments). (d-f) Live-cell imaging analysis of Sst-Cre/tdTomato and Sst-Cre/tdTomato/Dnmt1 loxP cells in E14.5 organotypic brain slices (350 µm coronal sections, time-period of imaging: 20 h). Analysis of the migrated path length and the radial migration velocity are shown in (d) and (e) , respectively. (f) DNMT1 does not impact the migratory morphology of tdTomato-positive cells investigated in organotypic brain slices (350 µm coronal sections). Nested t- test ( p < 0.01 **) with N = 3 embryos and N = 4 slices for control and KO animals, n = 185 cells for control, and n = 219 KO cells. (g-i) siRNA-mediated depletion of Dnmt1 in MGE-derived single cells in vitro (E14.5 + 1div) does not affect the cellular morphology compared to control siRNA-transfected cells. Scale bars: 10 µm. Quantification is shown in (i) . Student’s t -test, n = 60 cells each for control siRNA and Dnmt1 siRNA. siRNA: small-interfering RNA; LP: leading process, n.s.: not significant.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Knockdown, Control, Staining, Fluorescence, Live Cell Imaging, Imaging, Migration, Derivative Assay, In Vitro, Transfection, Small Interfering RNA

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Schematic illustration of a coronally sectioned E14.5 brain hemisphere, depicting the migration routes of cortical interneurons. (b, c) Exemplary microphotographs of tdTomato-positive interneurons in coronally sectioned (50 µm) hemispheres of Som-Cre/tdTomato (control) (b) and of Som-Cre/tdTomato/Dnmt1 loxP (KO) embryos (c) . Scale bars: 500 µm. (d) Quantitative analysis of the proportional distribution of tdTomato-positive cells in the basal telencephalon and the cerebral cortex in E14.5 coronal sections (Nested t -test , n = 12 sections for control and n = 8 sections for KO, N = 3 embryos per genotype). (e) Quantification of tdTomato-positive interneuron number within the cortex of control and KO embryos normalized to the area, analyzed in 50 µm coronal cryosections (Nested t -test n = 14 sections for control and n = 8 sections for KO, N = 3 embryos per genotype). (f, g) Magnified microphotographs of the cortices of control and KO embryos (E14.5) to illustrate the distribution of tdTomato + cells within the cortical zones (MZ: marginal zone, CP: cortical plate, IZ-VZ: intermediate zone to ventricular zone). White asterisks point to cells migrating along the MZ (superficial migratory stream) or deep stream. Yellow arrows indicate tdTomato + interneurons located in the CP. Scale bars: 100 µm. (h) Quantitative analysis of the proportional distribution of tdTomato + cells within the cortical zones normalized to the overall tdTomato + -cell count within the cortex of control and KO embryos, analyzed in 50 µm coronal cryosections (Nested t -test, n = 13 sections for control and n = 14 sections for KO, N = 3 embryos per genotype). (i, j) Temporal z-projections of migration tracks of tdTomato-positive cells in organotypic brain slice cultures (350 µm thickness, coronal) from E14.5 Som-Cre/tdTomato (control) and Som-Cre/tdTomato/Dnmt1 loxP (KO) embryos, captured over 20 h by live-cell imaging. Scale bars: 500 µm. k-n) Quantification of the live-cell recordings in organotypic brain slices. Quantitative analysis of the increase in fluorescence intensity in the cortical plate due to invasion of tdTomato cells (first 12 h, Nested t -test, n = 4 slices for control and n = 6 slices for KO embryos, N = 3 embryos per genotype) is shown in (k) , the average number of directional changes ( n = 134 cells across 5 slices for control ( N = 4 embryos), n = 74 cells across 3 slices for KO ( N = 3 embryos), Nested t -test) is depicted in (l) . The velocity of all cells migrating within the cortex ( n = 377 cells across 6 slices for control ( N = 5 embryos), n = 323 cells across 5 slices for KO ( N = 3 embryos)) is shown in (m), while the speed of cells within the area between CP and VZ is illustrated in (n) for control ( n = 193 cells across 6 slices, N = 5 embryos) and KO ( n = 114 cells across 3 slices, N = 3 embryos); Nested t -test. (o, p) Selected frames extracted from live-cell imaging recordings of organotypic brain slices (350 µm) derived from E14.5 control and KO embryos. Depicted are tdTomato-positive cells migrating through the cortex. For each genotype, three selected cells (dotted circles) were tracked. Scale bars: 50 µm. Error bars represent the standard error of the mean (SEM). p < 0.05 * , p < 0.01 ** , p < 0.001***; n.s.: not significant. CP: cortical plate, VZ: ventricular zone, SVZ: subventricular zone, MZ: marginal zone, IZ: intermediate zone.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Migration, Control, Cell Counting, Slice Preparation, Live Cell Imaging, Fluorescence, Derivative Assay

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Schematic illustration of the regions and extensions measured to analyze cortical extensions and areas of coronal, 50-µm-thick, fixated slices. Respective quantifications are depicted in (b) and (c) . Nested t -test, N = 3 for both genotypes, n = 13 Sst-Cre/tdTomato and n = 9 Sst-Cre/tdTomato/Dnmt1 loxP . MZ: marginal zone, IZ: intermediate zone, SVZ+VZ: subventricular and ventricular zone, not significant.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques:

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Exemplary microphotographs of tdTomato + interneurons in coronally sectioned (50 µm) hemispheres of E16.5 Som-Cre/tdTomato (control) and of Som-Cre/tdTomato/Dnmt1 loxP (KO) embryos; scale bars: 500 µm. TdTomato-positive cells are labeled in red and DAPI in blue. Quantitative analysis of the proportional distribution of tdTomato cells in the basal telencephalon and the cerebral cortex in E16.5 coronal sections (Nested t -test , n = 11 sections for control and n = 15 sections for KO, N = 4 embryos per genotype; error bars represent the standard error of the mean (SEM). (c, d) Magnified microphotographs of the cortices of control and KO embryos from E16.5 coronal brain sections (50 µm) to illustrate the distribution of tdTomato cells within the cortical zones. Scale bars: 100 µm. (e) Quantitative analysis of the proportional distribution of tdTomato cells within the E16.5 cortical zones normalized to the overall tdTomato-cell count within the cortex of control and KO embryos, analyzed in 50 µm coronal cryosections. Nested t -test , n = 13 sections for control and n = 14 sections for KO, N = 4 embryos per genotype. (f) Quantitative analysis of tdTomato cell density normalized to the given area of the respective cortical zones (E16.5, Nested t -test, n = 13 sections for control and n = 14 sections for KO, N = 4 embryos per genotype). (g, h) Exemplary microphotographs of tdTomato-positive interneurons in the cortex of coronally sectioned (50 µm) brains of E18.5 Som-Cre/tdTomato (control) and Som-Cre/tdTomato/Dnmt1 loxP (KO) embryos; scale bars: 100 µm. (i) Quantitative analysis of the proportional distribution of tdTomato cells within the E18.5 cortical zones normalized to the overall tdTomato-cell count within the cortex of control and KO embryos, analyzed in 50 µm coronal cryosections. Nested t -test, n = 11 sections for control and n = 9 sections for KO, N = 4 embryos per genotype, error bars represent the SEM. (j) Quantitative analysis of tdTomato cell density normalized to the given area of the respective cortical zones (E18.5, Nested t -test, n = 11 sections for control and n = 9 sections for KO, N = 4 embryos per genotype). MZ: marginal zone, CP: cortical plate, IZ-VZ: intermediate zone to ventricular zone. p < 0.05 * , p < 0.01 ** , p < 0.001 ***, n.s.: not significant.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Control, Labeling, Cell Counting

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a, d) Schematic illustration of the regions and extensions measured to analyze cortical dimensions and areas of coronal, 50-µm-thick, fixated slices at E16.5 (a) and E18.5 (d) . Respective quantifications are depicted in (b) , (c), and (e) . (b and c) Quantification of the cortical dimension of embryos at E16.5. Nested t -test, N = 4 embryos for both genotypes, n = 12 slices for Sst-Cre/tdTomato, and n = 15 slices for Sst-Cre/tdTomato/Dnmt1 loxP . (e) Quantification of the cortical dimension of embryos at E18.5. Nested t -test, N = 4 embryos for both genotypes, n = 12 slices for Sst-Cre/tdTomato, and n = 9 slices for Sst-Cre/tdTomato/Dnmt1 loxP . MZ: marginal zone, CP: cortical plate, IZ: intermediate zone, SVZ+VZ: subventricular zone, n.s.: not significant.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques:

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a) Schematic illustration of a coronally sectioned embryonic brain hemisphere, depicting the lateral and dorsal localization of the two bin selections, used to quantify the density and distribution of EOMES + – and TBR1 + -cells in E14.5 and E16.5 brain sections. (b-e) TBR1 and EOMES immunostaining in coronally sectioned (50 µm) brains of E14.5 Sst-Cre/tdTomato (control) and Sst-Cre/tdTomato/Dnmt1 loxP (KO) embryos. (b and d) Exemplary microphotographs of ( b ) TBR1 immunostaining (green) and (d) EOMES immunostaining (green); scale bars: 50 µm (DAPI is shown in blue). (c and e) Quantitative analysis of the TBR1 + postmitotic neuron numbers (c) and EOMES + intermediate progenitor cell (IPC) numbers (e) per bin in the E14.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections (two-way ANOVA with subsequent Bonferroni correction , n = 13 control and n = 9 KO sections from N = 3 embryos for both genotypes). (f) TBR1 immunostaining (green) in coronally sectioned (50 µm) brains of E16.5 control and KO embryos, scale bars: 50 µm. (g) Quantitative analysis of the TBR1-positive postmitotic neuron numbers per bin in the E16.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections (two-way ANOVA with subsequent Bonferroni correction , n = 8 for control and KO from N = 3 embryos for both genotypes). (h and j) Exemplary microphotographs of ( h ) EOMES immunostaining (green) and (j) TBR1 immunostaining (green) in coronal sections of E18.5 control and KO brains; scale bars: 50 µm (DAPI is shown in blue). (i and k) Quantitative analysis of the density of EOMES-positive IPCs (i) and TBR1-positive postmitotic neurons (k) in the E18.5 cortex of control and KO embryos, analyzed in 50 µm coronal cryosections(Nested t -test , n = 11 sections for both genotypes from N = 4 embryos per genotype). (l) Schematic illustration of the cortex of a coronally sectioned embryonic brain (E18.5), depicting the parameters analyzed for the quantification shown in ( m ). Three regions (lateral (1), dorsolateral (2), and dorsal (3)) were averaged to obtain the mean thickness respectively for each layer (nested t -test ; n = 12 control and n = 9 KO sections . N = 4 brains for both genotypes). (n and o) Panels depict significant ligand-receptor pairs involved in communication from SST + cINs to cortical progenitors, filtered by differentially expressed genes (DEG) from single-cell RNA-sequencing data. Communication probability is represented by dot color, and p -value by dot size, with p -values being computed using a one-sided permutation test. Panel ( n ) shows the communication with different apical progenitor (AP) populations at E14.5. Panel (o) illustrates communication with IPCs at E14.5. Panels (p-r) present microphotographs of cell pair combinations formed from cortical neurons isolated from E14.5 C57BL6/J embryos, plated at clonal density, after 24 hours. Cell clones were either treated with control-or recombinant efnB2-FC for 24 h, prior to immunocytochemical staining for nestin (magenta), β-III-tubulin (TUBB3, green), and DAPI (blue). Scale bars: 5 µm. (s) Quantification of the proportion of nestin/nestin, nestin/β-III-tubulin and β-III-tubulin/β-III-tubulin-positive cell pairs normalized to the overall number of these pairs is shown in (p-r) . Student’s t -test (control-Fc: 66 cell pairs, efnB2-Fc: 58 cell pairs, N = 3). p < 0.05 *, p < 0.01 ** , p < 0.001 ***. MZ: marginal zone, CP: cortical plate, IZ-VZ: intermediate zone-ventricular zone, n.s.: not significant, ctrl: control.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Immunostaining, Control, RNA Sequencing, Isolation, Clone Assay, Recombinant, Staining

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: Exemplary microphotographs of sagittal sections from a four-months-old Sst-Cre/tdTomato animal (Bregma 2.76). (a and b) Examples of immunohistochemical stainings using an anti-somatostatin antibody (green) and an anti-neuropeptide Y antibody (NPY, magenta). TdTomato-positive cells are depicted in red. (c and d) Examples of immunohistochemical stainings using an anti-somatostatin antibody (green) and an anti-calretinin antibody (magenta). TdTomato-positive cells are depicted in red. Scale bars in (a) and (c) : 100 μm. Scale bars in (b) and (d) : 20 μm (magnified views of sections outlined in (a) and (c) , respectively). (e) Mammalian phenotype ontology analysis for the enrichment of the genes upregulated in Sst-Cre/tdTomato/Dnmt1 loxP animals at E14.5. ShinyGO 0.80, Curated.MGI pathway database.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Immunohistochemical staining

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a-d) Immunostaining using an antibody directed against parvalbumin (PV) in 50 µm brain sections of six months old Sst-Cre/tdTomato (control) and Sst-Cre/tdTomato/Dnmt1 loxP (KO) mice show increased proportions of PV + Sst-Cre/tdTomato neurons upon Dnmt1 deletion. Exemplary microphotographs of immunostainings (tdTomato in red, PV in green, DAPI in blue) taken from the primary somatosensory cortex (S1) of coronal slices (Bregma 0.14, –0.22, and –0.34) from ( a ) control and ( b ) KO mice. Scale bars: 100 µm (left panels) and 20 µm (magnifications of marked selections). (c) Quantification of the proportion of PV-positive Sst-Cre/tdTomato cells over the whole S1 as well as in supra– and infragranular layers normalized to the total amount of all detected Sst-Cre/tdTomato cells. Nested t -test with n = 11 slices for control and n = 10 for KO from N = 3 brains for both genotypes. (d-g) Dnmt1 KO mice display changes in cortical layer thickness alongside alterations in Sst-Cre/tdTomato cell density. (d, e) Exemplary microphotographs of immunostainings of CUX1 and tdTomato combined with DAPI taken from S1 (sagittal slices of Bregma 1.32 and 1.44, six-month-old male mice). The CUX1-positive upper layers II-IV can be distinguished from deep layers V and VI. Scale bars: 100 µm. (f, g) Quantification of the tdTomato-positive cell density normalized to the given area of the respective layers ( f ), the cortical thickness, and the proportional thickness of the deep and upper layers ( g ). Nested t -test with n = 24 slices for control and n = 23 for KO from N = 3 brains for both genotypes. (h-q) Neuropixels recordings in S1 of adult Sst-Cre/tdTomato (control) and Sst-Cre/tdTomato/Dnmt1 loxP (KO) mice (n = 8 sessions from 2 mice per genotype), while animals were receiving either tactile stimulation by air puffs against their whiskers or visual stimuli (h) . (i) Average local field potential (LFP) in S1 across the whole cortical depth, following a 20 ms air puff stimulus (onset at white line) to the distal whisker pad. Blue colors indicate negative, and yellow colors positive LFP responses. Across all layers, LFP responses were stronger and more temporally precise in control mice (left) compared to KO mice (right). (j) Average current source density (CSD) in S1 calculated from the measured LFP. Blue colors indicate excitatory and yellow colors inhibitory currents. The earliest responses in control mice (left) occur in the granular layer at ∼400 µm cortical depth and then spread to layer II/III (100-300 µm) and layer V (500-900 µm). In contrast, no clear spatial response profile is visible in KO mice, suggesting a disruption of cortical network function. (k) Traces of LFP responses in S1 depicting averaged LFPs across all cortical layers (0 – 1 mm) for recordings in control (gray) and KO (orange) mice (shading indicates the standard error of the mean (SEM)) illustrate significantly larger and faster tactile responses in control mice compared to KO mice (peak amplitude control = –159.09 ± 34.37 μV, peak amplitude KO = –57.17 ± 7.49 μV; Wilcoxon rank-sum test, p = 0.0059; peak time control = 26.85 ± 26.85 ms, peak time KO = 30.80 ± 1.10 ms, p = 0.0108). (l-o) Quantification of the temporal precision of the spiking response of S1 neurons to tactile stimulation in control (gray) and KO (orange) mice. (l) Peristimulus time histogram (PSTH) for spiking response of S1 neurons to tactile stimulation in KO (orange) and control mice (gray). (m) The width of the initial response, measured as the full width at half maximum (FWHM), was significantly larger in KO compared to control mice (n = 298 clusters, Wilcoxon rank-sum test, p = 7×10 -31 ). (n) Quantification of early tactile response amplitude in control versus KO mice. In the first 30 ms after tactile stimulation, responses in control mice were significantly larger compared to KO mice (mean firing rate control = 9.09 ± 0.31 Hz, n = 436 clusters, mean firing rate KO = 7.22 ± 0.32 Hz, n = 298 clusters, Wilcoxon rank-sum test, p = 0.000045). (o) Late tactile responses in KO mice were significantly larger compared to control mice (mean firing rate control = 0.4 ± 0.10 Hz, n = 436 clusters, mean firing rate KO = 3.85 ± 0.43 Hz, n = 298 clusters, Wilcoxon ranksum test, p = 4×10 -37 ). (p) Absolute spectral power density (ASPD) of LFP recordings across all layers in primary visual cortex (V1) during full-field visual stimulation in control (left) and KO mice (right). Hot colors indicate higher spectral power, units are on a log -10 scale. Spectral power falls off with frequency but shows increased activity in the higher frequency range > 20Hz between 0.3– and 0.7-mm cortical depth for control mice. In contrast, no clear modulation of higher frequencies was found in KO mice. (q) Relative change in spectral power during visual stimulation, computed as the percent difference relative to baseline. In control mice (left), visual stimulation induced a strong increase in the gamma range (> 30Hz), especially at around 70 Hz between 0.3– and 0.7-mm cortical depth. In contrast, no clear modulation was found in KO mice (right). p < 0.05 *, p < 0.01 **, p < 0.001 ***, n.s.: not significant.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Immunostaining, Control, Whisker Assay, Disruption, Activity Assay

Journal: bioRxiv

Article Title: DNMT1-Mediated Regulation of Inhibitory Interneuron Migration Impacts Cortical Architecture and Function

doi: 10.1101/2024.09.04.611268

Figure Lengend Snippet: (a-e) Nest building test reveals a significant deterioration in using given material and forming proper nests in adult Sst-Cre/tdTomato/Dnmt1 loxP (KO) individuals compared to Sst-Cre/tdTomato (control) mice. With the help of the scoring index depicted in (a) and formerly described , all mice were ranked based on nest quality and amount of material used (d, e) . Representative photographs are shown in (b) for control and (c) for KO mice. Tests were performed overnight and twice within one week. Respective values were averaged for both trials. Unpaired, two-tailed t -test with subsequent Welch’s correction. N = 16 male adult mice for both genotypes. (f-h) Pentylenetetrazol (PTZ)-injections point to defects in cortical inhibition in KO mice. (f) Schematic illustration defining PTZ-induced seizures in three-month-old males, which received intraperitoneal injections of PTZ every ten minutes until displaying a final tonic-clonic seizure (event IV) within a time window of a maximum of 120 minutes. Footages captured from video monitoring of PTZ-induced convulsions defining criteria for small seizures (events I and II) and tonic-clonic seizures (III and IV). (g, h) Quantification of seizure profiles of control (N = 9) and KO individuals ( N = 6). Unpaired, two-tailed t- test with subsequent Welch’s correction. p < 0.05 *, p < 0.01 **, p < 0.001 ***.

Article Snippet: Transfection of MGE cells with siRNA oligos occurred after 5–6 h of incubation (37°C, 5% CO and 95% relative humidity) in Neurobasal medium with phenol red, 1x B27TM, and 0.25x GlutaMAX (Gibco, U.S.A.) for 24 h. For this, the medium was supplemented with Lipofectamine TM 3000 in accordance with the manufacturer’s instructions (Thermo Fisher, U.S.A.) together with Dnmt1 siRNA (30 nM, #sc-35203, Santa Cruz, U.S.A.) and a scrambled control siRNA construct (15 nM, BLOCK-iT TM Fluorescent Oligo, #2013, Thermo Fisher, U.S.A.).

Techniques: Control, Two Tailed Test, Inhibition