Journal: Cell metabolism

Article Title: Carbon source availability drives nutrient utilization in CD8 + T cells

doi: 10.1016/j.cmet.2022.07.012

Figure Lengend Snippet:

Article Snippet: Rabbit polyclonal anti-human/mouse beta-actin , Cell Signaling , #4967.

Techniques: Luciferase, shRNA, Plasmid Preparation, Recombinant, Staining, Cell Isolation, Selection, Protease Inhibitor, Software

Journal: Scientific Reports

Article Title: Inhibition of DYRK1B suppresses inflammation in allergic contact dermatitis model and Th1/Th17 immune response

doi: 10.1038/s41598-023-34211-x

Figure Lengend Snippet: DYRK1B inhibition regulates naïve CD4 + T cell differentiation by enhancing FOXO1 activity, which results in the suppression of Th1 and Th17 differentiation signaling. ( A ) Venn diagram showing the number of genes that are individually expressed in the DYRK1B inhibition (AZ-DYRK1B-33, 1 µM) and control subsets. ( B ) A volcano plot shows differentially expressed stimulated naïve CD4 + T cells compared between the absence and presence of a selective DYRK1B inhibitor. The data point above the significance threshold (FDR < 0.05) are marked in blue (− 1.2 < Log2 fold change < 1.2) and red (− 1.2 > Log2 fold change > 1.2). ( C,D ) A dot plot shows KEGG enrichment analysis of upregulated and downregulated pathways found after addition of a selective DYRK1B inhibitor compared to control, respectively (FDR < 0.05). The size of a point reflects the number of annotated genes. ( E ) A histogram shows significantly upregulated and downregulated Th1, Th17, Treg signature genes, and FOXO1 target genes after the addition of a selective DYRK1B inhibitor compared to control (FDR < 0.05).

Article Snippet: The following primary antibodies was used: mouse anti-human FOXO1 (1452T, Cell Signaling), rabbit anti-human pFOXO1 Ser-329 (PA5-38275, Invitrogen) and mouse anti-human β-actin (sc-47778, Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Cell Differentiation, Activity Assay

Journal: Scientific Reports

Article Title: Inhibition of DYRK1B suppresses inflammation in allergic contact dermatitis model and Th1/Th17 immune response

doi: 10.1038/s41598-023-34211-x

Figure Lengend Snippet: DYRK1B inhibition reduces phosphorylation of FOXO1 and enhances FOXP3 expression. ( A ) Human naïve CD4 + T cells stimulated under Treg-polarizing conditions in the absence or presence of a selective DYRK1B inhibitor (AZ-DYRK1B-33, 1 µM) for 6 or 12 h. The extracted proteins were analyzed by immunoblotting with anti-FOXO1 or anti-pFOXO1 Ser329 . ( B ) pFOXO1 Ser329 levels from ( A ) were quantified using Image Studio version 5.2 software and are summarized in a bar graph. ( C ) Relative mRNA expression levels of FOXO1 target genes in naïve CD4 + T cells stimulated under Treg-polarizing conditions in the absence or presence of a selective DYRK1B inhibitor (AZ-DYRK1B-33, 1 µM) for 24 h were analyzed by qRT-PCR. The results are summarized in the bar graphs, and the data are presented as mean ± SEM (* p < 0.05, *** p < 0.001).

Article Snippet: The following primary antibodies was used: mouse anti-human FOXO1 (1452T, Cell Signaling), rabbit anti-human pFOXO1 Ser-329 (PA5-38275, Invitrogen) and mouse anti-human β-actin (sc-47778, Santa Cruz Biotechnology, Inc.).

Techniques: Inhibition, Expressing, Western Blot, Software, Quantitative RT-PCR

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: The Expression of Plasmacytoid Dendritic Cells and TLR7/9-MyD88-IRAKs Pathway in Chronic Eczema Lesions

doi: 10.2147/CCID.S405491

Figure Lengend Snippet: Expressions of TLR7 in chronic eczema lesions and the perilesional normal skin at the edge (SP *400). ( a ) Epidermic of skin lesion. ( b ) Dermis of skin lesion. ( c ) Perilesional normal skin. ( a ) A few TLR7+ cells with brown cytoplasm were observed in the epidermal of chronic eczema lesions. ( b ) A few TLR7+ cells with brown cytoplasm were observed in the dermal papillary layer of chronic eczema lesion. ( c ) Few TLR7+ cells with brown cytoplasm were observed in both epidermal and papillary layer of perilesional normal skin of the lesion.

Article Snippet: Rabbit anti-human CD2AP monoclonal antibody and mouse anti-human CD123 monoclonal antibody were bought from Abcam, USA, and rabbit anti-human IRAK1 monoclonal antibody, rabbit anti-human IRF7 monoclonal antibody, mouse anti-human TLR7 monoclonal antibody, and rabbit anti-human TLR9 monoclonal antibody were bought from CST, USA.

Techniques:

Journal: Clinical, Cosmetic and Investigational Dermatology

Article Title: The Expression of Plasmacytoid Dendritic Cells and TLR7/9-MyD88-IRAKs Pathway in Chronic Eczema Lesions

doi: 10.2147/CCID.S405491

Figure Lengend Snippet: The Positive Rate and Intensity of TLR7 Expression in 17 Cases of Chronic Eczema and 13 Cases of Normal Skin Around Eczema

Article Snippet: Rabbit anti-human CD2AP monoclonal antibody and mouse anti-human CD123 monoclonal antibody were bought from Abcam, USA, and rabbit anti-human IRAK1 monoclonal antibody, rabbit anti-human IRF7 monoclonal antibody, mouse anti-human TLR7 monoclonal antibody, and rabbit anti-human TLR9 monoclonal antibody were bought from CST, USA.

Techniques: Expressing

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 loss induces SOD2 and other anti-oxidant enzymes deficiency in human and mouse PAECs. ( A ) An expression of the antioxidant enzymes measured by qPCR in human PAECs (HPAECs) transfected with siGATA6 or control scr siRNA. mRNA levels in scr siRNA transfected cells were set as one. Data are means ± SE, *p < 0.05 vs scr siRNA by Kruskal–Wallis test with post hoc Dunn’s test for multiple comparisons; n = 3–10 (see also Supplemental Table ). ( B ) Chromatin immune precipitation (ChiP) assay in HPAECs, n = 8 for SOD2 ; n = 5 for GPX1 . The experiments were repeated three times. Representative gel images and data quantification are shown. Values are means ± SE, **p < 0.01, ***p < 0.001 by Mann Whitney U test. ( C,D ) Control HPAECs transfected with siGATA6 or scr siRNA (siContr) were assayed by Western blot analysis to detect GATA6 and SOD2. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 5, **p < 0.01 by Mann Whitney U test. ( E,F ) Western blot analysis of whole lung tissue from Gata6 CKO and WT mice. Representative blots are shown. Values are means ± SE of the relative protein levels by densitometry, n = 4, *p < 0.05 by Mann Whitney U test. ( G ) SOD2, GPX, and Catalase activity were measured in Human HPAECs transfected with siGATA6 or scr siRNA, Values are means ± SE, n = 3, *p < 0.05 by Mann Whitney U test. ( H ) Mouse Sod2 (n = 5), Gpx (n = 10), and Cat (n = 9) activity were measured in mouse lung tissue in Gata6 CKO and WT mice. *p < 0.05, **p < 0.01 by Mann Whitney U test (Sod activity) and unpaired τ test (Gpx and Cat activities). ( I,J ) mRNA levels of the antioxidant enzymes measured by qPCR in whole lung tissues ( I ) and in PAEC from Gata6 CKO mice (J). Data are means ± SE. I: n = 12–13 for Sod , n = 10/group for Gpx1 , n = 9/group for Cat . J: n = 4 mice/group. *p < 0.05, **p < 0.01 by unpaired τ test (I) or Mann Whitney U test ( J ). The original blots are presented in Supplementary Fig. .

Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

Techniques: Expressing, Transfection, MANN-WHITNEY, Western Blot, Activity Assay

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: GATA6 is deficient in both PAEC and PASMC in human PAH lungs. ( A ) IHC analyses were performed to detect GATA6 (green), CD31 (red) and α-SMA (red) in small PAs from healthy controls (HC) and patients with PAH (SSc-PAH and IPAH) and analysis of nuclear GATA6 in CD31- and SMA-positive cells in small PAs was performed. Left: Images are representative from 3–4 subjects/control, SSc-PAH, and IPAH. Bar equals 50 μm. White arrowheads indicate GATA6-positive cells. Right: Data are in optical density units (OD); Data are means ± SE from 3–4 human subjects per control, 7–8 for PAH (SSc-PAH + IPAH) groups. *p < 0.05 by Mann Whitney U test. ( B,C ) Immunoblot analysis of early-passage PAEC and PASMC from healthy controls (HC) and subjects with IPAH to detect indicated proteins. Data are means ± SE from n = 5 subjects/group. **p < 0.01 by Mann Whitney U test. ( D ) Immunoblot analysis of nuclear fractions of PASMC from healthy control (HC) and IPAH subjects. Data are means ± SE from n = 4 subjects/group. *p < 0.05 by Mann Whitney U test. ( E ) Immunocytochemical analysis of PASMC from healthy control (HC) and IPAH subjects to detect GATA6 (red) and DAPI (blue). Bar equals 50 µm. Representative images from two subjects/group by Mann Whitney U test. ( F–J ) Human non-diseased PASMC were transfected with siRNA GATA6 or control scrambled siRNA ( − ). 48 h post-transfection, immunoblot (F, G) and cell count analysis (H) were performed. Data are means ± SE from n = 3 subjects/group. *p < 0.05 by Mann Whitney U test. ( I ) qPCR analysis of PASMC from healthy controls (HC) and patients with PAH to detect SOD2 expression. Data are means ± SE, n = 5 subjects/group. *p < 0.05 by Mann Whitney U test. ( K,L ) Immunoblot analysis of healthy control (HC) and IPAH PASMC to detect indicated proteins was performed on the same membrane using strip- re-probe approach. Correlation between GATA6 and SOD was examined (R2). Data are means ± SE, n = 5 subjects/group. **p < 0.01 by Mann Whitney U test ( K ), R2 = 0.81 by Spearman analysis with Holm–Sidak adjusted p values ( L ). The original blots are presented in Supplementary Fig. .

Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

Techniques: MANN-WHITNEY, Western Blot, Transfection, Cell Counting, Expressing, Stripping Membranes

Journal: Scientific Reports

Article Title: GATA6 coordinates cross-talk between BMP10 and oxidative stress axis in pulmonary arterial hypertension

doi: 10.1038/s41598-023-33779-8

Figure Lengend Snippet: Treatment with DMF restores expression of the BMP receptors, reverses oxidative stress and pulmonary hypertension in Gata6 CKO mice. (DMF or vehicle were administered daily via i.p. injection for 3 weeks. ( A ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of indicated BMP receptors. Data are means ± SE, n = 6–12, *p < 0.05. **p < 0.01 by Kruskal–Wallis test followed by Dunn’s multiple comparisons test ( BmpR2, ActRIIB, Alk1 ) and one-way ANOVA followed by post hoc Tukey’s multiple comparison ( Endoglin ). ( B ) qPCR analysis of whole lung tissue from WT and Gata6 CKO mice treated with DMF or vehicle to detect expression of the antioxidant enzymes and eNOS . Data are means ± SE, n = 6–17, *p < 0.05., **p < 0.01, ***p < 0.001 by one-way ANOVA followed by post hoc Tukey’s multiple comparisons test ( SOD2, GPX1, CAT ) and Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test ( eNOS ). ( C,D ) mRNA levels of indicated BMP receptors and antioxidant enzymes measured by qPCR in PAEC from WT and Gata6 CKO mice treated with DMF or vehicle. Data are means ± SE, n = 3–6 mice/group, *p < 0.05, **p < 0.01 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test. ( E–G ) RVSP, pulmonary acceleration time as a fraction of ejection time (PAT/ET) and Fulton index (RV/[LV + S]) were evaluated in WT and Gata6 CKO mice in the presence or absence of DMF. Data are means ± SE. n = 5–11 mice/group. *p < 0.05, **p < 0.01. ***p < 0.001 by Kruskal–Wallis test with post hoc Dunn’s multiple comparisons test (RVSP) and one-way ANOVA followed by post hoc Tukey’s multiple comparisons test (PAT/ET and RV/(LV + S).

Article Snippet: Antibodies were as follows: Goat anti-human GATA6 (R&D; AF1700, 1:500), mouse anti-human GATA6 (Santa-Cruz, 517554, 1:500), Rabbit anti-human/mouse SOD2 (Cell signaling technology; 13141, 1:1000; 13145, 1:5000), Goat anti-human ALK1 (R&D; AF370, 1:500), Rabbit anti-mouse ALK1 (ABGENT AP7807a, 1:1000), rabbit anti-human/mouse ActR2B (LS bio; LS-B7781, 1:1000), mouse anti-human BMPR2 (Novus; NBP2-37624 Clone 3F6; 1:1000), rabbit anti-mouse BMPR2 (Proteintech; 19087–1-AP; 1:1000), mouse anti-mouse/human/rat BMPR2 (Abcam, catalog #130206 1:500), rabbit anti-human/mouse Endoglin (Proteintech; 10862–1-AP; 1:1000), rabbit anti-mouse BiP (Abcam; ab53068; 1:1000), rabbit anti-mouse CHOP (Novus; NBP2-13172; 1:1000), mouse anti-beta-actin (Sigma; A1978; 1:2000), mouse anti-human PRPF4 (Abcam, 69878, 1:1000), rabbit anti-human MYEOV (Invitrogen, PA5-48938 1:1000), rabbit anti-human STING (Invitrogen, PA5-26751, 1:1000), rabbit anti-human/mouse/rat EAF1 (Invitrogen, PA5-100493, 1:1000), rabbit anti-human/mouse/rat Histone H3 (Cell Signaling, 9715 1:1000), rabbit anti-human/mouse/rat a-b Tubulin (Cell Signaling, 2148, 1:1000).

Techniques: Expressing, Injection

Journal: Free radical biology & medicine

Article Title: Suppressive effects of iron chelation in clear cell renal cell carcinoma and their dependency on VHL inactivation

doi: 10.1016/j.freeradbiomed.2018.12.013

Figure Lengend Snippet: ccRCC (786–0 and A704) and benign renal (RPTEC and HRCEp) cell lines were treated with and without 125 μM DFO for 48 hours (all cell lines) and 120 hours (A704 only), or with 125 μM DFX for 48 hours (A704 only). Treated cells were analyzed using Western blot to measure HIF1-α and HIF2-α protein levels, with β actin protein as a loading control; and using quantitative PCR (qPCR) to measure mRNA transcript levels of HIF-α transcriptional targets (CA9, OCT4, PDFGβ). (A) Representative Western blot of HIF-1α and HIF-2α proteins in ccRCC and benign renal cell lines with and without 48-hour DFO treatment. (B) Changes in gene expression for HIF-1α and HIF-2α/EPAS1 with and without 48-hour DFO treatment. (C) Representative western blots of HIF-2α protein levels in A704 cells treated with DFO for 0, 48 and 120 hours (left panel), or with and without DFO or DFX for 48 hours (right panel). (D) Changes in expression of HIF-α transcriptional target genes, CA9 (HIF1α), OCT4 (HIF-2α) and PDGF-β (HIF-1α/HIF-2α) after 48-hour DFO treatment. (E) Changes in OCT4 gene expression in A704 cells after extended (120-hour) DFO treatment. For gene expression data, mean levels in treated cells are normalized to those of untreated cells (dashed line). All data are from at least 3 independent experiments, with error bars denoting SEM. *, p<0.05; **, p<0.01 and ***, p<0.001 for comparison of treated and untreated cells using a one-sample t-test.

Article Snippet: Separated protein was transferred onto PVDF membranes (Bio-rad), which were then blocked for one hour at room temperature in Tris-buffered saline containing 0.1% tween (TBS-T) with 5% fat-free milk, followed by overnight incubation at 4°C with mouse anti-human HIF1-α antibody (Cell Signaling Technologies #14179S, Danvers, MA) (1:1000 dilution), goat anti-human HIF2-α antibody (1:1000 dilution) (R&D Systems #AF2997, Minneapolis, MN) or mouse anti-human β-actin antibody (1:10,000 dilution) (Cell Signaling Technology #3700S) in 5% fat-free milk with TBS-T. Membranes were washed in TBS-T and incubated for 20 minutes at room temperature with a 1:2000 dilution of horseradish peroxidase-conjugated rabbit anti-mouse antibody (Cell Signaling Technology #7076S), goat anti-rabbit antibody (Promega Life sciences #W401B), or donkey anti-goat antibody (R&D Systems #HAF109) in 5% milk with TBS-T.

Techniques: Western Blot, Real-time Polymerase Chain Reaction, Expressing