Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. Knockdown of TAp73 decreases, whereas knockdown of ΔNp73 increases, the expression of p21 and PUMA. The levels of p21, PUMA, and actin were measured in parental MDCK, MDCK-TAp73-KD, and MDCK-ΔNp73-KD cells by Western blotting. B. Generation of MDCK cell lines in which p21 was stably knocked down. Parental MDCK and MDCK-p21-KD cells were mock-treated or treated with camptothecin for 18h and the level of p21 and actin protein was measured by Western blotting. C. Representative microscopic images of parental MDCK and MDCK-p21-KD cells in 2-D cultures. D. Top panel: colony formation assay was performed with parental MDCK or MDCK-p21-KD cells. Bottom panel: the number of colonies was counted and presented as Mean ± S.D. from three separate experiments. E. Wound healing assay was performed with parental MDCK and MDCK-p21-KD cells. Top panel: cell migration was determined by visual assessment of cells migrating into the wound for 24 h using a phase-contrast microscopy. The dashed lines indicate the wound edge. Bottom panel: the time required for would closure was measured and presented as Mean ± s.d. from three separate experiments. F. Representative images of parental MDCK and MDCK-p21-KD cells in 3-D culture for 6 d or 12 d. Scale bar: 50 μM.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Expressing, Western Blot, Stable Transfection, Colony Assay, Wound Healing Assay, Migration, Microscopy

Journal: Oncotarget

Article Title: P73 tumor suppressor and its targets, p21 and PUMA, are required for madin-darby canine kidney cell morphogenesis by maintaining an appropriate level of epithelial to mesenchymal transition

doi:

Figure Lengend Snippet: A. - D. The levels of β-catenin, E-cadherin, Snail, Twist, and actin were determined by Western blotting with extracts from parental MDCK (A-D), MDCK-TAp73-KD A. , MDCK-p21-KD B. , MDCK-PUMA-KD C. , and MDCK-ΔNp73-KD D. cells. E. The levels of E-cadherin, Snail and Twist transcripts were measured by qRT-PCR in parental MDCK cells and MDCK cells with knockdown of TAp73, ΔNp73, p21 or PUMA. The level of Actin was measured as an internal control. F. A model for the role of p73, p21 and PUMA in MDCK cell morphogenesis.

Article Snippet: Antibodies used were purchased from Bethyl (anti-p73), ProSci (anti-PUMA), Santa Cruz Biotechnology (anti-p21 (H164), anti-β-catenin (E-5), anti-Snail-1, anti-Twist), BD Transduction Laboratories (anti-E-cadherin, San Jose, CA), Sigma (anti-actin, St. Louis, MO), and BioRad (secondary antibodies against rabbit or mouse IgG conjugated with HRP, Life Science Research, Hercules, CA).

Techniques: Western Blot, Quantitative RT-PCR

Journal: Oxidative Medicine and Cellular Longevity

Article Title: MicroRNA-29b Enhances Osteoclast Survival by Targeting BCL-2-Modifying Factor after Lipopolysaccharide Stimulation

doi: 10.1155/2019/6018180

Figure Lengend Snippet: Identification of target for LPS-induced miR-29b in OCs. BMMs were incubated with M-CSF (30 ng/ml) and RANKL (40 ng/ml) for 40 h, washed thoroughly, and incubated further with LPS (50 ng/ml) in the presence of M-CSF (30 ng/ml) for 48 h. (a) Cells were transfected with 30 nM of miR-29b mimic or con mimic in the presence of M-CSF (30 ng/ml) for 6 h. Total RNA was analyzed by qPCR to quantify the expression of BMF, PUMA, BAK1, BIM, and HRK. Expression levels with con mimic treatment were set at 1. Cell lysates were subjected to Western blot analysis with antibodies against BMF, PUMA, BAK1, BIM, HRK, and BCL-2. Antibodies against β -actin were used for normalization. (b) Without transfection, total RNA was analyzed by qPCR to quantify the expression of BMF, and cell lysates were subjected to Western blot analysis with anti-BMF Ab. (c) Total RNA and tissue lysate of tibiae from LPS-treated or vehicle-treated (V, PBS) mice were analyzed by qPCR to quantify the expression of BMF and were subjected to Western blot analysis with antibodies against BMF. (d) Cells were thoroughly washed, transfected with 30 nM of anti-miR-29b or con inh, and stimulated with LPS (50 ng/ml) in the presence of M-CSF. After 48 h, total RNA was analyzed by qPCR to quantify BMF expression. Expression levels with con inh treatment were set at 1.0. Cell lysates were subjected to Western blot analysis with anti-BMF and anti-BCL-2 Ab. (e) Cells were thoroughly washed, transfected with 50 nM of scRNA or siBMF, stimulated with LPS (50 ng/ml) in the presence of M-CSF for 48 h, and analyzed to measure TRAP-positive MNCs and annexin V-positive cells. siRNA-mediated silencing of BMF was confirmed by RT-PCR and qPCR. The Ct values of the genes were widely distributed between 17.33 and 30.92. ∗ p < 0.05; ∗∗ p < 0.01; ∗∗∗ p < 0.001 compared with each corresponding control. Similar results were obtained from three independent experiments.

Article Snippet: The antibodies used were acquired as follows: primary antibodies against BMF (NBP1-76658) and PUMA (NBP1-76639) from Novus Biologicals (Cambridge, UK), BAK (12105) and BCL-2 (3498) from Cell Signaling (Danvers, MA, USA), BIM (559685) from BD Pharmingen (San Diego, CA, USA), HRK (AHP1178) from Bio-Rad (Oxford, UK), and β -actin (A5441) from Sigma Chemical.

Techniques: Incubation, Transfection, Expressing, Western Blot, Reverse Transcription Polymerase Chain Reaction, Control

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Functional validation of HCAR2 as a receptor for heme measured by receptor signaling. (A) Titration of heme in the activity assay in agonist mode of HCAR2/GPR109A. (B) Titration of nicotinic acid (niacin) in the activity assay in agonist mode of HCAR2/GPR109A.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Functional Assay, Biomarker Discovery, Titration, Activity Assay

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: Biochemical validation of HCAR2 as a receptor for heme, illustrated by direct ligand/receptor binding. (A) Comparison by SPR of the interaction of immobilized recombinant HCAR2 compared to C3b (negative control) with different concentrations of heme (39 to 2500 nM). (B) Analysis of HCAR2-heme interaction by spectroscopy (0.35 to 10.2 μM). (C) Absorbance at the Soret peak (414 nM) as a function of the molar excess of heme over the protein. RU, response units.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Biomarker Discovery, Binding Assay, Comparison, Recombinant, Negative Control, Spectroscopy

Journal: Blood Advances

Article Title: HCAR2 is a novel receptor for heme ∗

doi: 10.1182/bloodadvances.2025016197

Figure Lengend Snippet: HCAR2 is overexpressed in hemolytic and SCD mice and its expression is regulated by heme/HO-1 axis. (A) Schematic overview of the experiment performed on mice. (B) Heat map of the normalized counts of the 3 main Hcar2 transcription factors of PHZ vs PBS (upper) and HbSS vs HbAA mice (lower). Normalized counts in each mouse are represented with row scale normalization. (C-F) Spearman correlation of Hcar2 and Hmox1 normalized gene expression levels in PHZ- and PBS-treated mice from RNAseq (C) and QuantiGene data (E) and HbSS and HbAA mice from RNAseq (D) and QuantiGene data (F). (G) QuantiGene analysis of Hcar2 gene expression levels in PHZ- and PBS-treated mice. (H) QuantiGene analysis of Hcar2 gene expression levels in HbSS and HbAA mice, either treated with PBS (left) or pretreated with SnMP, followed by an injection of 24-μM heme (right). (I-J) QuantiGene analysis of Hcar2 expression in HbAA mice injected with heme. (I) Level of expression of Hcar2 . (J) Spearman correlation of Hcar2 and Hmox1 normalized gene expression in the HbAA mice injected with PBS or heme. ∗∗∗∗ P < .0001; ∗ P < .05; Mann-Whitney test in panels G,I. ∗ P < .05; Kruskal-Wallis with Dunn test for multiple pairwise comparisons in panel H.

Article Snippet: Cells were harvested and washed with PBS 1× and further stained with live-dead (Invitrogen; L10119A) and anti-human HCAR2 primary antibody (Novus; NBP1-92180) and labeled with chicken anti-rabbit AF647 secondary antibody (Invitrogen; A21443).

Techniques: Expressing, Gene Expression, Injection, MANN-WHITNEY

Journal: Cell Reports Medicine

Article Title: Dietary vitamin B3 supplementation induces the antitumor immunity against liver cancer via biased GPR109A signaling in myeloid cell

doi: 10.1016/j.xcrm.2024.101718

Figure Lengend Snippet:

Article Snippet: HM74A APC , R&D Systems , Cat#FAB2760A; RRID: AB_573115.

Techniques: Control, Staining, Recombinant, Liposomes, Cell Isolation, CCK-8 Assay, TUNEL Assay, Apoptosis Assay, Activation Assay, Software