Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 1. Oncostatin M (OSM)–induced expression of CCL13. A and B, Normal dermal fibro- blasts (nDF), normal lung fibroblasts (nLF), normal cervical fibroblasts (nCF), and normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM for the indicated periods of time. A, Levels of CCL2, CCL13, and CCL11 mRNA were determined by RNase protection assay. Results are representative of 3 independent experiments. B, The amounts of CCL13 and CCL2 in culture supernatants were determined by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 3). P 0.05 versus control. C, Primary human blood neutrophils were stimulated with 10 ng/ml of granulocyte–macrophage colony-stimulating factor (GM-CSF) for 3 hours. The amounts of OSM in culture supernatants were determined by ELISA. Values are the mean SD (n 3). P 0.05 versus control. D, Culture supernatants (SNs) from untreated neutrophils or neutrophils stimulated with GM-CSF for 3 hours were transferred onto normal SFs. The contribution of OSM was evaluated by additional application of blocking antibodies to OSM or control antibodies (ctrl. Ab). The amounts of CCL13 produced in normal SF culture supernatants were then determined by ELISA. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus antibody control.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Rnase Protection Assay, Enzyme-linked Immunosorbent Assay, Control, Blocking Assay, Produced

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 2. Significance of OSM and other key cytokines involved in rheumatoid arthritis (RA) pathogenesis for CCL13 expression. A, Normal SFs were treated for 2 hours with 10 ng/ml of OSM, 200 units/ml of interleukin-6 (IL-6)/0.5 g soluble IL-6 receptor (sIL-6R), 10 ng/ml of IL-1, or 10 ng/ml of tumor necrosis factor (TNF). CCL13 mRNA levels were quantified, normalized to L32 levels, and compared with untreated cells. Values are the mean SD (n 4). P 0.05 versus specific control. B, Normal SFs were stimulated as described in A for 2 hours or 12 hours. Values are the mean SD (n 4). P 0.05 versus time-specific control. C, Normal SFs were treated for 3 hours with OSM, alone or in combination with IL-6/sIL-6R, IL-1, or TNF (at the concentrations described in A). Values are the mean SD (n 4). P 0.05 versus control. D, RASFs were stimulated with cytokines as described in A for 3 hours. Values are the mean SD (n 4). P 0.05 versus normal SF control; d P 0.05 versus control. E, OSM and CCL13 protein in the supernatant of unstimulated fibroblasts was determined by ELISA. Values are the mean SD (n 3). P 0.05 versus normal SF. F and G, Both normal SF and RASF supernatants were treated with OSM and either control or OSM-neutralization antibodies. Normal SFs and RASFs were restimulated with OSM-depleted supernatant for 3 hours. Values are the mean SD (n 3). and # P 0.05 versus control; d P 0.05 versus stimulated control. H, Supernatants of untreated RASFs were treated with control antibody, CCL13 antibody, or both CCL13 and OSM-neutralizing antibodies. Depleted supernatants were added to untreated normal SFs. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus control supernatant; # P 0.05 versus CCL13-depleted supernatant. w/o without; OASF osteoarthritis SF (see Figure 1 for other definitions).

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Expressing, Control, Enzyme-linked Immunosorbent Assay, Neutralization

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 4. Comparison of oncostatin M (OSM)–, interleukin-6 (IL-6)/soluble IL-6 receptor (sIL-6R)–, IL-1–, and tumor necrosis factor (TNF)–induced signal transduction, showing a potential role of phosphorylated (pY) STAT-5 in CCL13 induction. A, Normal synovial fibroblasts (nSF) were treated with 10 ng/ml of OSM, 200 units/ml of IL-6/0.5 g of sIL-6R, 10 ng/ml of IL-1, or 10 ng/ml of TNF. Lysates were subjected to Western blot analysis, using antibodies specific for the indicated proteins. The blots were stripped and reprobed with antibodies recognizing the proteins irrespective of their activation status or with loading control antibodies. B, Normal SFs were stimulated with OSM as indicated. Western blots of lysates were stained with the antibodies indicated. C, Normal SFs, normal cervical fibroblasts (nCF), normal lung fibroblasts (nLF), and normal dermal fibroblasts (nDF) were stimulated with 10 ng/ml of OSM. Cell lysates were analyzed as described in B. D, Normal SFs were transfected with control or STAT-5 small interfering RNA (siSTAT5). Forty-eight hours later, cells were stimulated with 10 ng/ml of OSM for 3 hours. CCL13 and CCL2 protein in normal SF culture supernatants was measured by enzyme-linked immunosorbent assay. Values are the mean SD (n 4). P 0.05 versus control; d P 0.05 versus stimulated siRNA control; w/o without.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Comparison, Transduction, Western Blot, Activation Assay, Control, Staining, Transfection, Small Interfering RNA, Enzyme-linked Immunosorbent Assay

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 5. Role of OSM-induced p38 activation in the stabilization of CCL13 mRNA through inhibition of tristetraprolin (TTP). A and C, Normal SFs and rheumatoid arthritis SFs (RASFs), respectively, were pretreated with 10 M AG490, 10 M SB202190, 10 M U0126, 10 M SP600125, 1 M JAK inhibitor I (JI-1), or the solvent DMSO, and then exposed to 10 ng/ml of OSM for 3 hours. Subsequently, CCL13 protein was quantified in culture supernatants by enzyme-linked immunosorbent assay (ELISA). Values are the mean SD (n 4). and # P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. B, Western blots from treated normal SFs were analyzed using the indicated antibodies. D, Normal SFs were stimulated with 10 ng/ml of OSM for 30 minutes. Then, cells were washed and cultivated for an additional 20 minutes in OSM-free medium containing actinomycin D (4 M). SB202190 (10 M) was added to the medium before cells were restimulated with OSM for the indicated periods of time. CCL13 and GAPDH mRNA levels were analyzed by reverse transcription–polymerase chain reaction. One of 3 representative independent experiments is shown. E, Normal SFs were transfected with TTP or control siRNA. Forty-eight hours later, cells were pretreated with 10 M SB202190 or DMSO for 20 minutes and then stimulated with 10 ng/ml of OSM for 3 hours. CCL13 was measured by ELISA. Values are the mean SD (n 3). P 0.05 versus control; d P 0.05 versus OSM-stimulated sample. See Figure 4 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Activation Assay, Inhibition, Solvent, Enzyme-linked Immunosorbent Assay, Control, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Transfection

Journal: Arthritis and rheumatism

Article Title: Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritis.

doi: 10.1002/art.24602

Figure Lengend Snippet: Figure 6. Contribution of STAT-5 and ERK-1/2 to CCL13 expres- sion. RASFs were transfected with STAT-5 or control small interfering RNA (siRNA). Forty-eight hours later, cells were preincubated with DMSO or U0126 for 30 minutes, prior to stimulation with 10 ng/ml of OSM for 3 hours. CCL13 protein was measured in culture superna- tants of RASFs, by ELISA. Values are the mean SD (n 4). P 0.05 versus unstimulated control; # P 0.05 versus OSM- stimulated DMSO control; d P 0.05 versus stimulated siRNA control. Inset, Knockdown efficiency was controlled by Western blot analysis of lysates, using a specific antiserum against tyrosine- phosphorylated STAT-5 (pY-STAT5). After stripping, the blots were reprobed with antiserum recognizing STAT-3. See Figure 5 for other definitions.

Article Snippet: Human CCL13 and OSM ELISA kits were purchased from R&D Systems (Minneapolis, MN) and used according to the manufacturer’s protocol.

Techniques: Transfection, Control, Small Interfering RNA, Enzyme-linked Immunosorbent Assay, Knockdown, Western Blot, Stripping Membranes

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 2. Activity of selected aptamer clones in a HEPG2 re- porter gene cell line. OSM signaling via the OSM receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Aptamer clones ADR58 (L), 120 (E), and 152 (M) have affinities of ,10 nM for OSM; ADR147 (f) is a negative control, and ADR66 (Œ) is an aptamer with an affinity .1000 nM for OSM. Aptamers were preincubated with 2 ng/ml OSM (which is approximately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The experiment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Activity Assay, Clone Assay, Negative Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 6. Effect of aptamers on LIF- and OSM-induced interleu- kin 6 release from KB cells. Shown are ADR58 (f) and ADR147 control (Œ). Aptamers were preincubated with either 1 ng/ml LIF (a) or 1 ng/ml OSM (b). The mixture was incubated with KB cells for 24 h. Interleukin 6 levels in the cell culture medium were then measured by ELISA. The experiment was carried out in triplicate. Error bars repre- sent S.D. The concentrations of LIF and OSM used were at the approx- imate ED50 for this assay. In the absence of aptamer, the IL-6 release induced by 1 ng/ml LIF was 1113 6 7.5 pg/ml, and the release induced by OSM was 4702 6 67 pg/ml. From cells incubated with medium alone, the IL-6 release was 49 6 9 pg/ml.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Cell Culture, Enzyme-linked Immunosorbent Assay

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 7. Effect of aptamer on IL-6 signaling in the HEPG2 re- porter gene cell line. IL-6 signaling via the IL-6 receptor in the HEPG2 reporter cell line results in production of secreted alkaline phosphatase. Shown are ADR58 (f) and ADR147 control (Œ). Aptamer and control RNA were preincubated with 4 ng/ml IL-6 (which is approx- imately the ED50 for this assay), and production of secreted alkaline phosphatase was monitored following addition of IL-6/aptamer mixture to the HEPG2 reporter cell line. In the absence of aptamer, cells incu- bated with IL-6 gave an A of 0.3, and in cells incubated with medium alone, the A was 0.06. ADR58 inhibition of OSM-induced secreted alkaline phosphatase (M) was included as a positive control. The exper- iment was carried out in triplicate. Error bars represent S.D.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Control, Incubation, Inhibition, Positive Control

Journal: Journal of Biological Chemistry

Article Title: The Generation and Characterization of Antagonist RNA Aptamers to Human Oncostatin M

doi: 10.1074/jbc.m002981200

Figure Lengend Snippet: FIG. 9. Functional activity of truncated ADR58. a, effect of trun- cated ADR58 on OSM signaling via the OSM receptor in the HEPG2 reporter cell line, measured by production of secreted alkaline phospha- tase (f). Aptamers were preincubated with 2 ng/ml OSM, and produc- tion of secreted alkaline phosphatase was monitored following addition of OSM/aptamer mixture to the HEPG2 reporter cell line. The A from cells incubated with OSM in the absence of aptamer was 0.443, and that from cells incubated with medium alone was 0.035. b, activity of trun- cated ADR58 in OSM-gp130 ELISA (f). The ability of truncated ADR58 to block the interaction of microtiter plate-immobilized OSM with gp130 is shown. gp130-IgG-Fc fusion binding was quantitated with an alkaline phosphatase-linked anti-IgG antibody. The experiment was carried out in triplicate. Error bars represent S.D. At the concentration of gp130-IgG-Fc used, the OSM was approximately at the ED50. The A values are corrected for nonspecific binding (where either OSM or gp130-IgG-Fc is excluded; A 5 0.06). The A in the presence of OSM and gp130-IgG-Fc but without aptamer was 1.29 6 0.3.

Article Snippet: Aptamers were preincubated with human OSM or LIF (R&D Systems, carrierfree) for 2 h at 37 °C in KB cell medium prior to addition to the cell monolayer.

Techniques: Functional Assay, Activity Assay, Incubation, Enzyme-linked Immunosorbent Assay, Blocking Assay, Binding Assay, Concentration Assay

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 3. Normal human epidermal keratinocytes (NKs) secrete oncostatin M (OSM) upon keratinocyte growth factor (KGF) stimulation inducing fibroblast activation. (a) The OSM messenger RNA (mRNA) expression analyzed in NKs after 24hours treatment with recombinant KGF compared with untreated NKs and kFCM and sFCM treatment compared with incubation with nFCM. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments with n ¼ 3 for each sample analyzed with analysis of variance (ANOVA) þ Kruskal–Wallis test. (b) OSM protein measured by ELISA secreted by NKs upon direct treatment with recombinant KGF (n ¼ 6) compared with untreated NKs (n ¼ 7) and upon incubation with kFCM and sFCM compared with nFCM (each n ¼ 5). The graph represents mean values of two independent experiments analyzed with ANOVAþ Tukey test. (c) Collagen type I-a1 (ColIa1) and (d) fibroblast activation protein (FAP) mRNA expression in normal fibroblasts (NFs), KFs, and scleroderma fibroblasts (SFs) with and without OSM treatment. (e) Fibroblast migration after treatment with recombinant OSM (10ng ml 1). (f) OSM mRNA expression upon KGF-treatment in NKs transfected with control small interfering RNA (CTRL siRNA) and siRNA against KGFR (each n ¼ 3). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (g) The OSM mRNA expression in keloid tissue (n ¼ 4) and scleroderma tissue (n ¼ 4) compared with normal skin (n ¼ 5). The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVA þ Kruskal–Wallis test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Recombinant, Incubation, Enzyme-linked Immunosorbent Assay, Migration, Transfection, Control, Small Interfering RNA, MANN-WHITNEY

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 4. STAT3 is activated in fibroblasts upon keratinocyte growth factor (KGF)–induced oncostatin M (OSM) secretion. Western blot analyis was performed for STAT3 and phosphorylated STAT3 (pSTAT3) using b-actin as a loading control. (a) STAT3 is constitutively expressed in normal fibroblasts (NFs) but phosphorylation only occurs upon direct OSM treatment or treatment with OSM-containing keratinocyte-conditioned media (KCMs) as confirmed by the OSM ELISA in Figure 3c. (b) Direct KGF treatment had no unspecific effect on STAT3 phosphorylation. NFs showed decreased STAT3 phosphorylation when treated with KCM:kFCM:siKGF compared with KCM:kFCM:siCTRL, but increased phosphorylation after treatment KCM:nFCM:KGF compared with KCM:nFCM:mock. (c) pSTAT3 and STAT3 expression status in NFs, keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (d) pSTAT3 and STAT3 expression in NFs treated with rOSM and KCM in the presence or absence of OSM-neutralizing antibodies ( þ antiOSM).

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Western Blot, Control, Phospho-proteomics, Enzyme-linked Immunosorbent Assay, Expressing, Recombinant

Journal: The Journal of investigative dermatology

Article Title: Increased KGF expression promotes fibroblast activation in a double paracrine manner resulting in cutaneous fibrosis.

doi: 10.1038/jid.2012.389

Figure Lengend Snippet: Figure 5. STAT3 mediates fibroblast activation. (a) Collagen type I-a1 (ColIa1) expression and (b) fibroblast migration cannot be induced upon oncostatin M (OSM) stimulation when pretreated with the STAT3 inhibitor S3I-201. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (c) The expression of the STAT3 target gene urokinase-type plasminogen activator (uPA) is induced upon direct OSM treatment and upon incubation with KCM (keratinocyte-conditioned medium):kFCM, KCM:sFCM, and KCM:rKGF. The graphs represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with analysis of variance (ANOVA)þ Kruskal–Wallis test and Mann–Whitney test, respectively. (d) uPA mRNA expression in normal fibroblasts (NFs), keloid fibroblasts (KFs), and scleroderma fibroblasts (SFs) upon treatment with recombinant OSM. (e) The induction of fibroblast migration measured upon OSM treatment and incubation with KCM:kFCM or KCM:sFCM is completely suppressed when the uPA expression is decreased by means of small interfering RNA transfection. The graphs in d and e represent mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test. (f) The uPA expression is significantly higher in keloid and scleroderma tissue compared with normal skin. The graphs represent mean values of four independent experiments, and error bars represent the mean±SD of these experiments analyzed with ANOVAþ Kruskal–Wallis test. (g) OSM-induced STAT3-mediated uPA expression can be suppressed by pretreatment with S3I-201. The graph represents mean values of three independent experiments, and error bars represent the mean±SD of these experiments analyzed with Mann–Whitney test.

Article Snippet: STAT3 phosphorylation following incubation with rOSM, KCM:kFCM, and KCM:sFCM was completely abolished when neutralizing antibodies against OSM (antiOSM; R&D Systems MAB295) were added to the KCM (1 mg ml 1) before transferring them onto the NFs (Figure 4d).

Techniques: Activation Assay, Expressing, Migration, MANN-WHITNEY, Incubation, Recombinant, Small Interfering RNA, Transfection

Journal: Journal of hematology & oncology

Article Title: Allosteric activation of the metabolic enzyme GPD1 inhibits bladder cancer growth via the lysoPC-PAFR-TRPV2 axis.

doi: 10.1186/s13045-022-01312-5

Figure Lengend Snippet: Fig. 4 GPD1 upregulates TRPV2 expression to promote Ca2+ influx, leading to apoptosis of bladder tumor cells. A RNA-seq analysis of control 5637 cells (CON), GPD1-OE 5637 cells (GPD1) and K120A GPD1-OE 5637 cells (K120A). Venn diagram showing the overlap of differentially expressed genes between the three groups. B Heatmap showing that the 1270 differentially expressed genes were shared by CON versus GPD1 and K120A versus GPD1. C GSEA of the genes associated with the NOD-like receptor signaling pathway. D RT–qPCR to validate the mRNA levels of differential genes (TRPV2, NLRP1, and ASC) associated with the NOD-like receptor signaling pathway identified by RNA-seq. E Western blotting to detect TRPV2 expression in control 5637 cells (CON), GPD1-OE 5637 cells (GPD1) and K120A GPD1-OE 5637 cells (K120A). F and G Flow cytometry was performed to detect TRPV2 expression on the membranes of 5637 and T24 cells overexpressing GPD1 or K120A GPD1. H and I Flow cytometry was performed to detect TRPV2 expression on the membranes of 5637 and T24 cells treated with G3P/NAD+. J and K Flow cytometry was performed to detect intracellular Ca2+ levels in 5637 cells and T24 cells overexpressing GPD1 or K120A GPD1. L and M Flow cytometry was performed to detect intracellular Ca2+ levels in 5637 cells and T24 cells treated with G3P/NAD+. N Western blots show the efficiency of siRNA knockdown of TRPV2 expression in T24 cells. O Flow cytometry was performed to detect intracellular Ca2+ levels of 5637 cells transfected with siRNAs in the presence or absence of G3P/NAD+. P Flow cytometry was performed to detect intracellular Ca2+ levels of T24 cells transfected with siRNAs in the presence of G3P/NAD+. Q Flow cytometry analysis of apoptosis in 5637 cells transfected with scramble or siRNAs in the presence or absence of G3P/NAD+. R Flow cytometry analysis of apoptosis in T24 cells transfected with scramble or siRNAs in the presence or absence of G3P/NAD+

Article Snippet: 5637 cells or T24 cells were incubated with anti-TRPV2 antibody (1:100 dilution, ACC-139, Alomone Labs) at 4 °C for 30 min.

Techniques: Expressing, RNA Sequencing, Control, Quantitative RT-PCR, Western Blot, Flow Cytometry, Knockdown, Transfection

Journal: Journal of hematology & oncology

Article Title: Allosteric activation of the metabolic enzyme GPD1 inhibits bladder cancer growth via the lysoPC-PAFR-TRPV2 axis.

doi: 10.1186/s13045-022-01312-5

Figure Lengend Snippet: Fig. 7 Wedelolactone inhibits bladder cancer growth in vitro and in vivo. A and B CCK-8 assay to evaluate the proliferation of 5637 and T24 cells treated with different compounds. C and D Colony formation of 5637 and T24 cells treated with wedelolactone. The number of colonies formed was counted by ImageJ. E and F Flow cytometry analysis of apoptosis in 5637 and T24 cells treated with wedelolactone. G and H Flow cytometry was performed to detect TRPV2 expression on the membranes of 5637 and T24 cells treated with wedelolactone. I and J. Flow cytometry was performed to detect intracellular Ca2+ levels in T24 cells treated with wedelolactone. K and L. Representative photographs of 5637 tumors implanted in mice treated with vehicle or wedelolactone. Tumor weight was analyzed (n = 8). M and N Representative photographs of T24 tumors implanted in mice treated with vehicle or wedelolactone. Tumor weight was analyzed (n = 7). O and P H&E staining of 5637 tumor tissues or T24 tumor tissues from tumor-bearing mice treated with wedelolactone or vehicle. Q and R TUNEL assay of 5637 tumor tissue or T24 tumor tissue from tumor-bearing mice treated with wedelolactone or vehicle

Article Snippet: 5637 cells or T24 cells were incubated with anti-TRPV2 antibody (1:100 dilution, ACC-139, Alomone Labs) at 4 °C for 30 min.

Techniques: In Vitro, In Vivo, CCK-8 Assay, Flow Cytometry, Expressing, Staining, TUNEL Assay