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Figure 2. Glucocorticoids alter the expression proﬁle of <t>ZBTB16</t> (A) RT-qPCR results for ZBTB16 and MAP2 across hCO development. (B) Western blots of ZBTB16 and ACTIN across hCO development. Each lane contains protein from a pool of three hCOs. (C) Representative images of day 30 baseline hCOs stained for DCX, SOX2, MAP2, ZBTB16, and DAPI. (D) Western blots of ZBTB16 and ACTIN in hCOs treated with 100 nM dex at day 43 and analyzed 7 days later at day 50. Each lane contains protein from a pool of three hCOs, and the six replicates were generated in two independent hCO batches. (E) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 protein expression in day 50 hCOs normalized over ACTIN. (F) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 mRNA levels normalized over endogenous genes and day 40 baseline ZBTB16 expression levels. RT-qPCR, quantitative reverse-transcription polymerase chain reaction; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone. For (E), signiﬁ- cance was tested with two-tailed Mann-Whitney comparison between treatment and vehicle. For (F), signiﬁcance was tested with one-way ANOVA with Ben- jamini, Krieger, and Yekutieli multiple testing correction (p = 0.0003). Box and whisker plots represent 25th to 75th percentile of the data with the center line representing the median and whiskers representing minima and maxima. Mann-Whitney p values for (E) or post hoc p values for (F): ****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns p > 0.05. Scale bars, 50 mm. See also Figure S3.

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1) Product Images from "Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression."

Article Title: Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

Journal: Neuron

doi: 10.1016/j.neuron.2024.02.005

Figure 2. Glucocorticoids alter the expression proﬁle of ZBTB16 (A) RT-qPCR results for ZBTB16 and MAP2 across hCO development. (B) Western blots of ZBTB16 and ACTIN across hCO development. Each lane contains protein from a pool of three hCOs. (C) Representative images of day 30 baseline hCOs stained for DCX, SOX2, MAP2, ZBTB16, and DAPI. (D) Western blots of ZBTB16 and ACTIN in hCOs treated with 100 nM dex at day 43 and analyzed 7 days later at day 50. Each lane contains protein from a pool of three hCOs, and the six replicates were generated in two independent hCO batches. (E) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 protein expression in day 50 hCOs normalized over ACTIN. (F) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 mRNA levels normalized over endogenous genes and day 40 baseline ZBTB16 expression levels. RT-qPCR, quantitative reverse-transcription polymerase chain reaction; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone. For (E), signiﬁ- cance was tested with two-tailed Mann-Whitney comparison between treatment and vehicle. For (F), signiﬁcance was tested with one-way ANOVA with Ben- jamini, Krieger, and Yekutieli multiple testing correction (p = 0.0003). Box and whisker plots represent 25th to 75th percentile of the data with the center line representing the median and whiskers representing minima and maxima. Mann-Whitney p values for (E) or post hoc p values for (F): ****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns p > 0.05. Scale bars, 50 mm. See also Figure S3.

Figure Legend Snippet: Figure 2. Glucocorticoids alter the expression proﬁle of ZBTB16 (A) RT-qPCR results for ZBTB16 and MAP2 across hCO development. (B) Western blots of ZBTB16 and ACTIN across hCO development. Each lane contains protein from a pool of three hCOs. (C) Representative images of day 30 baseline hCOs stained for DCX, SOX2, MAP2, ZBTB16, and DAPI. (D) Western blots of ZBTB16 and ACTIN in hCOs treated with 100 nM dex at day 43 and analyzed 7 days later at day 50. Each lane contains protein from a pool of three hCOs, and the six replicates were generated in two independent hCO batches. (E) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 protein expression in day 50 hCOs normalized over ACTIN. (F) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 mRNA levels normalized over endogenous genes and day 40 baseline ZBTB16 expression levels. RT-qPCR, quantitative reverse-transcription polymerase chain reaction; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone. For (E), signiﬁ- cance was tested with two-tailed Mann-Whitney comparison between treatment and vehicle. For (F), signiﬁcance was tested with one-way ANOVA with Ben- jamini, Krieger, and Yekutieli multiple testing correction (p = 0.0003). Box and whisker plots represent 25th to 75th percentile of the data with the center line representing the median and whiskers representing minima and maxima. Mann-Whitney p values for (E) or post hoc p values for (F): ****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns p > 0.05. Scale bars, 50 mm. See also Figure S3.

Techniques Used: Expressing, Quantitative RT-PCR, Western Blot, Staining, Generated, Reverse Transcription, Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Comparison, Whisker Assay

Figure 4. ZBTB16 is necessary for the effects of glucocorticoids on PAX6+EOMES+ basal progenitors (A) Treatment and analysis workﬂow in hCOs derived from edited No.1 iPSCs with either ZBTB16+/+ or ZBTB16+/ genotypes. (B) Western blots for ZBTB16 and ACTIN in ZBTB16+/+- or ZBTB16+/-derived hCOs at veh and dex. Each lane contains protein from a pool of three organoids. (C) Quantiﬁcation of western blot results. (D) Representative images of FCa of ZBTB16+/+-derived hCOs per treatment condition. TBR2 is an alternative name for EOMES. (E) Quantiﬁcation of the FCa results. (F) Representative images of FCa of ZBTB16+/-derived hCOs per treatment condition. (G) Quantiﬁcation of the FCa results. DMSO, dimethyl sulfoxide; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone; FCa, ﬂow cytometry analysis. For (C), (E), and (G), signiﬁcance was tested with two-way ANOVA with Benjamini, Krieger, and Yekutieli multiple testing correction (C: p.interaction = 0.03, E: p.interaction = 0.0068, G: p.interaction = 0.97). Data are represented as mean ± SEM. Post hoc p values: **p % 0.01, ns p > 0.05. See also Figure S5 and Table S5.

Figure Legend Snippet: Figure 4. ZBTB16 is necessary for the effects of glucocorticoids on PAX6+EOMES+ basal progenitors (A) Treatment and analysis workﬂow in hCOs derived from edited No.1 iPSCs with either ZBTB16+/+ or ZBTB16+/ genotypes. (B) Western blots for ZBTB16 and ACTIN in ZBTB16+/+- or ZBTB16+/-derived hCOs at veh and dex. Each lane contains protein from a pool of three organoids. (C) Quantiﬁcation of western blot results. (D) Representative images of FCa of ZBTB16+/+-derived hCOs per treatment condition. TBR2 is an alternative name for EOMES. (E) Quantiﬁcation of the FCa results. (F) Representative images of FCa of ZBTB16+/-derived hCOs per treatment condition. (G) Quantiﬁcation of the FCa results. DMSO, dimethyl sulfoxide; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone; FCa, ﬂow cytometry analysis. For (C), (E), and (G), signiﬁcance was tested with two-way ANOVA with Benjamini, Krieger, and Yekutieli multiple testing correction (C: p.interaction = 0.03, E: p.interaction = 0.0068, G: p.interaction = 0.97). Data are represented as mean ± SEM. Post hoc p values: **p % 0.01, ns p > 0.05. See also Figure S5 and Table S5.

Techniques Used: Derivative Assay, Western Blot, Cytometry

Image Search Results

Journal: Neuron

Article Title: Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

doi: 10.1016/j.neuron.2024.02.005

Figure Lengend Snippet: Figure 2. Glucocorticoids alter the expression proﬁle of ZBTB16 (A) RT-qPCR results for ZBTB16 and MAP2 across hCO development. (B) Western blots of ZBTB16 and ACTIN across hCO development. Each lane contains protein from a pool of three hCOs. (C) Representative images of day 30 baseline hCOs stained for DCX, SOX2, MAP2, ZBTB16, and DAPI. (D) Western blots of ZBTB16 and ACTIN in hCOs treated with 100 nM dex at day 43 and analyzed 7 days later at day 50. Each lane contains protein from a pool of three hCOs, and the six replicates were generated in two independent hCO batches. (E) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 protein expression in day 50 hCOs normalized over ACTIN. (F) Quantiﬁcation of the effect of 100 nM dex over 7 days on ZBTB16 mRNA levels normalized over endogenous genes and day 40 baseline ZBTB16 expression levels. RT-qPCR, quantitative reverse-transcription polymerase chain reaction; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone. For (E), signiﬁ- cance was tested with two-tailed Mann-Whitney comparison between treatment and vehicle. For (F), signiﬁcance was tested with one-way ANOVA with Ben- jamini, Krieger, and Yekutieli multiple testing correction (p = 0.0003). Box and whisker plots represent 25th to 75th percentile of the data with the center line representing the median and whiskers representing minima and maxima. Mann-Whitney p values for (E) or post hoc p values for (F): ****p % 0.0001, ***p % 0.001, **p % 0.01, *p % 0.05, ns p > 0.05. Scale bars, 50 mm. See also Figure S3.

Article Snippet: More specifically, the human ZBTB16 ORF (NM_006006.5, 2034 bp) sequence was amplified from a plasmid delivered fromGenScript and the F2A-GFP from the Snap25-LSL-2A-GFP vector (Addgene, #61575).

Techniques: Expressing, Quantitative RT-PCR, Western Blot, Staining, Generated, Reverse Transcription, Polymerase Chain Reaction, Two Tailed Test, MANN-WHITNEY, Comparison, Whisker Assay

Journal: Neuron

Article Title: Human cortical neurogenesis is altered via glucocorticoid-mediated regulation of ZBTB16 expression.

doi: 10.1016/j.neuron.2024.02.005

Figure Lengend Snippet: Figure 4. ZBTB16 is necessary for the effects of glucocorticoids on PAX6+EOMES+ basal progenitors (A) Treatment and analysis workﬂow in hCOs derived from edited No.1 iPSCs with either ZBTB16+/+ or ZBTB16+/ genotypes. (B) Western blots for ZBTB16 and ACTIN in ZBTB16+/+- or ZBTB16+/-derived hCOs at veh and dex. Each lane contains protein from a pool of three organoids. (C) Quantiﬁcation of western blot results. (D) Representative images of FCa of ZBTB16+/+-derived hCOs per treatment condition. TBR2 is an alternative name for EOMES. (E) Quantiﬁcation of the FCa results. (F) Representative images of FCa of ZBTB16+/-derived hCOs per treatment condition. (G) Quantiﬁcation of the FCa results. DMSO, dimethyl sulfoxide; hCOs, human cerebral organoids; Veh, vehicle; Dex, dexamethasone; FCa, ﬂow cytometry analysis. For (C), (E), and (G), signiﬁcance was tested with two-way ANOVA with Benjamini, Krieger, and Yekutieli multiple testing correction (C: p.interaction = 0.03, E: p.interaction = 0.0068, G: p.interaction = 0.97). Data are represented as mean ± SEM. Post hoc p values: **p % 0.01, ns p > 0.05. See also Figure S5 and Table S5.

Techniques: Derivative Assay, Western Blot, Cytometry

Figure 1. Endometrial stromal and glandular cells display enhanced ZBTB16 immunoreactivity in women administered depo-medroxyprogesterone acetate (DMPA). (A) Representative ZBTB16 im- munostaining (brown) in parafﬁn sections from paired endometria obtained from pre- and 3 months post-DMPA administration. Original magniﬁcation ×40. Enhanced ZBTB16 immunoreactivity was observed in post-DMPA endometrial compared to pre-DMPA. A strong ZBTB16 immunoreactivity is seen in post-DPMA vascular endothelium (arrows) in the inset micrographs. Original magniﬁcation ×100. (B) HSCOREs for ZBTB16 immunoreactivity conﬁrmed signiﬁcantly higher ZBTB16 immunore- activity in both endometrial stromal (185.4 ± 9.2 vs. 92.1 ± 13.8) and glandular cells (181.3 ± 20.4 vs. 47.7 ± 14.7), as well as endothelial cells (185.3 ± 19.9 vs. 45.4 ± 16.3; p < 0.001) in post- vs. pre-DMPA endometria. Bars represent the mean ± SEM; n = 7/each; *** p < 0.001 vs. pre-DMPA analyzed by the t-test.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 1. Endometrial stromal and glandular cells display enhanced ZBTB16 immunoreactivity in women administered depo-medroxyprogesterone acetate (DMPA). (A) Representative ZBTB16 im- munostaining (brown) in parafﬁn sections from paired endometria obtained from pre- and 3 months post-DMPA administration. Original magniﬁcation ×40. Enhanced ZBTB16 immunoreactivity was observed in post-DMPA endometrial compared to pre-DMPA. A strong ZBTB16 immunoreactivity is seen in post-DPMA vascular endothelium (arrows) in the inset micrographs. Original magniﬁcation ×100. (B) HSCOREs for ZBTB16 immunoreactivity conﬁrmed signiﬁcantly higher ZBTB16 immunore- activity in both endometrial stromal (185.4 ± 9.2 vs. 92.1 ± 13.8) and glandular cells (181.3 ± 20.4 vs. 47.7 ± 14.7), as well as endothelial cells (185.3 ± 19.9 vs. 45.4 ± 16.3; p < 0.001) in post- vs. pre-DMPA endometria. Bars represent the mean ± SEM; n = 7/each; *** p < 0.001 vs. pre-DMPA analyzed by the t-test.

Article Snippet: Transient Transfection of ZBTB16 Overexpression To overexpress ZBTB16 in HESCs, the expression vector containing the human fulllength ZBTB16 gene open reading frame (pCMV6-ZBTB16) was purchased from Origene Inc. (Rockville, MD, USA).

Techniques: Activity Assay

Figure 2. MPA administration induces endometrial ZBTB16 expression in guinea pigs. (A) Repre- sentative images of ZBTB16 immunostaining in endometria obtained from ovariectomized guinea pigs treated 21 days with placebo as the control (Cont; n = 5), or estradiol (E2; n = 4), or medrox- yprogesterone acetate (MPA; n = 5), or E2 + MPA (n = 6). Original magniﬁcation: ×20. The insert represents negative control staining. (B) HSCORE analysis of ZBTB16 immunoreactivity in stromal and glandular cells of endometria. Bars represent the mean ± SEM; * p < 0.05 vs. the control or E2 treatment in stromal cells, and * p < 0.05 vs. the control or E2 treatment in glandular epithelial cells analyzed by one-way ANOVA followed by the Holm–Sidak method.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 2. MPA administration induces endometrial ZBTB16 expression in guinea pigs. (A) Repre- sentative images of ZBTB16 immunostaining in endometria obtained from ovariectomized guinea pigs treated 21 days with placebo as the control (Cont; n = 5), or estradiol (E2; n = 4), or medrox- yprogesterone acetate (MPA; n = 5), or E2 + MPA (n = 6). Original magniﬁcation: ×20. The insert represents negative control staining. (B) HSCORE analysis of ZBTB16 immunoreactivity in stromal and glandular cells of endometria. Bars represent the mean ± SEM; * p < 0.05 vs. the control or E2 treatment in stromal cells, and * p < 0.05 vs. the control or E2 treatment in glandular epithelial cells analyzed by one-way ANOVA followed by the Holm–Sidak method.

Techniques: Expressing, Immunostaining, Control, Negative Control, Staining

Figure 3. Increased ZBTB16 expression during decidualization of cultured HESCs. (A) ZBTB16 mRNA levels in HESCs treated with 10−8 M E2 + 10−7 MPA + 5 × 10−5 cyclic AMP (EMC) for 0, 3, or 6 days by qPCR. The data represent the fold change as the mean ± SEM; n = 5/each; *** p < 0.001 vs. Day 0 analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. (B) MPA mediated upregulation of ZBTB16 mRNA levels in HESCs treated with vehicle (control; Cont) or E2 or MPA or cAMP for 6 days. Bars represent mean ± SEM; n = 4/each; * p < 0.05 vs. Cont or E2 or cAMP analyzed by one way ANOVA followed by Student-Newman-Keuls method.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 3. Increased ZBTB16 expression during decidualization of cultured HESCs. (A) ZBTB16 mRNA levels in HESCs treated with 10−8 M E2 + 10−7 MPA + 5 × 10−5 cyclic AMP (EMC) for 0, 3, or 6 days by qPCR. The data represent the fold change as the mean ± SEM; n = 5/each; *** p < 0.001 vs. Day 0 analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. (B) MPA mediated upregulation of ZBTB16 mRNA levels in HESCs treated with vehicle (control; Cont) or E2 or MPA or cAMP for 6 days. Bars represent mean ± SEM; n = 4/each; * p < 0.05 vs. Cont or E2 or cAMP analyzed by one way ANOVA followed by Student-Newman-Keuls method.

Techniques: Expressing, Cell Culture, Control

Figure 4. pLARCs induce ZBTB16 mRNA and protein levels in cultured HESCs. ZBTB16 mRNA (A) and protein (B) levels were analyzed by qPCR and immunoblotting, respectively, in HESCs treated with 10−8 M E2 ± 10−7 M ORG, ETO, LNG, MPA, or DEX for 7 days. Bars represent the mean ± SEM; (A) n = 4 for mRNA fold change; * p < 0.05 vs. E2 alone; and (B) n = 3 for protein levels after normalization to β-actin; * p < 0.05 vs. E2 alone analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. E2: estradiol, ORG: Organon 2058, ETO: etonogestrel, LNG: levonorgestrel, MPA: medroxyprogesterone acetate, DEX: dexamethasone. (C) NR3C1, ZBTB16, and FKBP5 mRNA levels in HESCs transfected with either the nonspeciﬁc control (Cont) or NR3C1- speciﬁc siRNA and treated with EMC for 3 days. Bars represent the mean ± SEM; n = 3; *** p < 0.001 vs. control siRNA by the t-test.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 4. pLARCs induce ZBTB16 mRNA and protein levels in cultured HESCs. ZBTB16 mRNA (A) and protein (B) levels were analyzed by qPCR and immunoblotting, respectively, in HESCs treated with 10−8 M E2 ± 10−7 M ORG, ETO, LNG, MPA, or DEX for 7 days. Bars represent the mean ± SEM; (A) n = 4 for mRNA fold change; * p < 0.05 vs. E2 alone; and (B) n = 3 for protein levels after normalization to β-actin; * p < 0.05 vs. E2 alone analyzed by one-way ANOVA followed by the Student–Newman–Keuls method. E2: estradiol, ORG: Organon 2058, ETO: etonogestrel, LNG: levonorgestrel, MPA: medroxyprogesterone acetate, DEX: dexamethasone. (C) NR3C1, ZBTB16, and FKBP5 mRNA levels in HESCs transfected with either the nonspeciﬁc control (Cont) or NR3C1- speciﬁc siRNA and treated with EMC for 3 days. Bars represent the mean ± SEM; n = 3; *** p < 0.001 vs. control siRNA by the t-test.

Techniques: Cell Culture, Western Blot, Transfection, Control

Figure 5. ZBTB16 overexpression induces decidualization markers, as well as F3, IL-8, and PTGS2 levels in HESCs. (A) Decidualization markers’, PRL and IGFBP1, mRNA levels in ZBTB16-vector (ZBTB16-v) or control-vector (Cont-v)-transfected HESCs treated with or without 10−8 M estra- diol + 10−7 M medroxyprogesterone acetate + 5 × 10−5 M cAMP (EMC) for 3 days. Bars represent the mean ± SEM; n = 4; *** p < 0.05 vs. control-v-EMC; + p < 0.05 vs. ZBTB16-v + EMC. (B) Tissue factor (F3) mRNA levels in control- (Cont-v) or ZBTB16-vector (ZBTB16-v)-transfected HESCs treated with or without EMC for 3 days ± 1 U/mL of thrombin (THR) for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. (C) Interleukin 8 (IL-8) and prostaglandin endoperoxide synthase 2 (PTGS2 aka cyclooxygenase 2; COX2) mRNA levels in the control- or ZBTB16-vector-transfected HESCs treated with EMC for 3 days ± 1 U/mL THR for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. The data were analyzed by one-way ANOVA followed by the Student–Newman–Keuls method.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 5. ZBTB16 overexpression induces decidualization markers, as well as F3, IL-8, and PTGS2 levels in HESCs. (A) Decidualization markers’, PRL and IGFBP1, mRNA levels in ZBTB16-vector (ZBTB16-v) or control-vector (Cont-v)-transfected HESCs treated with or without 10−8 M estra- diol + 10−7 M medroxyprogesterone acetate + 5 × 10−5 M cAMP (EMC) for 3 days. Bars represent the mean ± SEM; n = 4; *** p < 0.05 vs. control-v-EMC; + p < 0.05 vs. ZBTB16-v + EMC. (B) Tissue factor (F3) mRNA levels in control- (Cont-v) or ZBTB16-vector (ZBTB16-v)-transfected HESCs treated with or without EMC for 3 days ± 1 U/mL of thrombin (THR) for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. (C) Interleukin 8 (IL-8) and prostaglandin endoperoxide synthase 2 (PTGS2 aka cyclooxygenase 2; COX2) mRNA levels in the control- or ZBTB16-vector-transfected HESCs treated with EMC for 3 days ± 1 U/mL THR for 6 h. Bars represent the mean ± SEM; n = 4; * p < 0.05 vs. Cont-v or ZBTB16-v; + p < 0.05 vs. Cont-v; # p < 0.05 vs. Cont-v + THR. The data were analyzed by one-way ANOVA followed by the Student–Newman–Keuls method.

Techniques: Over Expression, Plasmid Preparation, Control, Transfection

Figure 6. Schematic demonstration of the role of elevated ZBTB16 expression in the pathogenesis of pLARC-induced AUB. Administration of progestin-only, long-acting, reversible contraception (pLARCs) reduce uterine blood ﬂow, which causes local hypoxia [8,9] and induces HESC decidu- alization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inﬂammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis, which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombin- induced endometrial angiogenesis and inﬂammation by increasing IL8 and PTGS2 levels in HESCs.

Journal: International journal of molecular sciences

Article Title: Enhanced ZBTB16 Levels by Progestin-Only Contraceptives Induces Decidualization and Inflammation.

doi: 10.3390/ijms241310532

Figure Lengend Snippet: Figure 6. Schematic demonstration of the role of elevated ZBTB16 expression in the pathogenesis of pLARC-induced AUB. Administration of progestin-only, long-acting, reversible contraception (pLARCs) reduce uterine blood ﬂow, which causes local hypoxia [8,9] and induces HESC decidu- alization, which increases tissue factor [15,23] and ZBTB16 levels. Increased ZBTB16 levels induce excess tissue factor expression, which generates thrombin. The resulting excess thrombin binds to protease-activated receptors (PARs), which increase the expression of several inﬂammatory factors and angiogenic factors, such as VEGF or IL-8 [19,20,48]. These factors promote excess angiogenesis, which results in abnormal uterine bleeding. pLARC-induced excess ZBTB16 exacerbates thrombin- induced endometrial angiogenesis and inﬂammation by increasing IL8 and PTGS2 levels in HESCs.

Techniques: Expressing

Figure 1. PLZF is downregulated in the majority of GC cells. (A) Relative protein levels of PLZF in a panel of GC cells were assessed by western blot analysis. (B) Relative mRNA levels of PLZF in a panel of GC cells were determined by qRT‑PCR. **P<0.01 for GC cells vs. GES1. Error bars indicated the means ± SD from three independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; GES1, gastric epithelium cell line.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 1. PLZF is downregulated in the majority of GC cells. (A) Relative protein levels of PLZF in a panel of GC cells were assessed by western blot analysis. (B) Relative mRNA levels of PLZF in a panel of GC cells were determined by qRT‑PCR. **P<0.01 for GC cells vs. GES1. Error bars indicated the means ± SD from three independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; GES1, gastric epithelium cell line.

Article Snippet: PLZF (ZBTB16) (NM_006006) Human Tagged ORF Clone (cat. no. RG206745) and pCMV6‐AC‐GFP Tagged Cloning Vector (cat. no. PS100010) were purchased from OriGene Tech‐ nologies, Inc. (Rockville, MD, USA).

Techniques: Western Blot

Figure 2. Overexpression of PLZF inhibits the proliferation, migration and invasion of GC cells. (A) Transfected efficiencies of overexpressed PLZF in SGC7901 and BGC823 cells. (B) Cellular proliferation was determined by the MTS assay after SGC7901 and BGC823 cells were stably transfected with PLZF plasmid and vectors. (C) The representative results from 3 independent experiments and statistical data of colony formation assays using SGC7901 and BGC823 cells stably transfected with PLZF plasmid and vectors. (D) Cell invasion was assessed by Transwell assay, and cell lines were treated the same as aforementioned. (E) Cell migration was monitored by the wound healing assay, and cell lines were treated the same as aforementioned. *P<0.05 and **P<0.01 for cells transfected with PLZF plasmid vs. cells transfected with vectors. Error bars indicated the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 2. Overexpression of PLZF inhibits the proliferation, migration and invasion of GC cells. (A) Transfected efficiencies of overexpressed PLZF in SGC7901 and BGC823 cells. (B) Cellular proliferation was determined by the MTS assay after SGC7901 and BGC823 cells were stably transfected with PLZF plasmid and vectors. (C) The representative results from 3 independent experiments and statistical data of colony formation assays using SGC7901 and BGC823 cells stably transfected with PLZF plasmid and vectors. (D) Cell invasion was assessed by Transwell assay, and cell lines were treated the same as aforementioned. (E) Cell migration was monitored by the wound healing assay, and cell lines were treated the same as aforementioned. *P<0.05 and **P<0.01 for cells transfected with PLZF plasmid vs. cells transfected with vectors. Error bars indicated the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer.

Techniques: Over Expression, Migration, Transfection, MTS Assay, Stable Transfection, Plasmid Preparation, Transwell Assay, Wound Healing Assay

Figure 3. Lack of PLZF expression promotes the proliferation of GC cells in vivo. (A) Transfection efficiencies of overexpressed PLZF in SGC7901 cells. (B) SGC7901 cells stably transfected with PLZF plasmid and vectors were injected into nude mice (n=5) that were sacrificed by carbon dioxide euthanasia on 20th day after injection. Tumor volumes were calculated every 3 days beginning 5 days after injection. (C) Tumor weights were represented as the means of tumor weights ± SD. (D) The tumor sections underwent IHC staining using antibodies against Ki‑67. *P<0.05 and **P<0.01 for cells transfected with PLZF plasmid vs. cells transfected with vectors. Error bars indicated the means ± SD. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; IHC, immunohistochemistry.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 3. Lack of PLZF expression promotes the proliferation of GC cells in vivo. (A) Transfection efficiencies of overexpressed PLZF in SGC7901 cells. (B) SGC7901 cells stably transfected with PLZF plasmid and vectors were injected into nude mice (n=5) that were sacrificed by carbon dioxide euthanasia on 20th day after injection. Tumor volumes were calculated every 3 days beginning 5 days after injection. (C) Tumor weights were represented as the means of tumor weights ± SD. (D) The tumor sections underwent IHC staining using antibodies against Ki‑67. *P<0.05 and **P<0.01 for cells transfected with PLZF plasmid vs. cells transfected with vectors. Error bars indicated the means ± SD. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; IHC, immunohistochemistry.

Techniques: Expressing, In Vivo, Transfection, Stable Transfection, Plasmid Preparation, Injection, Immunohistochemistry

Figure 4. Negative association of PLZF and ANRIL in GC cells. (A) Transfection efficiencies of a panel of lncRNA siRNAs were tested by qRT‑PCR in BGC823 cells. (B) The effects of knockdown of a panel of lncRNAs on the expression of PLZF mRNA. (C) Negative association between PLZF and ANRIL in SGC7901 and BGC823 cells. The left panel represents the relative expression of ANRIL mRNA, and the right panel represents the relative expression of PLZF mRNA. (D) The effect of ANRIL knockdown on the expression of PLZF mRNA was confirmed. The left panel indicates the ANRIL siRNA transfection efficiency, and the right panel indicates the expression of PLZF mRNA upon ANRIL knockdown. **P<0.01. Error bars indicate the means ± SD from 3 inde- pendent experiments. PLZF, promyelocytic leukemia zinc finger; ANRIL, CDKN2B antisense RNA 1; GC, gastric cancer; lncRNAs, long non‑coding RNAs.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 4. Negative association of PLZF and ANRIL in GC cells. (A) Transfection efficiencies of a panel of lncRNA siRNAs were tested by qRT‑PCR in BGC823 cells. (B) The effects of knockdown of a panel of lncRNAs on the expression of PLZF mRNA. (C) Negative association between PLZF and ANRIL in SGC7901 and BGC823 cells. The left panel represents the relative expression of ANRIL mRNA, and the right panel represents the relative expression of PLZF mRNA. (D) The effect of ANRIL knockdown on the expression of PLZF mRNA was confirmed. The left panel indicates the ANRIL siRNA transfection efficiency, and the right panel indicates the expression of PLZF mRNA upon ANRIL knockdown. **P<0.01. Error bars indicate the means ± SD from 3 inde- pendent experiments. PLZF, promyelocytic leukemia zinc finger; ANRIL, CDKN2B antisense RNA 1; GC, gastric cancer; lncRNAs, long non‑coding RNAs.

Techniques: Transfection, Knockdown, Expressing

Figure 5. ANRIL induces DNA methylation of PLZF. (A) Transfection efficiencies of overexpressed PLZF in BGC823 and SGC7901 cells were determined by qRT‑PCR. (B) Transfection efficiencies were analyzed by western blot analysis. (C) Relative ANRIL enrichment was assessed by the RIP experiment using PLZF antibody with an IgG antibody as a control. (D) Methylated PLZF was assessed by MPCR using methylated PLZF primers and unmethylated PLZF primers in GC cells BGC823 and SGC7901 and normal cells GES1. (E) BGC823 and SGC7901 cells were treated with 5‑Aza and methylated PLZF was reduced. (F) In BGC823 and SGC7901 cells, the protein levels of PLZF were analyzed by western blot analysis after SAHA and 5‑Aza treatments for 48 h. **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. ANRIL, CDKN2B antisense RNA 1; PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; GES1, gastric epithelium cell line.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 5. ANRIL induces DNA methylation of PLZF. (A) Transfection efficiencies of overexpressed PLZF in BGC823 and SGC7901 cells were determined by qRT‑PCR. (B) Transfection efficiencies were analyzed by western blot analysis. (C) Relative ANRIL enrichment was assessed by the RIP experiment using PLZF antibody with an IgG antibody as a control. (D) Methylated PLZF was assessed by MPCR using methylated PLZF primers and unmethylated PLZF primers in GC cells BGC823 and SGC7901 and normal cells GES1. (E) BGC823 and SGC7901 cells were treated with 5‑Aza and methylated PLZF was reduced. (F) In BGC823 and SGC7901 cells, the protein levels of PLZF were analyzed by western blot analysis after SAHA and 5‑Aza treatments for 48 h. **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. ANRIL, CDKN2B antisense RNA 1; PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer; GES1, gastric epithelium cell line.

Techniques: DNA Methylation Assay, Transfection, Western Blot, Control, Methylation

Figure 6. PLZF is negatively associated with EZH2 and H3K27me3 in GC cells. (A) Relative ANRIL enrichment was assessed by an RIP experiment using PRC2 component EZH2 and SUZ12 antibodies with an IgG antibody as a control. (B) Relative mRNA level of EZH2 was confirmed by qRT‑PCR in GC and control cells. (C) Protein levels of EZH2 and H3K27me3 were analyzed by western blot analysis. (D) Transfection efficiency of EZH2 siRNA. (E) Relative expression of PLZF upon EZH2 knockdown. **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 6. PLZF is negatively associated with EZH2 and H3K27me3 in GC cells. (A) Relative ANRIL enrichment was assessed by an RIP experiment using PRC2 component EZH2 and SUZ12 antibodies with an IgG antibody as a control. (B) Relative mRNA level of EZH2 was confirmed by qRT‑PCR in GC and control cells. (C) Protein levels of EZH2 and H3K27me3 were analyzed by western blot analysis. (D) Transfection efficiency of EZH2 siRNA. (E) Relative expression of PLZF upon EZH2 knockdown. **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; GC, gastric cancer.

Techniques: Control, Western Blot, Transfection, Expressing, Knockdown

Figure 7. Absence of PLZF induces EMT of GC cells and is correlated with poor prognosis. (A) Relative expression of EMT markers in BGC823 cells stably transfected with PLZF plasmid and vectors. (B) Relative expression of EMT markers in SGC7901 cells stably transfected with PLZF plasmid and vectors. (C) Relative mRNA expression of PLZF and epithelial marker E‑cadherin in GC tissues with paracarcinoma tissue as a control. (D) Relative mRNA expression of PRC2 components and mesenchymal markers in GC tissues with paracarcinoma tissue as a control. (E) Relative mRNA expression of PLZF in GC tissues with corresponding paracarcinoma tissues as controls (n=60). (F) Survival times of patients with low PLZF expression were decreased compared to those of patients with high PLZF expression. *P<0.05 and **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; EMT, epithelial‑ mesenchymal transformation; GC, gastric cancer.

Journal: Oncology reports

Article Title: Tumor suppressor PLZF regulated by lncRNA ANRIL suppresses proliferation and epithelial mesenchymal transformation of gastric cancer cells.

doi: 10.3892/or.2018.6866

Figure Lengend Snippet: Figure 7. Absence of PLZF induces EMT of GC cells and is correlated with poor prognosis. (A) Relative expression of EMT markers in BGC823 cells stably transfected with PLZF plasmid and vectors. (B) Relative expression of EMT markers in SGC7901 cells stably transfected with PLZF plasmid and vectors. (C) Relative mRNA expression of PLZF and epithelial marker E‑cadherin in GC tissues with paracarcinoma tissue as a control. (D) Relative mRNA expression of PRC2 components and mesenchymal markers in GC tissues with paracarcinoma tissue as a control. (E) Relative mRNA expression of PLZF in GC tissues with corresponding paracarcinoma tissues as controls (n=60). (F) Survival times of patients with low PLZF expression were decreased compared to those of patients with high PLZF expression. *P<0.05 and **P<0.01. Error bars indicate the means ± SD from 3 independent experiments. PLZF, promyelocytic leukemia zinc finger; EMT, epithelial‑ mesenchymal transformation; GC, gastric cancer.

Techniques: Expressing, Stable Transfection, Transfection, Plasmid Preparation, Marker, Control, Transformation Assay

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 1: Plzf expression is elevated in aged mouse testes. A, qPCR analysis of Plzf mRNA in young and aged testes normalized to Ddx4. Graphs represent mean value ± s.e.m. n = 9 mice per group. *P < 0.05. B, representative images of IHC for Plzf in young and aged testes. Images are representative of testes from 3 mice examined. Arrow, low Plzf. Arrowhead, high Plzf. *, degenerative seminiferous tubule. C, representative flow profile of detecting Plzf-expressing cells by intracellular staining and FACS by using anti-Plzf antibody (PE- conjugated). D, quantification of the percentage of Plzf-positive cells analyzed by FACS in panel C. Graphs represent mean value ± s.e.m. n = 6 mice per group. *P < 0.05. E, Plzf intensity in Plzf-expressing cells from young versus aged mice. F, Quantification of Plzf expression levels in young and aged testes. Graphs represent mean value ± s.e.m. n = 4 mice per group. *P < 0.05.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Expressing, Staining

Figure 5: Plzf expression inhibits RA-induced Stra8 transcription in F9 cells and correlates with a lack of Stra8- expressing cells in aged testes. A, qPCR analysis for Stra8 mRNA in F9 cells normalized to β-actin. Graphs represent mean value ± s.e.m. n = 3 independent cultures per group. *P < 0.05. B, western blotting analysis of Stra8 in F9 cells. Data are representative of 2 independent sets of experiments. C, dual-IF staining for Plzf and Stra8 in young (left) and aged (right) testes. Arrow, Plzf-expressing cells. Arrowhead, Stra8-expressing cells. D, IHC for Stra8 in aged testes. *, degenerative seminiferous tubule.

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 5: Plzf expression inhibits RA-induced Stra8 transcription in F9 cells and correlates with a lack of Stra8- expressing cells in aged testes. A, qPCR analysis for Stra8 mRNA in F9 cells normalized to β-actin. Graphs represent mean value ± s.e.m. n = 3 independent cultures per group. *P < 0.05. B, western blotting analysis of Stra8 in F9 cells. Data are representative of 2 independent sets of experiments. C, dual-IF staining for Plzf and Stra8 in young (left) and aged (right) testes. Arrow, Plzf-expressing cells. Arrowhead, Stra8-expressing cells. D, IHC for Stra8 in aged testes. *, degenerative seminiferous tubule.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Expressing, Western Blot, Staining

Figure 6: Schematics of Plzf overexpression and spermatogonia aging. Loss of Plzf exhausts spermatogonia pool by promoting their differentiation at the expense of self-renewal. On the contrary, age-related increase in Plzf expression causes spermatogonia hypermaintenance and aging by preventing their differentiation.

Journal: Oncotarget

Article Title: Hypermaintenance and hypofunction of aged spermatogonia: insight from age-related increase of Plzf expression.

doi: 10.18632/oncotarget.4045

Figure Lengend Snippet: Figure 6: Schematics of Plzf overexpression and spermatogonia aging. Loss of Plzf exhausts spermatogonia pool by promoting their differentiation at the expense of self-renewal. On the contrary, age-related increase in Plzf expression causes spermatogonia hypermaintenance and aging by preventing their differentiation.

Article Snippet: Plzf expression plasmid was obtained from Origene (RC206745).

Techniques: Over Expression, Expressing

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