Journal: bioRxiv

Article Title: Human endometrial KISS1R inhibits stromal cell decidualization in a manner associated with a reduction in ESR1 levels

doi: 10.1101/2022.11.20.517219

Figure Lengend Snippet: Quantification of mRNA levels of hallmark molecular markers of decidualization (A) FOXO1 , (B) IGFBP1 , (C) PRL and ( D ) representative western blot analysis of endometrial FOXO1 protein levels with tubulin serving as the housekeeping control before and after six days of decidualization (see Fig. S2 for additional blots). Quantification of mRNA levels of known regulators of ESC decidualization ( E ) DCN , ( F ) LEFTY2 , and ( G ) WNT4 before and after six days of decidualization. qPCR analysis of mRNA expression of ( H ) KISS1 and ( I and J ) KISS1R before and after one, two, four, and six days of decidualization. Statistics (biological replicates: n=3-6 biopsies and technical replicates: n=3-6 for each group): student’s unpaired two-tailed t-test and one-way ANOVA with Bonferroni Multiple Comparison post hoc test. * p<0.05; ** p<0.01; and *** p<0.001. D0: day zero; D1: day one; D2: day two; D4: day four; D6: day six of decidualization; BM: basal medium; and EPC: decidualization medium.

Article Snippet: The amplified cDNA was diluted to 10 ng/μl, and quantitative PCR was performed using 40 ng of cDNA with the following TaqMan probes (Applied Biosystems/Life Technologies), KISS1 (Hs00560041_m1), and KISS1R (Hs00173906_m1).

Techniques: Western Blot, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Human endometrial KISS1R inhibits stromal cell decidualization in a manner associated with a reduction in ESR1 levels

doi: 10.1101/2022.11.20.517219

Figure Lengend Snippet: Quantification of mRNA and protein levels in empty vector control and KISS1R expressing primary HESCs following six days of in vitro decidualization. qPCR analysis of ( A) KISS1 , ( B ) KISS1R and phenotypic markers of decidualization ( C ) DESMIN, KRT7 , and KRT18 mRNA levels. Representative images of the immunofluorescence analysis of cytokeratin 7 protein levels in empty vector control ( D1-3 ) and KISS1R ( D4-6 ) expressing primary HESCs. ( E ) qPCR analysis of decidualization and ESR1 signaling markers FOXO1, IGFBP1, PRL, DCN, LEFTY2, WNT4, ESR1, PGR, MMP2 , and MMP7 mRNA levels. Representative western blot analysis of endometrial FOXO1 and ESR1 protein levels with tubulin serving as the housekeeping control before and after six days of decidualization in empty vector and KISS1R expressing HESCs (see Fig. S3 for additional blots). Statistics (biological replicates: n=3-5 biopsies and technical replicates: n=3-6 for each group): student’s unpaired two-tailed t-test. * p<0.05; ** p<0.01; and *** p<0.001. EV: Empty vector control; KISS1R or KR: KISS1R expression; BM: basal medium; and EPC: decidualization medium.

Article Snippet: The amplified cDNA was diluted to 10 ng/μl, and quantitative PCR was performed using 40 ng of cDNA with the following TaqMan probes (Applied Biosystems/Life Technologies), KISS1 (Hs00560041_m1), and KISS1R (Hs00173906_m1).

Techniques: Plasmid Preparation, Expressing, In Vitro, Immunofluorescence, Western Blot, Two Tailed Test