1. Dienogest inhibits nerve growth factor expression induced by tumor necrosis factor-α or interleukin-1β 
Shizuka Mita, M.S., Yutaka Shimizu, Ph.D., Ayumi Sato, M.S., Tatsuto Notsu, Ph.D., Kazunori Imada, M.S., Satoru Kyo, M.D., Ph.D. 
Fertility and Sterility 
Volume 101, Issue 2, Pages e1 (February 2014)
DOI: /j.fertnstert
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

2. Figure 1
Nerve growth factor (NGF) up-regulation by tumor necrosis factor-α (TNF-α) or interleukin-1β (IL-1β) in EM-PR cells. The NGF messenger RNA (mRNA) levels in EM-PR cells were evaluated 24 hours after administration of TNF-α or IL-1β. The results are presented as the means ± SEM of three separate experiments. Statistical significance versus vehicle treatment is indicated by the symbol: #P<.05 using Dunnett's test.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

3. Figure 2
Effect of dienogest (DNG) on the up-regulation of nerve growth factor (NGF) messenger RNA (mRNA). The EM-PR cells were treated with stimulants and drugs, and NGF mRNA levels were evaluated 24 hours after treatment (A and B) and at 3, 6, 9, and 24 hours after treatment (C and D). (C and D) The final drug concentration of DNG and P (P4) in the culture medium was 10−7 mol/L. The results are presented as the means ± SEM of three separate experiments. Statistical significance versus control is indicated by the symbol: *P<.05 using Student's t-test or #P<.05 using Dunnett's test. IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

4. Figure 3
Effect of dienogest (DNG) on nerve growth factor (NGF) neurite outgrowth activity and protein expression. The EM-PR cells were treated with the indicated stimulants and drugs, and the culture medium was collected 24 hours after treatment. (A and B) The NGF neurite outgrowth activity in the culture medium as evaluated with the PC-12 bioassay. (C and D) The NGF protein concentration in the culture medium. The final drug concentration of DNG and P (P4) in the culture medium was 10−7 mol/L. The results are presented as the means ± SEM of three (A and B) or four (C and D) separate experiments. Statistical significance versus control is indicated by the symbol: *P<.05 using Student's t-test or #P<.05 using Aspin-Welch's t-test. IL-1β = interleukin-1β; TNF-α = tumor necrosis factor-α.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions

5. Supplemental Figure 1
Effect of protein synthesis inhibition on nerve growth factor (NGF) inhibitory effect of dienogest (DNG). The EM-PR cells were treated with stimulants and DNG (10−7 mol/L) in either the presence or absence of an inhibitor of protein synthesis cycloheximide (CHX) at indicated concentration. The NGF messenger RNA (mRNA) levels were evaluated 3 hours after treatment. Stimulants used were tumor necrosis factor-α (TNF-α) (1 ng/mL) in A and interleukin-1β (IL-1β) (1 ng/mL) in B. The results are presented as percentages of inhibition calculated using the formulas: % inhibition = − [(Sample relative mRNA level) − (Averaged relative mRNA level of stimulant alone)] / [(Averaged relative mRNA level of stimulant alone) − (Averaged mRNA level of vehicle treatment)] × 100 and the means ± SEM of four different wells. NS = not significant versus without CHX using Dunnett's test. The NGF mRNA inhibition by treatment with DNG was not affected by cotreatment with CHX, suggesting that DNG inhibits NGF mRNA expression without de novo protein synthesis.
Fertility and Sterility  , e1DOI: ( /j.fertnstert )
Copyright © 2014 American Society for Reproductive Medicine Terms and Conditions