Fig 2: MAPK/ERK signaling and PDGFR-β mediate effects of PDGFD on ESCs. a Heatmap showing the differentially expressed genes in shPdgfd ESCs related to MAPK/ERK signaling. b Western blot result showing reduced ERK phosphorylation in shPdgfd ESCs while STAT3 phosphorylation was not changed. c Western blot result showing PDGFD-induced ERK and PDGFR-β phosphorylation as well as PDGFR-β upregulation in ESCs. d Western blot result showing that administration of PDGFR-β neutralizing antibody (nab) abolished PDGFD-induced ERK phosphorylation in ESCs. e AP staining showing that administration of an ERK pathway inhibitor PD0325901 abolished PDGFD overexpression (PDGFD OE)-induced inhibition of colony formation in ESCs. Scale bar 300 μm. f, g qRT-PCR results showing that administration of an ERK pathway inhibitor PD0325901 abolished PDGFD overexpression (PDGFD OE)-induced down-regulation of Sox2 and up-regulation of Cdh5 in ESCs. Densitometric quantification normalized to HSP90 (b, d) and α-TUBULIN (c) and expressed relative to Control group is shown beneath the blots. Data are presented as mean ± SD, n = 3 each group. Statistical significance was determined using one-way ANOVA in f, g and one-sample t-test in b–d. *p < 0.05, **p < 0.01, ***p < 0.001

Fig 3: Effect of altrenogest and/or thermal treatment on endometrial transcript abundance of heat shock proteins. Endometrium from pregnant gilts either supplemented with altrenogest or not (postinsemination), and maintained under thermal neutral or heat stress conditions during the peri-implantation period were assessed for changes in specific HSP transcript abundance using qPCR. Neither ALT supplementation nor thermal treatment demonstrated an effect on (A) HSP90AA1 transcript abundance. (B) HSP90AB1 abundance was reduced as a result of ALT supplementation under TN conditions; an effect reversed by HS in spite of ALT supplementation. HS also increased (C) HSPA1A and (D) HSPA6 transcript abundance, in spite of ALT supplementation. No effects of either treatment were observed on (E) HSPD1, (F) HSPB1, and (G) HSPB8 transcript abundances. Normalized transcript abundances are reported as fold change. Different lowercase letters indicate statistical significance between treatment groups; P ≤ 0.05.

Fig 4: Assessment of heat shock protein abundance in the endometrial samples by Western blotting. Representative figure of immunoblotting of endometrial protein lysates for each treatment group using antibodies directed toward HSP90AA1, HSP90AB1, HSPA1A, HSPA6, HSPD1, and HSPB1. Ponceau S stained blots were imaged to ascertain uniform loading and bands thus obtained were utilized for loading control normalization of specific target proteins. The samples from different treatment groups were uniformly distributed across two gels and pooled samples were used on both gels to allow normalization and unbiased comparisons of band densities across both gels.

Fig 5: Identification of HSP90AB1 as potential Cyclin-F target.(A) Overview of CCNF proximity partners and interaction candidates with decreased diGly sites in CCNF KO and mutant cells. Proteins found enriched or decreased by proximity proteomics (APEX2-CCNF) and/or immunoprecipitation mass spectrometry (HA-FLAG-CCNF IP) are marked red and blue, respectively. Unchanged proteins are marked in grey, whereas proteins that were not found are indicated with white boxes. (B, C) Lysates from parental (mock), HA-FLAG-FBXO28, or HA-FLAG-CCNF overexpressing SH-SY5Y cells were subjected to HA immunoprecipitation (IP), SDS–PAGE and immunoblotting. Arrows indicate specific protein bands. (D) CCNF KO SH-SY5Y cells re-expressing HA-FLAG-CCNF WT, V335M, or D628V or left untreated (mock) were lysed and incubated with HA-agarose followed by SDS–PAGE and immunoblotting. Arrows indicate specific protein bands.