Journal: iScience

Article Title: Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity

doi: 10.1016/j.isci.2022.105293

Figure Lengend Snippet: Prdm14 depends on Stat3 activity to promote mESC self-renewal (A) Heatmap showing the expression of direct targets of Stat3 isolated from the high-throughput sequencing of transcriptomes regulated by Prdm14 upregulation. Genes were ranked according to the level of log2-fold change. (B) qRT‒PCR analysis of the expression of candidate genes in (A). The data are presented as the mean ± SD (N = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01 versus EV, as determined by Student’s t test. (C) Western blot analysis of Socs3, Stat3, and phospho-Stat3 Y705 (pStat3) in 46C mESCs overexpressing EV or PB- Prdm14 . β-Tubulin was used as a loading control. (D) Densitometric analysis of the relative protein levels of pStat3 and Socs3 was performed with ImageJ software (C). Protein levels were normalized to β-Tubulin. ∗∗p < 0.01 versus EV, as determined by Student’s t test. (E) qRT-PCR analysis of the expression of Stat3 direct genes in EV- and PB- Prdm14 -expressing cells cultured in serum-containing medium without LIF for 8 days in the presence or absence of 5 μM JAK inhibitor I (JAKi). The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus EV, # p < 0.05, ## p < 0.01 versus PB- Prdm14 , as determined by one-way ANOVA with Sidak’s multiple comparisons test. (F) AP staining of EV- and PB- Prdm14 -expressing cells treated with or without JAKi for 8 days in the absence of LIF. Bar,100 μM. (G) Quantification of AP-positive colonies in (F). (H) qRT‒PCR analysis of the expression levels of undifferentiated ( Oct4 , Esrrb , and Nanog ) and differentiated genes ( T , Cdx2 , and Sox17 ) in EV- and PB- Prdm14 -expressing cells treated with or without JAKi. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus EV, # p < 0.05 versus PB- Prdm14 , as determined by one-way ANOVA with Sidak’s multiple comparisons test. (I) Immunofluorescence of Sox2 in the indicated cells. Bar,100 μM. (J) Western blot analysis of Flag, pStat3 and Stat3 protein levels in PB- Prdm14 -expressing mESCs infected with scramble or Stat3 shRNA lentiviruses. (K) Quantification of AP-positive colonies in (L). (L) AP staining of Stat3 knockdown 46C mESCs overexpressing Prdm14 in the absence of LIF. Bar,100 μM. (M) Immunofluorescence staining of Sox2 in Stat3 knockdown 46C mESCs overexpressing Prdm14 and cultured in serum-containing medium without LIF for 8 days. Bar,100 μM. See also Figures S2 and , Tables S2 and .

Article Snippet: The medium used for routine maintenance was DMEM (Biological Industries, Israel) supplemented with 15% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma) and 1000 U/ml LIF (LIF1010, Millipore, USA).

Techniques: Activity Assay, Expressing, Isolation, Next-Generation Sequencing, Western Blot, Control, Software, Quantitative RT-PCR, Cell Culture, Staining, Immunofluorescence, Infection, shRNA, Knockdown

Journal: iScience

Article Title: Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity

doi: 10.1016/j.isci.2022.105293

Figure Lengend Snippet: Upregulation of Socs3 inhibits the self-renewal-promoting effect of Prdm1 4 (A) qRT‒PCR analysis of the expression levels of Prdm14 and Socs3 in 46C mESCs cultured in serum-containing medium treated with LIF for 1 h or 24 h. The data are presented as the mean ± SD (N = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01 versus NT. NT, no treatment, as determined by one-way ANOVA with Dunnet’s multiple comparisons test. ns: nonsignificant. (B) qRT‒PCR analysis of Socs3 transcripts in 46C mESCs infected with scramble or Socs3 shRNA lentiviruses. The data are presented as the mean ± SD (N = 3 biological replicates). ∗∗p < 0.01 versus scramble, as determined by one-way ANOVA with Dunnet’s multiple comparisons test. (C) AP staining of scramble and Socs3 shRNA 46C mESCs cultured in serum-containing medium without LIF for eight days. Bar,100 μM. (D) Quantification of AP-positive colonies in (C). (E) Western blot analysis of HA, Stat3, and pStat3 in PB- Prdm14 -expressing mESCs overexpressing EV or HA-tagged Socs3 . β-Tubulin was used as a loading control. (F) Densitometric analysis of the relative protein levels of pStat3 was performed with ImageJ software (E). Protein levels were normalized to β-Tubulin. ∗∗p < 0.01 versus EV, as determined by Student’s t test. (G) qRT‒PCR analysis of the expression of undifferentiation ( Sox2 , Nanog , Klf4 , Esrrb , and Tfcp2l1 ) and differentiation-associated genes ( T , Pax6 , Cdx2 , and Fgf5 ) in PB- Prdm14- expressing mESCs overexpressing EV or HA-tagged Socs3 . The data are presented as the mean ± SD (N = 3 biological replicates). ∗p < 0.05, ∗∗p < 0.01 versus EV, as determined by Student’s t test. (H and I) Immunofluorescence staining of Sox2 and AP staining of PB- Prdm14 -expressing mESCs overexpressing EV or HA-tagged Socs3 and cultured in serum-containing medium without LIF treatment for eight days. Bar, 100 μM. (J) Quantification of AP-positive colonies in (I). See also and and Tables S1–S3 .

Article Snippet: The medium used for routine maintenance was DMEM (Biological Industries, Israel) supplemented with 15% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma) and 1000 U/ml LIF (LIF1010, Millipore, USA).

Techniques: Expressing, Cell Culture, Infection, shRNA, Staining, Western Blot, Control, Software, Immunofluorescence

Journal: iScience

Article Title: Prdm14 promotes mouse ESC self-renewal and PGCLC specification through enhancement of Stat3 activity

doi: 10.1016/j.isci.2022.105293

Figure Lengend Snippet:

Article Snippet: The medium used for routine maintenance was DMEM (Biological Industries, Israel) supplemented with 15% FBS (FND500, ExCell Bio, Australia), 1× MEM nonessential amino acids (N1250, Solarbio, China), 0.1 mM β-mercaptoethanol (M3148, Sigma) and 1000 U/ml LIF (LIF1010, Millipore, USA).

Techniques: Recombinant, Protease Inhibitor, Staining, Reporter Assay, Isolation, Reverse Transcription, Plasmid Preparation, Purification, Software, Transfection