Fig 2: SEC mediates the rapid initiation of differentiation in response to PKC signaling.a qRT-PCR comparing the relative mRNA expression of differentiation-activating TFs as well as ATF3, DUSP1, and RND3 with 1 hour or 3 hours of TPA treatment relative to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). b qRT-PCR shows that the PKC inhibitors, gouml6983 (Gouml) or bisindolylmaleimide I (Bis), consistently suppress the upregulation of representative genes induced by TPA treatment (1 hour) as compared with the DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). c, d qRT-PCR comparing gene expression induced by TPA alone or in combination with KL where panel c is TPA relative to a DMSO control and d is KL + TPA relative to KL alone. (n = 3 technical replicates, data are presented as mean values ± standard deviation). e qRT-PCR showing gene upregulation by TPA (1 hour) in combination with CDK9 inhibitors, flavopiridol (Flav) or NVP2, relative to DMSO control (n = 3 technical replicates, data are presented as mean values ± standard deviation). f Venn diagram (left) and heatmap (right) comparing 3-hour KL RNA-seq and 1-hour TPA RNA-seq differentially expressed (fold change ≥2, P < 0.05, two-tailed, Wald test) genes (Fisher’s exact test, two-tail, P = 2×10−5). Source data are provided as a Source Data file.

Fig 3: TMEM106C regulates apoptosis pathway and LINC00238 is transcriptionally regulated by ATF3 in HCC. (A) The Enriched KEGG pathway analysis showed that TMEM106C suppressed the apoptosis signaling pathway. (B) The results of western blotting showed that protein expression of ASP7, TIMP2, PDCD4 and ATF3 in HCCLM3 and MHCC-97h cells was increased by LINC00238 overexpression while decreased by TMEM1106C overexpression. *P<0.05 vs. NC group; #P<0.05 vs. pc-LINC00238 group. (C) Recognition motif of ATF3 from the JASPAR database. (D) Venn diagram showing ATF3 is upstream transcription factor of LINC00238 of LINC00958 predicted by PROMO and KEGG. (E) The results of western blotting showed that ATF3 protein expression in HCCLM3 and MHCC-97h cells was decreased by ATF3 siRNAs. (F) The results of RT-qPCR showed that LINC00238 expression in HCCLM3 and MHCC-97h cells was decreased by ATF3 siRNAs. (G) The results of RT-qPCR showed that ATF3 expression was significantly lower in HCC tissues than that in corresponding normal tissues. (H) Scatter plots comparing ATF3 expression in HCC samples (n=396) and normal tissue samples (n=160) showed that ATF3 was downregulated in HCC tumor samples compared with normal samples. (I) Kaplan-Meier analysis indicated an association between high TMEM106 and poor overall survival in patients with HCC. (J) The correlation analysis among ATF3, LINC00238 and TMEM106C showed that the ATF3 expression was positively correlated with LINC00238 and negatively correlated with TMEM106C expression. *P<0.05. TMEM106C, transmembrane protein 106C; LINC, long intergenic non-protein coding RNA; HCC, hepatocellular carcinoma; KEGG, Kyoto Encyclopedia of Genes and Genomes; ATF3, activating transcription factor 3; CASP7, caspase-7; TIMP2, tissue inhibitor of metalloproteinase 2; PDCD4, programmed cell death 4; pc-, plasmid; si, short interfering; RT-qPCR, reverse transcription-quantitative PCR.

Fig 4: ATF3 overexpression reverses the promoting effect of LINC00238 silencing on the procession of HCC. (A) RT-qPCR showed that LINC00238 expression was significantly decreased by LINC00238 silencing. (B) RT-qPCR showed that ATF3 expression was significantly decreased by ATF3 overexpression. (C) MTS assay showed that ATF3 overexpression reduced the proliferation ability of HCCLM3 and MHCC-97h cells and eliminated the promoted effect of LINC00238 silence on HCC proliferation. (D) Flow cytometry showed that ATF3 overexpression increased the apoptosis of HCCLM3 and MHCC-97h cells and eliminated the inhibitory effect of LINC00238 silence on HCC apoptosis. (E) Wound healing assay showed that ATF3 overexpression inhibited the migration capacity of HCCLM3 and MHCC-97h cells and eliminated the promoted effect of LINC00238 silence on HCC migration (magnification, ×100). (F) Transwell assay showed that ATF3 overexpression inhibited the invasion capacity of HCCLM3 and MHCC-97h cells and eliminated the promoted effect of LINC00238 silence on HCC invasion (magnification, ×200). *P<0.05 vs. NC group; #P<0.05 vs. si-LINC00238 group. ATF3, activating transcription factor 3; LINC, long intergenic non-protein coding RNA; HCC, hepatocellular carcinoma; RT-qPCR, reverse transcription-quantitative PCR.

Fig 5: The SEC rapid-response gene ATF3 drives keratinocyte differentiation.a–c Western blot showing the overexpression (OE) of ATF3, DUSP1, or RND3 in keratinocytes using the pTRIPZ vector post DOX induction. d–f qRT-PCR showing the mRNA expression levels of differentiation-activating TFs with ATF3, DUSP1, or RND3 overexpression (n = 3 technical replicates, data are presented as mean values ± standard deviation). g–j Representative images and quantification of clonogenic assays comparing human keratinocytes expressing ATF3, DUSP1, or RND3 versus the empty-vector control. Colonies >1 mm2 are included for quantification (n = 3/group, ****P < 0.001, **P < 0.05, ns = not significant, ATF3 P < 0.0001, DUSP1 P = 0.3489, RND3 P = 0.0406, two-tailed, unpaired t test, data are presented as mean values ± standard deviation). Source data are provided as a Source Data file.