Fig 2: Schematic illustration of the MAPKAR. Two tandem ERK1 monomers were inserted between the N-terminal and C-terminal fragments of click beetle luciferase, and coupled by a flexible glycine-serine (G/S) linker. MAPK activation (phosphorylation and ERK1 interaction) keeps the split-luciferase halves apart and no emission of luminescence occurs. MAPK inactivation triggers a conformational change that brings the split luciferase halves together and a luminescent signal is emitted.

Fig 3: Cbx3.VD enables modular and potent gene repression.a Relative EGFP levels of HEK293T-SV40 EGFP reporter cells stably transduced with different KRAB domain-containing repressors (KOX1-dCas9, ZIM3-dCas9, and Cbx3.VD-ZIM3-dCas9) and a repressor lacking KRAB domains, obtained by directly fusing Cbx3.VD to dCas9 (Cbx3.VD-dCas9). Histograms show the mean from three independent experiments ±1 SD of EGFP levels relative to untransduced reporter cells. b Repression of endogenous CD81. CRISPRi repressors were recruited to CD81 TSS and CD81 surface expression levels were measured by flow cytometry 4 days after antibiotic selection of repressor-transduced cells. Left panel shows CD81 surface levels relative to HEK293T cells transduced only with gRNA targeting CD81 promoter (guide only). Data are mean from three independent measurements ± 1 SD, asterisks indicate p values (two-tailed unpaired t-test, **p = 0.002, ***p = 0.0001). Some error bars are too small to be visible. Right panel shows representative flow cytometry profiles of CD81 expression detected with an anti-CD81 PE-conjugated antibody. c–e Comparison of the repressive potency of Cbx3.VD-ZIM3-dCas9 with that of ZIM3-dCas9 and dCas9. CRISPRi repressors were recruited to ERK1 and APRC2 loci and their protein expression levels in c HEK293T, d U20S, and e HeLa cells were measured by western blot. The percentage relative expression was determined by densitometry and was normalized to the band intensity of untransduced cells (mean ± 1 SD; from two biological replicates). See Supplementary Fig. 12 for representative western blots.

Fig 4: RAGE presence caused a decrease in p38 MAPK activation in AFBs due to diabetic-conditioned media while ERK activation TLR4 expression remained the same. Protein expression was quantified of TLR4 (95 kDa: (A)), ERK 1/2 (42/44 kDa: (B)) and p38 MAPK (38 kDa: (C)). All protein levels were normalized to β-tubulin and (D) representative western blot images. Data was and graphed as mean ± SEM with n = 3–6 of independent replicates. A two-way ANOVA with Fisher’s LSD post hoc was performed (* p < 0.05) on each graph individually. All data were analyzed together with a two-way ANOVA to give the following: TLR4 (Interaction; p = 0.9501, Genotype; p < 0.0001, and Treatment; p = 0.5220), ERK 1/2 (Interaction; p = 0.9976, Genotype; p < 0.0001, and Treatment; p = 0.9408), and p38 MAPK (Interaction; p = 0.9292, Genotype; p < 0.0001, and Treatment; p = 0.2917).

Fig 5: ERK2 function is required for normal TOP2B behavior and transcriptional activation.a qRT-PCR results presenting the effects of ERK2 catalytic inhibition on EGR1 and HSP70 transcription. HEK293 cells were not synchronized (n = 3). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. b qRT-PCR results presenting the effects of ERK2 catalytic inhibition on the EGR1 and FOS transcription (n = 3). HEK293 cells were synchronized at G0 (S0) before they were serum-induced to early G1 (S15). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. c ChIP-qPCR results showing S2 Pol II occupancy changes in the EGR1 gene with or without functional ERK2 (n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. d ChIP-qPCR results showing a dramatic increase of TOP2B at EGR1 upon the catalytic inhibition of ERK2 (n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. e Immunoblots of ERK1 (K1KD), ERK2 (K2KD), and ERK1/ERK2 double KD (DoubleKD) HEK293 cells. SCR, scrambled siRNA; Tubulin, a reference. f qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on EGR1 transcription (left) and TOP2B occupancies at the EGR1 TSS (right), respectively (n = 3 independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. g qRT-PCR and ChIP-qPCR results presenting the effects of ERK KDs on FOS transcription (left) and TOP2B occupancies at the FOS TSS (right), respectively (n = independent experiments). Data are presented as mean values and SD. P values for the bar graphs were calculated with the unpaired, one sided Student’s t test. Source data are provided as a Source Data file.