Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 depletion causes the nucleolar stress response in MCF10A and A549 cells. A , schematic of the nucleolar stress response in human cells. Disruption of ribosome biogenesis causes accumulation of free 5S RNP which inhibits the ubiquitin ligase MDM2, leading to accumulation of TP53 and increased transcription of CDKN1A . B , TP53 stabilization after SPRR3 depletion in MCF10A cells. Representative images and quantification of TP53 Western blots after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. TP53 levels were normalized to total protein (trichloroethanol stain), then siNT. C , CDKN1A mRNA levels are elevated after SPRR3 depletion in MCF10A cells. After 72 h of siSPRR3 treatment, CDKN1A mRNA transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. These data were normalized to 7SL RNA abundance, then to siNT for comparison using the ΔΔC T method. D , siSPRR3 reduces SPRR3 mRNA levels in A549 cells. After 72 h of siSPRR3 treatment, SPRR3 transcript levels were detected by RT-qPCR. The data were normalized as in ( C ). E , siSPRR3 reduces SPRR3 protein levels in A549 cells. After 72 h of treatment with siSPRR3, SPRR3 protein levels were detected by Western blot. SPRR3 levels were normalized as in ( B ). F , SPRR3 depletion lowers 47S/45S pre-rRNA levels in A549 cells. Primers to the 5′ ETS of the 47S/45S rRNA were used to detect pre-rRNA levels after 72h of treatment with siSPRR3. siNOL11 was used as a positive control. The data were normalized as in ( C and D ). G , TP53 stabilization after SPRR3 knockdown in A549 cells. Representative images and quantification of TP53 Western blots after 48h or 72h of treatment with siSPRR3 are shown. Protein levels were normalized as in ( B and E ). H , CDKN1A levels are elevated after SPRR3 depletion in A549 cells. After 72 h of treatment with siSPRR3, CDKN1A transcript levels were detected with RT-qPCR using primers for CDKN1A mRNA. siNOL11 was used as a positive control. Data were normalized as in ( C ). The mean ± SEM are shown alongside individual data points. For graphs with two conditions, data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. For graphs with more than two conditions, data were analyzed by ordinary one-way ANOVA with multiple comparisons against siNT and Holm-Šídák correction in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001.

Article Snippet: A549 human lung carcinoma cells (#CCL-185, American Type Culture Collection) were cultured in DMEM with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin.

Techniques: Disruption, Ubiquitin Proteomics, Western Blot, Positive Control, Staining, Quantitative RT-PCR, Comparison, Knockdown

Journal: The Journal of Biological Chemistry

Article Title: A high-throughput screen for nucleolar function reveals a role for the signaling protein, SPRR3, in ribosome biogenesis

doi: 10.1016/j.jbc.2026.111132

Figure Lengend Snippet: SPRR3 drives AKT phosphorylation and maintains POLR1A levels. A , AKT phosphorylation at serine 473 (pAKT) is decreased after a 72h SPRR3 depletion in MCF10A cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). Blots were probed for pAKT, then stripped and re-probed for total AKT. Signal was measured in Bio-Rad Image Lab. pAKT levels were normalized to total protein and total AKT. Total AKT was normalized to total protein, ensuring no significant difference in overall AKT levels upon SPRR3 depletion. B , phosphorylated AKT (pAKT) levels are decreased after 72h SPRR3 depletion in A549 cells. Representative images and quantification are shown for pAKT, total AKT, and total protein (measured by trichloroethanol stain). C , POLR1A levels are decreased upon 72h SPRR3 depletion. Representative image of Western blotting and quantification of POLR1A levels in MCF10A cells. siPOLR1A was used as a positive control. D , summary of effects of siSPRR3 depletion that we have confirmed in both MCF10A cells and A549 cells. For all graphs in this figure, the mean ± SEM are shown alongside individual data points. Data were analyzed by unpaired two-sided Welch's t -tests in GraphPad Prism. ∗, p < 0.05; ∗∗, p < 0.01; ∗∗∗, p < 0.001; ns, not significant.

Article Snippet: A549 human lung carcinoma cells (#CCL-185, American Type Culture Collection) were cultured in DMEM with 10% Fetal Bovine Serum (FBS) and 1% Penicillin-Streptomycin.

Techniques: Phospho-proteomics, Staining, Western Blot, Positive Control