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

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: IFNs mediate immunotherapy-associated gene expression in tumors and activate immune cells in healthy PBMCs. A qPCR was used to detect the PD-L1 expression in A549 (5 × 10 5 in a 6-well plate) treated with IFNα and IFNγ (20 ng/mL for each, incubated for 2 h). B The expression of the top 8 up-regulated genes from RNAseq analysis (Table S2), including ICAM1, BATF, IRF1, SOCS1, HAPLN3, TAP1, PSMB9, and MAFF, were validated by qPCR analysis in A549 treated with IFNα and IFNγ (20 ng/mL for each, incubated for 2 h). C The healthy PBMCs (1 × 10 6 in a 6-well plate) treated with IFNs (20 ng/mL for each, incubated for 24 h), co-cultured with A549, IFNα- and IFNγ (3 h)-pretreated A549 at a 20:1 ratio, and IFNα, IFNγ, and A549 concurrently treated for 24 h were analyzed by flow cytometry to (D and E) detect the activation marker CD107a levels in CD4 + T, CD8 + T cells, and CD45 + CD3 − (nonT) PBMCs (n = 3). CD4 + T and CD8 + T were gated by staining with anti-CD45-Pacific blue, anti-CD3-APC/Cy7, anti-CD8-Alexa488, and anti-CD4-PE. CD107a was detected using anti-CD107a-BV605. (F) In addition, the activation markers IFNG, and cytotoxic marker granzyme B (GZMB) were detected by qPCR in the collected PBMCs after individual treatments. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Gene Expression, Expressing, Incubation, RNA sequencing, Cell Culture, Flow Cytometry, Activation Assay, Marker, Staining, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy increases IFNs and promotes immunogenic activity but distinct gene expression in the IFNγ- and 8 Gy-treated A549 cells. A A549 cells (5 × 10 5 in a 6-well plate) treated with irradiation (0, 1, 2, 4, 8 Gy) and further incubated for 24 h were collected to detect the p53-downstream gene CDKN1A, MDM2, and tumor marker MKI67 by qPCR. Statistical analysis was achieved by one-way ANOVA. B In addition, IFNA and IFNG and their downstream PD-L1 expression in the irradiated A549 cells and C MDM2 inhibitor Nutlin-3a-treated A549 cells were measured by qPCR. D The healthy PBMCs (1 × 10 6 in a 6-well plate) treated with IFNα and IFNγ (20 ng/mL for each) for 2 h and 24 h were collected and analyzed for the immune activation marker IFNG and cytotoxic marker granzyme B (GZMB) expression using qPCR. E M1 markers TNFA and CXCL10, and M2 markers ARG1 and IL-10 were also investigated in the collected PBMCs with the individual treatments by qPCR. F The healthy PBMCs (1 × 10 6 in a 6-well plate) incubated with irradiation-treated A549 (0, 4, 8 Gy) at a ratio of 20:1 for 24 h were collected and investigated for the immune activation marker IFNG and cytotoxic marker GZMB expression using qPCR. G Meanwhile, M1 markers TNFA, CXCL10, and M2 markers ARG1, IL-10 were also investigated in the collected PBMCs with the individual treatments by qPCR. (H) RNAseq was used to search for the differential genes in the A549 cells (5 × 10 5 in a 6-well plate) treated with IFNγ (20 ng /mL, incubated for 2 h) and x-ray irradiation (8 Gy was selected based on the highest CDKN1A and MDM2 induction, 24 h post-irradiation). There were 75 up-regulated and 12 down-regulated genes selected in IFNγ-treated A549 and 88 up-regulated and 202 down-regulated genes selected in 8 Gy-treated A549 according to log2 fold change > 1 or < -1 with p value < 0.05 (Table S2-4). There was no overlapped gene between IFNγ and 8 Gy treatment. I The 75 and 88 up-regulated genes in IFNγ- and irradiated A549 cells were analyzed by NetworkAnalyst ( https://www.networkanalyst.ca/ ) based on the KEGG dataset, revealing that the differential genes were involved in STATs and p53 signaling pathways, respectively. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001. ns, non-significant

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Activity Assay, Gene Expression, Irradiation, Incubation, Marker, Expressing, Activation Assay, RNA sequencing, Protein-Protein interactions, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy specifically suppresses TP53-wild type tumors. A Flow cytometry based on fluorescent Annexin V staining was used to detect apoptosis in the irradiation (0, 4, and 8 Gy)-treated TP53-wild type A549, HCT116, and TP53null HCT116 cells (5 × 10 5 in a 6-well plate) post 24 h culture. n = 2. B - C qPCR was used to validate the 13 irradiation-mediated genes from RNAseq (Table S3 and Table S4), including the increased MDM2, CYFIP2, STOM, and the decreased MKI67, CENPE, ARGHGAP11A, BRCA1, ASPM, ALAN, TOP2A, FANCI, TOPBP1, and ECT2, in (B) A549 treated with 8 Gy (24 h post-irradiation), C A549 treated with MDM2 inhibitor Nutlin-3a (10 µg/mL for 24 h). D and E qPCR was also used to detect p53-downstream CDKN1A and the selected 13 genes in TP53-wild type and TP53null HCT116 treated with 8 Gy of irradiation (24 h post-irradiation). n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Flow Cytometry, Staining, Irradiation, RNA sequencing, Two Tailed Test

Journal: Cancer Immunology, Immunotherapy : CII

Article Title: Radiotherapy enhances M1 macrophage immunogenic activity through IFNs induction and stimulation in TP53-wild type tumors

doi: 10.1007/s00262-026-04300-7

Figure Lengend Snippet: Radiotherapy induces IFNs in TP53-wild type tumors. A qPCR was used to detect the expression of IFNs in TP53-wild type HepG2, HCT116, TP53-mutant PLC5, and TP53null HCT116 (5 × 10 5 in a 6-well plate) treated with 0, 4, 8 Gy of irradiation (24 h post-irradiation). B The IFNγ-mediated up-regulated genes from RNAseq analysis (Table S2), including PD-L1, ICAM1, BATF, IRF1, SOCS1, HAPLN3, TAP1, PSMB9, and MAFF, were investigated by qPCR in TP53-wild type and TP53null HCT116 (5 × 10 5 in a 6-well plate) treated with 8 Gy of irradiation (24 h post-irradiation). C The cultured medium from the irradiated A549 (0, 4, 8 Gy, post 24 h) was collected to treat parental A549 (5 × 10 5 in a 6-well plate) for 2 h. qPCR was used to measure the selected gene expression. n = 3 and error bars were presented by SD in qPCR analysis. Statistical analysis was achieved by an unpaired two-tailed Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001

Article Snippet: The human lung cancer cell lines A549, HepG2, and PLC5 used in this study were purchased from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Mutagenesis, Irradiation, RNA sequencing, Cell Culture, Gene Expression, Two Tailed Test