Fig 2: SMYD3‐mediated methylation enhances the formation of HER2 homodimer. (A) HeLa cells were transfected with FLAG‐HER2 and HA‐HER2, with Mock vector or SMYD3‐expressing vector. After 24 h of incubation, cells were treated with 0 or 100 ng/mL of EGF. Cell extracts were immunoprecipitated with anti‐FLAG ® M2 affinity gel, and immunoblotted with anti‐HA (Y‐11), anti‐FLAG (F7425), anti‐HER2 (29D8), anti‐EGFR (D38B1), anti‐SMYD3 (D2Q4V), and anti‐ACTB (#4967). (B and C) 293T cells were transfected with HA‐HER2‐WT, and FLAG‐HER2‐WT or FLAG‐HER2‐K175A in the presence of SMYD3 expression vector and incubated for 48 h. Cell lysates were immunoprecipitated with anti‐HA‐agarose. (B) or anti‐FLAG ® M2 affinity gel. (C), then immunoblotted with anti‐FLAG (F7425), anti‐HA (Y‐11), anti‐SMYD3 (D2Q4V), and anti‐ACTB (#4967). (D) 293T cells were cotransfected with FLAG‐HER2‐WT or FLAG‐HER2‐K175A, and Mock vector or SMYD3‐expressing vector. After 48 h of incubation, cell lysates were immunoprecipitated with anti‐FLAG ® M2 affinity gel and immunoblotted with anti‐FLAG (F7425), anti‐phospho HER2 (Tyr 1248) (#2247), anti‐SMYD3 (D2Q4V), and anti‐ACTB (#4967). The signal intensities of phosphorylated HER2 were quantified, and normalized by each FLAG level.

Fig 3: PIKfyve inhibition in Inpp4b-deficient cells disrupts lysosome gene expression.A, Inpp4b+/+ or Inpp4b−/− MEFs transiently expressing pEGFP-TFEB and treated with vehicle or apilimod 10 nM for 48 h. DNA was stained with Hoechst 33342. B, TFEB nuclear translocation measured as percentage of cells demonstrating nuclear EGFP-TFEB for (A). The scale bar represents 25 μm. C, Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h followed by qRT-PCR analysis of lysosome gene expression normalized against Actb. Shown is mean ± SD from three independent experiments. D, immunoblot of Inpp4b+/+ or Inpp4b−/− MEFs treated with vehicle or apilimod 10 nM for 48 h followed by assessment of select lysosomal proteins as indicated. E, analysis of protein levels from (D) shown as mean ± SD from three independent experiments. Data represent ± SD from five independent experiments with 30 to 35 cells assessed per treatment condition per experiment for (A and B). F, Inpp4b+/+ or Inpp4b−/− MEF treated with vehicle or apilimod 10 nM for 48 h followed by Magic red incubation for 1 h. The scale bar represents 25 μm. G, quantification of Magic red intensity from (F) as measured through flow cytometry and mean ± SD from three independent experiments. Statistical significance was measured by ANOVA and multiple Student’s t test and represented as ∗ (p < 0.05). INPP4B, inositol polyphosphate 4-phosphatase type II; MEF, mouse embryonic fibroblast; PIKfyve, Phosphoinositide Kinase, FYVE-Type Zinc Finger Containing; TFEB, Transcription Factor EB.

Fig 4: Growth kinetics of HiBiT-tagged rIBVs in culture cells. (A) Viral protein synthesis in Vero cells infected with the rIBVs in a time course infection. Vero cells were infected with rIBV-p65, rS-HiBiT, or rM-HiBiT at MOI ~ 2. Cell lysates were harvested by three freeze/thaw cycles at the indicated time points and subjected to SDS-PAGE, followed by Western blot analysis using antisera against IBV S2, M, and N. Beta-actin was used as the loading control. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. (B) Growth kinetics of the three rIBVs in Vero cells. Vero cells were infected as in (A). Cell lysate and supernatant samples harvested at the indicated time points were subjected to virus titration using the TCID50 method. IBV titers were expressed in the unit of log TCID50 per ml. The experiment was repeated three times with similar results, and the result of one representative experiment is shown. (C) Quantification of HiBiT protein by luminescent assays in rIBV-infected cells. Vero cells were infected as in (A). 30 μL cell lysate or supernatant samples were subjected to luminescent analysis using the Nano-Glo HiBiT lytic detection system. The background (medium only) was subtracted from the measured relative light unit (RLU) values, and luminescent intensities were expressed in the unit of log RLU per 30 μL. The experiment was repeated three times. The average values and standard deviations of the three experiments were shown. (D) Quantification of HiBiT protein in other cell lines infected with rS-HiBiT. DF1, H1299, and BHK cells were infected with rIBV-p65 or rS-HiBiT at MOI ~ 2. Quantification of S-HiBiT was done as in (C). The experiment was repeated three times. The average values and standard deviations of the three experiments were shown.

Fig 5: SH3BGRL drives autophagy through PIK3C3 translation by interaction ribosome. (A) GST affinity isolation of SH3BGRL with ribosomal subunits. GST affinity isolation was performed with lysates of MCF-7 and MDA-MB-453 cells, followed by MS peptide sequencing. (B) Heatmap of RNA-seq and Polyribosome-profile analyses of ULK1, PIK3C3, BECN1, ATG7, ATG5, ATG3, MAP1LC3B and SQSTM1. (C) Immunoblots of SH3BGRL and PIK3C3 in indicated cells. ACTB was used as an internal loading control. Statistical analysis of PIK3C3 expression in indicated cells by four independent immunoblots. (D) Immunoblots of PIK3C3 protein in the indicated cells treated with or without 50 µg/ml cycloheximide (CHX) for 6 h. PIK3C3 expression was statistically analyzed based on three independent experiments. (E,F) Polyribosome profiling of PIK3C3 mRNA in MCF-7 (E) or MDA-MB-453 (F) cells. ACTB was used as an internal negative loading control. The marked numbers are equivalent to the collected fractions after gradient centrifuge. Fraction 1 represents the top (10%) of the gradient, fraction 10 represents the bottom (50%) of the gradient. (G) Polyribosome profiling of ATG12 mRNA in indicated cells as (E). All experiments were independently repeated at least three times. (H) Immunoblots of ULK1, BECN1, ATG7, ATG5 and ATG3 in the indicated cells. ACTB was used as a loading control. All significant differences are shown as *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.