Fig 2: HPV16 E2 expression causes nuclear accumulation of Rint1. (A and B) Validation of the Rint1 antibody used for detection of endogenous Rint1 was performed by Western blot analysis of untransfected (UT) C33a cell lysates and lysates from cells transfected with increasing amounts of an HA-Rint1-expressing plasmid (A) and IF detection in the absence (−) or presence (+) of a Rint1 antibody blocking peptide (B). (C) Localization of E2 and endogenous Rint1 was determined in C33a cells that were either untransfected or transfected with an HPV16 E2 expression plasmid. Cells were fixed and stained with a Rint1-specific antibody (red/gray), E2 TVG261 antibody (green), and the appropriate Alexa Fluor-conjugated secondary antibodies. Enlarged images are shown on the right. Colocalization was quantified by using ImageJ to calculate Manders' overlap coefficient; 95.4% of the E2 protein colocalized with Rint1 (SD, ±5.3%; n = 56). (D) Rint1 localization (red) and DNA staining (blue) in HPV16 episome-containing W12E cells. (E) Nuclear accumulation of the Rint1 protein in untransfected and E2-expressing cells was determined by subcellular fractionation followed by Western blotting of Rint1 and E2 proteins alongside markers for the cytoplasmic (C) (Grb2), membrane (M) (ERp72), and chromatin-bound (CB) (histone H3) fractions. (F) Untransfected or E2-transfected cells were costained with E2 (green)-, Rint1 (red)-, and Nopp140 (magenta)-specific antibodies to observe nucleolar localization. The white arrows indicate Nopp140-positive nucleoli that also contain the Rint1 protein, and the white arrowheads indicate E2/Rint1-positive nuclear foci that do not costain with Nopp140. Enlarged images are shown on the right. Bars, 10 μm.

Fig 3: TMED2 regulates SMO abundance at the PM in a SHH-dependent manner.(A, B) Activation of HH signaling promotes SMO cellular redistribution. (A) FM images of SMO–HA in WT NPCs treated for 24 hours with PMP or SHH and in Tmed2−/− cells. Scale bar = 10 μm. (B) Three-dimensional color distribution of HA–SMO in 3D-STORM experiments performed in WT NPCs treated with SHH for 24 hours and in Tmed2−/− NPCs. Scale bar = 5 μm. (C–F) SHH treatment and Tmed2 mutation promotes SMO trafficking form ER–Golgi compartments. Dual color 3D-STORM analysis showing ERP72 (C), RCAS1 (D) and RAB7 (E) distribution in green and the SMO colocalizing domains in gray. (F) Plot showing percentage of SMO–HA colocalizing events normalized to marker distribution upon SHH treatment and Tmed2 mutation. Asterisks denote statistical significance for difference from the WT untreated samples. (G) Tmed2 regulates SMO abundance at the PM. Cells were treated with NHS-SS-Biotin (Biotin) to label PM proteins, and with SHH as indicated. Western analysis of PM proteins and input before purification (1/50 of pull-down) are shown. (H) Percentage of cells expressing OLIG2 (left) and NKX2.2 (right) relative to total cell count in neuralized EBs. Neuralized EBs derived from WT cells and from ESCs overexpressing SMO–HA with and without a Tmed2 mutation were treated with or without SHH. Asterisks denote statistical significance for difference between indicated samples. Individual EBs are plotted (n = 10). (I) SHH treatment disrupts the SMO–TMED2 complex in NPCs. Western analysis of co-immunopurification of endogenous TMED2 with HA–SMO (top) and input (1/25 of IP, bottom) in NPCs expressing N-term HA tagged SMO. Cells were treated with recombinant SHH for the indicated amount of time. Actin is shown as loading control. (J) Summary of the proposed mechanism for TMED2-regulated SMO secretion from the ER–Golgi compartment. The data underlying all the graphs shown in the figure are included in the S1 Data file. EB, embryoid body; EGFR, epidermal growth factor receptor; ER, endoplasmic reticulum; ESC, embryonic stem cell; FM, fluorescence microscopy; HA, hemagglutinin; HH, hedgehog; LE, late endosome; NPC, neural progenitor cell; NT, not treated; PM, plasma membrane; PMP, purmorphamine; SHH, Sonic hedgehog; SMO, Smoothened; WT, wild-type.

Fig 4: Response over time to TBG-RNAi-CK2 treatment in PC3-LN4 xenograft tumors(A) Verification of nuclear and cytosol protein fractionation from tumor tissue is shown by comparative immunoblot analysis of CK2αα′, lamin A/C, ERp72, and actin. T1, T2, and T3 labels indicate different tumors. Antibody sourcing information is listed in Materials and Methods. (B) Immunoblot analysis of fractionated PC3-LN4 tumor lysates following intravenous treatments of 0.01 mg/kg TBG-RNAi-CK2 or TBG-RNAi-F7 as indicated above the blots. The signals for three mice per group are shown, the proteins detected are indicated on the right, and the cell fraction and size markers are indicated on the left. Actin signal was used as the loading control. T1, T2, and T3 labels indicate different tumors within each treatment group. Antibody sourcing information is listed in Materials and Methods. (C) The percentage of Ki-67-positive tumor cells was determined from Ki-67-stained tumor sections. The percentage of Ki-67-positive cells on each day was expressed relative to day 5 (setting day 5 as 100%) and is shown graphically for each treatment. Mean and standard deviation are shown. Sample sizes: n = 6 for all groups except n = 7 for TBG-RNAi-F7 day 7.

Fig 5: SMO interacts with TMED2 in the Golgi compartment.(A–C) Super-resolution SMO colocalization with (A) ERP72, (B) RCAS1, and (C) RAB7. SMO–HA cells were differentiated into NPCs and SMO–HA cellular distribution (red) was compared to organelle marker staining (green) in dual-color 3D-STORM experiments. Colocalizing events are shown in independent plots and labeled in gray; scale bar = 5 μm. Magnified areas are shown on the left; scale bar = 2 μm. (D) SMO colocalizes with TMED2 in NPCs in dual-color 3D-STORM experiments. Colocalization between SMO–HA (red) and TMED2 (green) is shown in the top left panel and compared to the one detected in parental cells with a Tmed2 mutation (bottom right); scale bar = 5 μm. Magnified area is shown on the left; scale bar = 500 nm. Colocalizing events are labeled in gray. (E) Quantification of the colocalizing events in SMO–HA NPCs WT and depleted for Tmed2. (F) TMED2 binds SMO in ESCs. Western analysis of coimmunopurification of endogenous TMED2 and TMED10 with SMO–HA (left) and input (1/25 of IP, right) from extracts of 2 independent ESCs clones expressing SMO–HA. (G) TMED2 binds SMO in NPCs. Western analysis of coimmunopurification of endogenous TMED2 with SMO–HA (left) and input (1/25 of IP, right). (H, I) Endogenous SMO colocalizes with TMED2 in NPCs. (H) Scheme showing the CRISPR/Cas-9 strategy used to endogenously tag the Smo gene with a 3xHA epitope in ESCs. (I) FM colocalization between endogenous 3xHA–SMO and TMED2 in experiments. Scale bar = 5 μm. Magnified area is shown on the bottom. The data underlying all the graphs shown in the figure are included in the S1 Data file. ESC, embryonic stem cell; FM, fluorescence microscopy; gRNA, guide RNA; HA, hemagglutinin; NPC, neural progenitor cell; SMO, Smoothened; WT, wild-type.