Cell lines and cell culture

Human cell lines T24, UM-UC-3, 5637, BIU87, and human embryonic kidney HEK293T were cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and 0.5% penicillin/streptomycin at 37 °C with 5% CO2. The patient-derived cell line (PDC) SYBC1 was cultured under the same conditions in RPMI 1640 with 10% FBS and 0.5% penicillin/streptomycin. Cell lines T24, UM-UC-3, 5637, and HEK293T were acquired from the American Type Culture Collection (ATCC), whereas the BIU87 cell line was obtained from the Kunming Cell Bank at the Chinese Academy of Sciences. The SYBC1 cell line was generously provided by Dr. Zhuowei Liu of the Sun Yat-sen University Cancer Center. All human cell lines were confirmed to be mycoplasma-negative and authenticated by short tandem repeat (STR) profiling analysis.

Plasmid construction, virus transduction

Human KLF16, MYC, DUSP16, and WBP11 cDNAs were amplified by PCR and cloned into the pSIN or pcDNA3.1 vectors, with or without tags (3xFlag, SFB, HA, EGFP, mCherry). The full-length mRNA of KLF16 was cloned into pCDH vectors. Mutants of SFB-DUSP16-C244S were generated from WT SFB-DUSP16 constructs using a one-step cloning kit (Vazyme, C113-02). Constructs for tagBFP-Pol II-CTD, MED1-IDR, BRD4, and CDK7 were obtained as previously described [53]. All shRNAs targeting human KLF16 and MYC were cloned into the PLKO.1 vector. sgRNAs were designed using GUIDES web tool (http://guides.sanjanalab.org/) and cloned into the lentiCRISPR v2 vector. The sequences of all shRNAs and sgRNAs used are provided in Supplementary Material 2.

Lentivirus packaging was performed in HEK293T cells by cotransfecting with the targeted plasmids, psPAX2, and pMD2.G using polyethyleneimine (PEI). After transfection for 6 to 8 h, the cell culture medium was replaced, and the virus-containing medium was collected after an additional 48 h. Transduced cell populations were typically selected 48 h post-infection.

qPCR

Total RNA was isolated using the RNAprep Pure Cell/Bacteria Kit (TIANGEN) and reverse transcribed with HiScript® II Q RT SuperMix for qPCR (+ gDNA wiper) (Vazyme) according to the manufacturer’s instructions. qPCR was conducted using the ChamQ Universal SYBR qPCR Master Mix (Vazyme) with the LightCycler 480 Instrument II system (Roche). The qPCR primers used are provided in Supplementary Material 2.

Western blotting and coimmunoprecipitation (Co-IP)

Cells were lysed with RIPA buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 0.5% NP-40; 5 mM EDTA; protease inhibitor cocktails and phosphatase inhibitors). The supernatant was collected via centrifugation (30 min, 4 °C, 12,000 rpm) and then boiled at 100 °C for 10 min after adding 1 × SDS sample buffer. Proteins were resolved by 8%-12% SDS–PAGE and transferred onto polyvinylidene difluoride membranes (Millipore). The membranes were blocked with 5% nonfat milk (1 h at room temperature) and incubated with the indicated primary antibodies (overnight at 4 °C), followed by incubation with horseradish peroxidase–conjugated secondary antibody (1 h at room temperature). The chemiluminescent signaling was detected by using ECL reagents (Tanon). The antibodies used and their concentrations are listed in Supplementary Material 3.

For co-IP, the clarified cell lysates were incubated with antibody-coated beads or agarose for 2 h or overnight at 4 °C. For endogenous co-IP assay, the clarified cell lysates were first incubated with specific primary antibodies overnight at 4 °C, followed by the addition of protein A/G agarose beads and further incubation for 4 h at 4 °C. Afterwards, the immune-precipitates were washed four times with cold RIPA buffer and then boiled at 100 °C for 10 min after adding 1 × SDS sample buffer.

Colony formation assay

Stable cells were seeded in six-well plates at a density of 500 cells per well. After 7–10 days, when visible clones had formed, they were washed with phosphate-buffered saline (PBS), fixed in methanol, and stained with 0.1% crystal violet. Colonies with more than 50 cells were quantified using ImageJ software.

OptoDroplets assay

The optoDroplets assay was performed as described previously [35]. Briefly, T24 and HEK293T cells were transfected with KLF16-mCherry-Cry2 and MYC-mEGFP-Cry2 separately or together. Cells were illuminated with a 488-nm laser, and images were captured for mEGFP, mCherry, or both signals at the indicated time using a spinning disk confocal microscope (Nikon CSU-W1).

MTT assay

BLCA stable cells were seeded into 96-well plates at a density of 2,000 cells (T24, UM-UC-3, SYBC1 stable cells) or 4,000 cells (BIU87 stable cells) per well and incubated for the indicated time. Subsequently, MTT solution was added to each well at a final concentration of 0.5 mg mL−1 followed by incubation for 4 h at 37 °C. The formazan crystals were dissolved in DMSO, and the optical density (OD) was measured at 490 nm using a microplate reader. The IC50 values were calculated using GraphPad software with Nonlinear regression, Dose–response inhibition, log (inhibitor) versus normalized response Variable slope methods.

Apoptosis assay

BLCA stable cells were seeded into 6-well plates and incubated for 24 h, then treated with the indicated drugs for 24–48 h. Apoptotic cells were collected and subjected to apoptosis assay using the Annexin V-FITC/PI Apoptosis Detection Kit (KGA107, KeyGEN BioTECH) following the manufacturer’s instructions.

Dual-luciferase reporter assay

Cells were seeded in 24-well plates and incubated for 24 h. They were then cotransfected with 0.5 µg of a promoter-luciferase plasmid and 10 ng of pRL-CMV (Renilla luciferase) using Lipofectamine 3000. After 48 h of transfection, luciferase activity was measured using a Dual-luciferase Reporter Assay kit (Promega) following the manufacturer’s instructions. Renilla luciferase was utilized as an internal control to normalize luminescence levels.

RNA–protein interaction detection based on TurboID and MS

The following procedures were performed as described previously [27, 28]. To detect proteins interacting with full-length KLF16 mRNA, we first generated two constructs: a puromycin-labeling doxycycline-inducible λN-3 × HA-turboID construct and a G418-labeled pCGN-boxB-KLF16-FL-boxB-GFP construct. These expression vectors were transiently transfected into HEK293T cells for viral packaging. T24 cells were then infected with the λN-3 × HA-turboID lentivirus, and stable cell lines expressing λN-3 × HA-turboID were established through puromycin selection. A secondary infection was performed using the boxB-KLF16-FL-boxB lentivirus or a boxB-vector control. G418 selection was added to obtain the stable cell line of interest. Subsequently, stable cells were treated with doxycycline (100 ng mL−1) for 24 h, followed by the addition of 50 μM biotin to the culture medium for 20 min. The cells were lysed and sonicated in RIPA-SDS buffer (50 mM Tris–HCl, pH 8.0; 150 mM NaCl; 0.1% SDS; 0.5% sodium deoxycholate; 1% Triton X-100). The clarified cell lysates were incubated with streptavidin beads at 4 °C for 2 h, followed by sequential washes: twice with lysis buffer, once each with 1 M KCl buffer, 0.1 M Na2CO3 buffer, 2 M urea in 10 mM Tris buffer, and twice more with lysis buffer. After suspension in 1 × loading buffer and boiling at 100 °C for 10 min, the beads were subjected to mass spectrometry (MS) analysis.

RNA interference treatment

BLCA cells, at 20–30% confluence, were transfected with 100 nM small interfering RNAs (siRNAs) using Lipofectamine RNAiMAX in accordance with the manufacturer’s instructions. The sequences of all siRNAs used in this study are listed in Supplementary Material 2.

Ubiquitination assay

UM-UC-3 stable cells were seeded in 10 cm plates and transfected with a His-tagged ubiquitin (His-Ub) plasmid for 24 h. Cells were treated with bortezomib (1 μM) for 8 h and subsequently lysed with buffer A (0.1 M Na2HPO4/NaH2PO4; 6 M guanidine-HCl; 10 mM imidazole, pH 8.0). After sonication, the cell lysates were incubated with Ni–NTA beads (Beyotime) at room temperature for 2–4 h. His-ubiquitinated precipitates were washed three times with buffer A, twice with buffer A&Ti (3 parts buffer Ti and one part buffer A), and once with buffer Ti (20 mM imidazole and 25 mM Tris–HCl, pH 6.8). The His-ubiquitinated precipitates were then resuspended in buffer Ti and boiled at 100 °C for 10 min after adding 1 × SDS sample buffer.

Immunohistochemical staining (IHC) and fluorescence IHC

Paraffin-embedded tissue slides were heated at 60 °C for 2 h, followed by deparaffinization, dehydration, and antigen retrieval. Subsequently, the tissue slides were blocked with goat serum (30 min, room temperature) and then incubated with the indicated primary antibodies against KLF16 (sc-377519, SantaCruz) and MYC (ab32072, Abcam) at 4 °C overnight. The tissue slides were then washed three times with PBST and treated with 3% H2O2 for 10 min to block endogenous peroxidase activity. Afterwards, the tissue slides were incubated with anti-mouse/rabbit IgG secondary antibody, treated with DAB reagent (Dako Omnis), and counterstained with hematoxylin staining solution (Beyotime Biotechnology) according to the manufacturer’s instructions. The expression of KLF16 and MYC was evaluated using the H-score, which combines staining intensity (0, no evidence of staining; 1, weak; 2, moderate; 3, strong) with the percentage of stained cells at each intensity level (0% to 100%). The final H-score was calculated by multiplying the staining intensity by the percentage of cells, as previously reported [54]. The median IHC score of KLF16 served as the cutoff value to distinguish between the high and low KLF16 expression groups. The IHC scores of MYC and KLF16 are listed in Supplementary Material 5. The paraffin-embedded pathologic specimens from 80 patients with BLCA were obtained from Sun Yat-sen University Cancer Center, and additional details are provided in Supplementary Material 6.

For fluorescence IHC, slides were subjected to deparaffinization, antigen retrieval, and blocking. They were then incubated with primary antibodies against KLF16 and MYC overnight at 4 °C. Afterward, the slides were treated with Alexa Fluor 594/488-conjugated secondary antibody (Invitrogen) for 2 h at room temperature. Cell nuclei were counterstained with Hoechst 33,342 (Thermo Fisher, H3570) for 2 min. The antibodies used and their concentrations are detailed in Supplementary Material 3.

RNA-seq analysis

T24 cells stably expressing KLF16-targeted sgRNAs/shRNAs or a control were collected, and total RNA was extracted using TRIzol reagent (Life Technologies, 15,596,026). RNA sequencing assays were performed by Novogene (Beijing, China). Genes with an adjusted P-value < 0.05 were considered differentially expressed genes. For GSEA, the MYC signaling pathway gene set was generated based on the gene list from the KEGG. Normalized expression data were then analyzed and visualized using GSEA software (version 3.0, http://www.broadinstitute. org/gsea/). The RNA-seq analysis results for T24 cells expressing KLF16-targeted shRNAs are detailed in Supplementary Material 7.

Chromatin immunoprecipitation (ChIP)-qPCR

Cells were cross-linked with 1% formaldehyde (10 min, room temperature) and stopped with glycine (125 mM) for 5 min at room temperature. After four times washes with cold PBS, cells were scraped from the dishes and centrifuged at 800 × g at 4 °C for 5 min. Fixed pellet cells were lysed with cell lysis buffer (20 mM Tris–HCl, pH 8.0; 85 mM KCl; 0.5% NP-40; protease inhibitor cocktails) on ice for 15 min. Supernatants were removed, and the isolated nuclei were resuspended in nuclei lysis buffer (10 mM Tris–HCl, pH 7.5; 1% NP40; 0.5% deoxycholate and 0.1% SDS; protease inhibitor cocktails). After sonication, the samples were immunoprecipitated with MYC antibody (2 μg, ab32072, Abcam) or normal rabbit IgG (CST) overnight at 4 °C. ChIP-grade Protein A/G magnetic beads (Millipore) were added and incubated for 2 h. The immunoprecipitates were then washed twice, each with low-salt, high-salt, LiCl buffer, and TE buffer. Eluted DNA was reverse-crosslinked and purified using a PCR purification kit (Qiagen), and analyzed by qPCR on the LightCycler 480 Instrument II system (Roche). The specific primers used are provided in Supplementary Material 2.

CUT&Tag assays

CUT&Tag assays were performed essentially as described previously [55]. Briefly, 1 × 105 T24 cells were incubated with 10 μL of concanavalin A-coated magnetic beads (Bangs Laboratories) for 10 min at room temperature. Bead-bound cells were then suspended in dig wash buffer (20 mM HEPES, pH 7.5; 150 mM NaCl; 0.5 mM Spermidine; 1 × Protease inhibitor cocktail; 0.05% Digitonin; and 2 mM EDTA) and incubated overnight at 4 °C with a 1:50 dilution of antibodies against MYC (ab32072, Abcam) and FLAG (14,793, CST). After removal of primary antibody using a magnet stand, cells were incubated with a secondary antibody (1:100 Anti-Rabbit IgG antibody, Goat monoclonal: Millipore AP132) for one hour at room temperature, followed by incubation with the pA-Tn5 adapter complex for an additional hour. After three washes with Dig-med buffer (0.01% digitonin; 20 mM Hepes, pH 7.5; 300 mM NaCl; 0.5 mM spermidine; and 1 × protease inhibitor cocktail), cells were resuspended in Tagmentation buffer (10 mM MgCl2 in Dig-med Buffer) and incubated at 37 °C for 1 h. Genomic DNA was extracted using the TIANamp Genomic DNA Kit (TIANGEN) according to the manufacturer’s instructions. Subsequently, sequencing libraries were purified using XP beads (Beckman Counter). The sequencing procedure was carried out on the Illumina NovaSeq 6000 platform, utilizing PE150 sequencing as recommended by the manufacturer. The antibodies used and their concentrations are listed in Supplementary Material 3.

Animal experiments

Male athymic BALB/c nude mice were purchased from the GemPharmatech. For the BLCA subcutaneous transplantation model, T24 (3 × 106) and BIU87 (4 × 106) stable cells were resuspended in PBS and mixed in equal volumes with matrigel. The mixture was then injected subcutaneously into the flanks of the nude mice at the age of 5 weeks (n = 7–8 per group). Seven days after injection, tumor sizes were measured using a caliper every three days, and tumor volume was calculated using the formula: tumor volume = 1/2 (length × width2). Mice were sacrificed at the final time point, and the xenograft tumors were dissected and weighed.

For assays involving DDP/gemcitabine and OTX015 combination treatments, mice bearing T24 tumors were randomly assigned to the experimental groups. The groups received either vehicle, DDP (5 mg kg−1, intraperitoneally twice weekly), gemcitabine (50 mg kg−1, intraperitoneally twice weekly), OTX015 (25/50 mg kg−1, intragastric administration daily), or a combination of DDP/gemcitabine plus OTX015.

For combination treatment assays with DDP and ABBV-744, mice bearing T24 tumors were randomly assigned to 6 groups (n = 7 per group). The groups received either vehicle, DDP (3/5 mg kg−1, intraperitoneally twice weekly), ABBV-744 (10 mg kg−1, intragastric administration daily), or a combination of DDP and ABBV-744. The vehicle was prepared and administered in the same manner as the drug treatments, with the exception that no drug was added. Doses of 100 μL were administered by intraperitoneal injection or intragastric administration. Tumor sizes were measured and tumor volume was calculated. Mice were sacrificed at the final time point, and the xenograft tumors were dissected and weighed.

Statistical analysis

All results were obtained from three independent experiments. Statistical analyses were performed with GraphPad Prism 10.2.0 using Student’s two-tailed unpaired t-tests for comparisons between two groups. KLF16 and MYC expression and cancer survival curves were assessed by Kaplan–Meier plots and compared by a log-rank test. To test differences in mouse tumor growth, we conducted a two-way ANOVA analysis followed by Tukey’s multiple comparisons test. To determine if age, gender, grade, TNM stage, and treatment are independent risk parameters from clinical features, we performed multivariate analyses using the Cox proportional hazards model and data stratification. Statistical research was carried out using the SPSS 26.0 software (SPSS, Inc., Chicago, IL, USA). Data are presented as the mean ± SD from a minimum of three independent experiments. For correlation analysis, the Spearman correlation coefficient was calculated for crosstabs and the P-value was determined by Pearson's chi-squared test.